An in vitro workflow to create and modify infectious clones using replication cycle reaction.

Reverse genetics systems are critical tools in combating emerging viruses which enable a better understanding of the genetic mechanisms by which viruses cause disease. Traditional cloning approaches using bacteria are fraught with difficulties due to the bacterial toxicity of many viral sequences, resulting in unwanted mutations within the viral genome. Here, we describe a novel in vitro workflow that leverages gene synthesis and replication cycle reaction to produce a supercoiled infectious clone plasmid that is easy to distribute and manipulate. We developed two infectious clones as proof of concept: a low passage dengue virus serotype 2 isolate (PUO-218) and the USA-WA1/2020 strain of SARS-CoV-2, which replicated similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. Results indicate that our workflow is a viable method to generate and manipulate infectious clones for viruses that are notoriously difficult for traditional bacterial-based cloning methods.

Hydrogen Bonding (Base Pairing) in Antiviral Activity.

Base pairing based on hydrogen bonding has, since its inception, been crucial in the antiviral activity of arabinosyladenine, 2'-deoxyuridines (i.e., IDU, TFT, BVDU), acyclic nucleoside analogues (i.e., acyclovir) and nucleoside reverse transcriptase inhibitors (NRTIs). Base pairing based on hydrogen bonding also plays a key role in the mechanism of action of various acyclic nucleoside phosphonates (ANPs) such as adefovir, tenofovir, cidofovir and O-DAPYs, thus explaining their activity against a wide array of DNA viruses (human hepatitis B virus (HBV), human immunodeficiency (HIV) and human herpes viruses (i.e., human cytomegalovirus)). Hydrogen bonding (base pairing) also seems to be involved in the inhibitory activity of Cf1743 (and its prodrug FV-100) against varicella-zoster virus (VZV) and in the activity of sofosbuvir against hepatitis C virus and that of remdesivir against SARS-CoV-2 (COVID-19). Hydrogen bonding (base pairing) may also explain the broad-spectrum antiviral effects of ribavirin and favipiravir. This may lead to lethal mutagenesis (error catastrophe), as has been demonstrated with molnutegravir in its activity against SARS-CoV-2.

THE SPIKE OF SARS-CoV-2 VARIANTS ALLOWS FOR MORE EFFICIENT COUNTERACTION OF BST2

Background: BST2/Tetherin is an interferon-stimulated gene with antiviral activity against enveloped viruses. Particularly, BST2 tethers virions at their site of assembly, preventing their release and spread. In addition to this primary role, BST2 is as an important bridge between the innate and adaptive immune system, since (i) BST2 routes tethered particles to lysosomes, which generates viral breakdown products that engage pattern recognition receptors;and (ii) trapped virions facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). In turn, viruses have evolved mechanisms to bypass BST2, either by targeting BST2 for proteasomal/lysosomal degradation or by removing BST2 from sites of virion assembly. However, the role of BST2 in SARS-CoV-2 replication, spread, evolution, and pathogenesis remains largely unknown. Method(s): The antiviral potential of BST2 against SARS-CoV-2 was investigated by infecting different SARS-CoV-2 isolates (Hong Kong, Alpha, Beta, Delta, and Omicron) in BST2+ and BST2- cells. Culture supernatants were collected to assess virion production by ELISA and infectivity by TCID50. Infected cells were analyzed by western blot and flow cytometry to examine viral and cellular protein levels, including BST2. Transfection of individual SARS-CoV-2 ORFs and mutagenesis studies allowed us to identify the genes that the virus uses to downregulate BST2. Immunoprecipitation assays revealed protein-protein interactions and changes in ubiquitination patterns. Experiments with proteasomal and lysosomal inhibitors furthered our mechanistic understanding of how SARS-CoV-2 counteracts BST2. Finally, fluorescence microscopy studies uncovered changes in the subcellular distribution of BST2 in SARS-CoV-2 infected cells. Result(s): While BST2 reduces virion release, SARS-CoV-2 has evolved to counteract this effect. Specifically, SARS-CoV-2 uses the Spike to interact with BST2, sequester the protein at perinuclear locations, and ultimately route it for lysosomal degradation. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that each variant is more efficient than the previously circulating strain at downregulating BST2 and facilitating virion production, and that mutations in the Spike account for their enhanced BST2 antagonism. Conclusion(s): As part of its adaptation to humans, SARS-CoV-2 is improving its capacity to counteract BST2, highlighting that BST2 antagonism is important for SARS-CoV-2 infectivity and transmission.

Determining the Effect of KmtR Mutants, E101Q, H102Q, and H111Q on the Metal Binding Affinities

Mycobacterium tuberculosis is the second leading infectious killer after COVID-19. The bacteria utilizes several metal transport systems to help it survive in the host.With an increase in the number of multiresistant, extensively resistant and totally drug-resistant strains, the development of new therapeutic strategies that target other essential pathways in the bacteria is critical. The bacteria contain several metal transport systems which are necessary for its survival. Additionally, the bacteria has two metalloregulators that are associated with nickel and cobalt export, NmtR and KmtR. The focus of this research is on KmtR, which represses the expression of the genes, cdf (which encodes the export protein) and kmtR. The goal of our research is to identify the residues that are responsible for binding the cognate metals, nickel and cobalt, as well as the noncognate metal, zinc, to KmtR. Mutagenesis studies coupled with metal binding experiments will be used to determine how KmtR binds these metals. The E101Q, H102Q, and H111Q mutants, among others, have been made, expressed, and purified in our lab. Data obtained from Isothermal Titration Calorimetry determined that all three mutant proteins bind cobalt with nanomolar affinities and the H111Q mutant KmtR proteins binds cobalt an order of magnitude weaker than the other two mutant proteins. Research reported as supported fully by the RI Institutional Development Award (IDeA) Network for Biomedical Research Excellence (RI-INBRE) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #P20GM103430.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Comparison of the Old and New - Novel Mechanisms of Action for Anti-coronavirus Nucleoside Analogues

Over the past two and a half years the world has seen a desperate scramble to find a treatment for SARS-CoV-2 and COVID. In that regard, nucleosides have long served as the cornerstone to antiviral treatments due to their resemblance to the naturally occurring nucleosides that are involved in numerous biological processes. Unlike other viruses however, it was found early on during the search for drugs to treat SARS-1 and later MERS, that the coronaviruses possess a unique repair enzyme, an exonuclease (ExoN)[3] which rendered nucleoside analogues useless, thus negating their use.[4] During the current outbreak however, as both well-known and new nucleoside analogues were investigated or reinvestigated as a possible cure for SARS-CoV-2, several novel and/or lesser-known mechanisms of action were uncovered. This review briefly describes these mechanisms.

Study of SARS-nCoV2 Indian Isolates Gaining Insights into Mutation Fre-quencies, Protein Stability and Prospective Effect on Pathogenicity

Background: SARS-nCOV-2 is a variant of the known SARS coronavirus family. The mutations in viruses are very rapid and can play a crucial role in the evolution or devolution of the organism. This has a direct impact on "host jumping" and the pathogenicity of the virus. Objective(s): The study aims to understand the frequency of genomic variations that have occurred in the virus affecting the Indian sub-population. The impact of variations translating to proteins and its consequences affecting protein stability and interaction were studied. Method(s): Phylogenetic analysis of the 140 genomes from the India region was performed, followed by SNP and Indel analysis of both CDS and non-CDS regions. This effort was followed by a prediction of mutations occurring in 8 proteins of interest and the impact on protein stability and prospective drug interactions. Result(s): Genomes showed variability in origin, and major branches can be mapped to the 2002 outbreak of SARS. The mutation frequency in CDS regions showed that 241 C >T, 3037 C >T, 2836 C >T, and 6312 C >A occurred in 81.5% of genomes mapping to major genes. Corresponding mutations were mapped to protein sequences. The effect of mutations occurring in spike glycoprotein, RNA dependent RNA polymerase, nsp8, nucleocapsid and 3c protease was also depicted. Conclusion(s): Whilst the mutations in spike glycoprotein showcased an increase in protein stability, the residues undergoing mutations were also a part of drug binding pockets for hydroxychloro-quine. Mutations occurring in other proteins of interest led to a decrease in protein stability. The mutations were also a part of drug binding pockets for Favipiravir, Remdesivir and Dexametha-sone. The work allows analyzing larger datasets to understand mutation patterns globally.Copyright © 2021 Bentham Science Publishers.

Current pandemic COVID-19 vaccine strategies and development: a comprehensive review

The coronavirus disease-19 pandemic with the characteristics of asymptomatic condition, long incubation period and poor treatment has influenced the entire globe. Coronaviruses are important emergent pathogens, specifically, the recently emerged sever acute respiratory syndrome coronavirus 2, the causative virus of the current COVID-19 pandemic. To mitigate the virus and curtail the infection risk, vaccines are the most hopeful solution. The protein structure and genome sequence of SARS-CoV-2 were processed and provided in record time;providing feasibility to the development of COVID-19 vaccines. In an unprecedented scientific and technological effort, vaccines against SARS-CoV-2 have been developed in less than one year. This review addresses the approaches adopted for SARS-CoV-2 vaccine development and the effectiveness of the currently approved vaccines.Copyright © 2023, Finlay Ediciones. All rights reserved.

Comprehensive deep mutational scanning reveals the pH induced stability and binding differences between SARS-CoV-2 spike RBD and human ACE2.

The SARS-CoV-2 spike (S) glycoprotein with its mobile receptor-binding domain (RBD), binds to the human ACE2 receptor and thus facilitates virus entry through low-pH-endosomal pathways. The high degree of SARS-CoV-2 mutability has raised concern among scientists and medical professionals because it created doubt about the effectiveness of drugs and vaccinations designed specifically for COVID-19. In this study, we used computational saturation mutagenesis approach, including structure-based free energy calculations to analyse the effects of the missense mutations on the SARS-CoV-2 S-RBD stability and the S-RBD binding affinity with ACE2 at three different pH (pH 4.5, pH 6.5, and pH 7.4). A total of 3705 mutations in the S-RBD protein were analyzed, and we discovered that most of these mutations destabilize the RBD protein. Specifically, residues G404, G431, G447, A475, and G526 were important for RBD protein stability. In addition, RBD residues Y449, Y489, Y495, Q498, and N487 were critical for the RBD-ACE2 interaction. Next, we found that the distribution of the mean stability changes and mean binding energy changes of RBD due to mutations at both serological and endosomal pH correlated well, indicating the similar effects of mutations. Overall, this computational analysis is useful for understanding the effects of missense mutations in SARS-CoV-2 pathogenesis at different pH.Communicated by Ramaswamy H. Sarma.

A Rapid Method for Generating Infectious SARS-CoV-2 and Variants Using Mutagenesis and Circular Polymerase Extension Cloning.

The appearance of SARS-CoV-2 variants in late 2020 raised alarming global public health concerns. Despite continued scientific progress, the genetic profiles of these variants bring changes in viral properties that threaten vaccine efficacy. Thus, it is critically important to investigate the biologic profiles and significance of these evolving variants. In this study, we demonstrate the application of circular polymerase extension cloning (CPEC) to the generation of full-length clones of SARS-CoV-2. We report that, combined with a specific primer design scheme, this yields a simpler, uncomplicated, and versatile approach for engineering SARS-CoV-2 variants with high viral recovery efficiency. This new strategy for genomic engineering of SARS-CoV-2 variants was implemented and evaluated for its efficiency in generating point mutations (K417N, L452R, E484K, N501Y, D614G, P681H, P681R, Δ69-70, Δ157-158, E484K+N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (ΔORF7A) and insertion (GFP). The application of CPEC to mutagenesis also allows the inclusion of a confirmatory step prior to assembly and transfection. This method could be of value in the molecular characterization of emerging SARS-CoV-2 variants as well as the development and testing of vaccines, therapeutic antibodies, and antivirals. IMPORTANCE Since the first emergence of the SARS-CoV-2 variant in late 2020, novel variants have been continuously introduced to the human population, causing severe public health threats. In general, because these variants acquire new genetic mutation/s, it is critical to analyze the biological function of viruses that such mutations can confer. Therefore, we devised a method that can construct SARS-CoV-2 infectious clones and their variants rapidly and efficiently. The method was developed based on a PCR-based circular polymerase extension cloning (CPEC) combined with a specific primer design scheme. The efficiency of the newly designed method was evaluated by generating SARS-CoV-2 variants with single point mutations, multiple point mutations, and a large truncation and insertion. This method could be of value for the molecular characterization of emerging SARS-CoV-2 variants and the development and testing of vaccines and antiviral agents.

A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport.

The ongoing SARS-CoV-2/COVID-19 pandemic caused a global public health crisis. Yet, everyone's response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson's coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXXΦ motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187-188) displayed a similar phenotype to the YXXΦ deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson's coefficients ≥0.8). Structural analyses further suggest that the diG motif supports a type-II ß-turn between the anti-parallel ß4 and ß5 sheets and connects to the YXXΦ motif via hydrogen bonds between two monomers. The diG- YXXΦ interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.

In-silico investigation of systematic missense mutations of middle east respiratory coronavirus spike protein.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe pneumonia-like symptoms and is still pose a significant threat to global public health. A key component in the virulence of MERS-CoV is the Spike (S) protein, which binds with the host membrane receptor dipeptidyl peptidase 4 (DPP4). The goal of the present investigation is to examine the effects of missense mutations in the MERS-CoV S protein on protein stability and binding affinity with DPP4 to provide insight that is useful in developing vaccines to prevent coronavirus infection. We utilized a saturation mutagenesis approach to simulate all possible mutations in the MERS-CoV full-length S, S Receptor Binding Domain (RBD) and DPP4. We found the mutations in MERS-CoV S protein residues, G552, C503, C526, N468, G570, S532, S451, S419, S465, and S435, affect protein stability. We identified key residues, G538, E513, V555, S557, L506, L507, R511, M452, D537, and S454 in the S protein RBD region are important in the binding of MERS-CoV S protein to the DPP4 receptor. We investigated the effects of MERS-CoV S protein viral mutations on protein stability and binding affinity. In addition, we studied all DPP4 mutations and found the functional substitution R336T weakens both DPP4 protein stability and S-DPP4 binding affinity. We compared the S protein structures of MERS-CoV, SARS-CoV, and SARS-CoV-2 viruses and identified the residues like C526, C383, and N468 located in equivalent positions of these viruses have effects on S protein structure. These findings provide further information on how mutations in coronavirus S proteins effect protein function.

Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin.

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

SKI-1/S1P Facilitates SARS-CoV-2 Spike Induced Cell-to-Cell Fusion via Activation of SREBP-2 and Metalloproteases, Whereas PCSK9 Enhances the Degradation of ACE2.

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2, which requires cleavage of its spike protein (S) at two sites: S1/S2 and S2'. This study investigates the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. The assays used were cell-to-cell fusion in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increased cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduced cell-to-cell fusion by promoting the cellular degradation of ACE2. SKI-1 activity led to enhanced S2' formation, which was attributed to increased metalloprotease activity as a response to enhanced cholesterol levels via activated SREBP-2. However, high metalloprotease activity resulted in the shedding of S2' into a new C-terminal fragment (S2″), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2″ formation abolished S2' and cell-to-cell fusion, as well as pseudoparticle entry, indicating that the formation of S2″ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested the binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms.

Favipiravir: An Antiviral Drug

Favipiravir is an antiviral drug that has been shown to treat a variety of life-threatening infections, including Ebola, Lassa, and the COVID-19 virus. It's a pyrazine carboxamide derivative with antiviral action that targets RNAdependent RNA polymerase enzymes, which are required for viral genome transcription and replication. Favipiravir is an antiviral previously indicated for influenza and Ebola, which has shown some promise in early trials for treatment of COVID-19. The nucleoside analogue favipiravir is rapidly metabolized in host cells which disrupts viral synthesis and leads to mutagenesis The mechanism of action of the Favipiravir and Side effects like QTc prolongation or teratogenicity pose risk to extensive community application discusses in this review. In this article, we have tried to provide a comprehensive, evidence-based review of this drug about synthesis, Pharmacology, Mechanism of Action, Antiviral activity, Consequences.

Computational analysis of the novel Thailand-specific mutations in SARS-CoV-2 spike glycoprotein sequences

As of 14 January 2022, 2.3 million people in Thailand had been reportedly infected with SARS-CoV-2, and 21,883 people had died. Spike glycoprotein, on the SARS-CoV-2 membrane, is a key factor for viral infection. Some scientists have demonstrated that some amino acid mutations in the protein increase infectivity and transmissibility of the virus. However, many studies concerning mutations in the spike glycoprotein, particularly in Thailand, were not comprehensive enough to illustrate the impacts of the mutations on the spike glycoprotein. To narrow this gap, we examined approximately 8.3 million spike glycoprotein sequences retrieved from GISAID Initiative and NCBI Virus databases to identify novel mutations. Limiting our scope to the Thai samples, we demonstrated how local SARS-CoV-2 strains changed over 2021. In addition, we found that T95I had emerged and become one of the main characteristics of delta strains in Thailand. We further detected 28 Thailand-specific novel mutations, which were then analyzed with amino acid-based analysis tools to gain insights into their impacts on the spike glycoprotein. Upon closer examination, we found that 2 novel mutations, L249E and R457W, were likely to diminish the interactions between the spike glycoprotein and neutralizing antibodies in silico. This finding suggests that both mutations may hinder the neutralization, allowing the virus to escape the antibodies. Additionally, our study highlights the significant effects of some novel mutations on the stability and functionality of the spike glycoprotein, which may be useful for COVID-19 diagnosis and vaccine development. [ FROM AUTHOR]

An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites-a case study with Omicron Spike DNA.

Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as 4 other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required.

Bacterial drug-resistance and viability phenotyping upon disinfectant exposure revealed by single-nucleotide resolved-allele specific isothermal RNA amplification.

Disinfectant abuse poses a risk of bacterial evolution against stresses, especially during the coronavirus disease 2019 (COVID-19) pandemic. However, bacterial phenotypes, such as drug resistance and viability, are hard to access quickly. Here, we reported an allele specific isothermal RNA amplification (termed AlleRNA) assay, using an isothermal RNA amplification technique, i.e., nucleic acid sequence-based amplification (NASBA), integrated the amplification refractory mutation system (ARMS), involving the use of sequence-specific primers to allow the amplification of the targets with complete complementary sequences. AlleRNA assay enables rapid and simultaneous detection of the single nucleotide polymorphism (SNP) (a detection limit, a LOD of 0.5 % SNP) and the viability (a LOD of 80 CFU) of the quinolone resistant Salmonella enterica. With the use of AlleRNA assay, we found that the quinolone resistant S. enterica exhibited higher survival ability during exposure toquaternary ammonium salt, 75 % ethanol and peracetic acid, which might be attributed to the upregulation of stress response-associated genescompared with the susceptible counterparts. Additionally, the AlleRNA assay indicated the potential risk in a high-frequency occurrence of viable but nonculturable (VBNC) quinolone resistant S. enterica induced by disinfectants due to the depression of ATP biosynthesis. The excessive usage of disinfectants during the COVID-19 pandemic should be carefully evaluated due to the latent threat to ecological and human health.

Vaccinia Virus-Based Reverse Genetics for Feline Coronaviruses

For decades, the genetic modification of coronavirus genomes and the generation of recombinant coronaviruses have been hampered mostly due to the extraordinary large size of the coronaviral genome. The very first reverse genetic system for feline coronaviruses (FCoVs) was established in the early 2000s;the respective approach exclusively enabled the manipulation of the 3'-third of the viral genome. Later on, vaccinia virus-and bacterial artificial chromosome (BAC)-based systems have been developed. Both systems have the advantage that the entire FCoV genome is amenable for mutagenesis. The main focus of this chapter is the vaccinia virus-based reverse genetic system for FCoVs. Here we present protocols for (1) the generation of a full-length cDNA clone, (2) the manipulation of the FCoV genome, and (3) the rescue of recombinant FCoVs. Copyright © Springer Science+Business Media New York 2016.

Databases, DrugBank, and virtual screening platforms for therapeutic development

The upsurge of the severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has turned into a global health disaster. Many remodeled medications were suggested for treatment in the early stages of this pandemic, but these dosages afterward came across with distinct offshoots. Thus, these consequences compelled the scientists to develop new drugs using various antiviral, antiinflammatory, antibacterial, and phytochemical compounds. A handful of drugs have been scrutinized in silico, in vitro, plus through human trials such as anti-SARS-CoV-2 agents and made available as various databases by various scientific communities. The SARS-CoV-2 pandemic databases are designed to allay difficulties associated with this scenario. Some of the popular databases are GESS (global evaluation of SARS-CoV-2/HCoV-19 sequences) which gives a thorough study of data based on tenfold of thousands of complete coverage and quality of SARS-CoV-2 genomes, CORona Drug InTERactions (CORDITE) database for SARS-CoV-2 which profoundly combines the understanding of potential drugs and make it available for scientists and medicos. SARSCOVIDB set one’s sights to merge all differential gene expression data, at mRNA and protein levels, helping to accelerate analysis and research on the molecular impact of covid-19. This chapter aims to provide a piece of complete information about the SARS-CoV-2 virus databases, potentially available drugs, and virtual screening methods. And also provides a different webserver to reach out for information related to the COVID-19 pandemic and its future. © 2022 Elsevier Inc. All rights reserved.

Structural Insights Into Tautomeric Dynamics in Nucleic Acids and in Antiviral Nucleoside Analogs.

DNA (2'-deoxyribonucleic acid) and RNA (ribonucleic acid) play diverse functional roles in biology and disease. Despite being comprised primarily of only four cognate nucleobases, nucleic acids can adopt complex three-dimensional structures, and RNA in particular, can catalyze biochemical reactions to regulate a wide variety of biological processes. Such chemical versatility is due in part to the phenomenon of nucleobase tautomerism, whereby the bases can adopt multiple, yet distinct isomeric forms, known as tautomers. For nucleobases, tautomers refer to structural isomers that differ from one another by the position of protons. By altering the position of protons on nucleobases, many of which play critical roles for hydrogen bonding and base pairing interactions, tautomerism has profound effects on the biochemical processes involving nucleic acids. For example, the transient formation of minor tautomers during replication could generate spontaneous mutations. These mutations could arise from the stabilization of mismatches, in the active site of polymerases, in conformations involving minor tautomers that are indistinguishable from canonical base pairs. In this review, we discuss the evidence for tautomerism in DNA, and its consequences to the fidelity of DNA replication. Also reviewed are RNA systems, such as the riboswitches and self-cleaving ribozymes, in which tautomerism plays a functional role in ligand recognition and catalysis, respectively. We also discuss tautomeric nucleoside analogs that are efficacious as antiviral drug candidates such as molnupiravir for coronaviruses and KP1212 for HIV. The antiviral efficacy of these analogs is due, in part, to their ability to exist in multiple tautomeric forms and induce mutations in the replicating viral genomes. From a technical standpoint, minor tautomers of nucleobases are challenging to identify directly because they are rare and interconvert on a fast, millisecond to nanosecond, time scale. Nevertheless, many approaches including biochemical, structural, computational and spectroscopic methods have been developed to study tautomeric dynamics in RNA and DNA systems, and in antiviral nucleoside analogs. An overview of these methods and their applications is included here.