Deep mining of the Sequence Read Archive reveals major genetic innovations in coronaviruses and other nidoviruses of aquatic vertebrates.

Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.

Label-free drug interaction screening via Raman microscopy.

Development of a simple, label-free screening technique capable of precisely and directly sensing interaction-in-solution over a size range from small molecules to large proteins such as antibodies could offer an important tool for researchers and pharmaceutical companies in the field of drug development. In this work, we present a thermostable Raman interaction profiling (TRIP) technique that facilitates low-concentration and low-dose screening of binding between protein and ligand in physiologically relevant conditions. TRIP was applied to eight protein-ligand systems, and produced reproducible high-resolution Raman measurements, which were analyzed by principal component analysis. TRIP was able to resolve time-depending binding between 2,4-dinitrophenol and transthyretin, and analyze biologically relevant SARS-CoV-2 spike-antibody interactions. Mixtures of the spike receptor-binding domain with neutralizing, nonbinding, or binding but nonneutralizing antibodies revealed distinct and reproducible Raman signals. TRIP holds promise for the future developments of high-throughput drug screening and real-time binding measurements between protein and drug.

The harms of promoting the lab leak hypothesis for SARS-CoV-2 origins without evidence.

Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.

ICTV Virus Taxonomy Profile: Coronaviridae 2023.

The family Coronaviridae includes viruses with positive-sense RNA genomes of 22-36 kb that are expressed through a nested set of 3' co-terminal subgenomic mRNAs. Members of the subfamily Orthocoronavirinae are characterized by 80-160 nm diameter, enveloped virions with spike projections. The orthocoronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome-related coronavirus are extremely pathogenic for humans and in the last two decades have been responsible for the SARS and MERS epidemics. Another orthocoronavirus, severe acute respiratory syndrome coronavirus 2, was responsible for the recent global COVID-19 pandemic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Coronaviridae which is available at www.ictv.global/report/coronaviridae.

Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5.

The 5' cap structures of higher eukaryote mRNAs have ribose 2'-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2'-O-methylation of mRNA remains elusive. Here we show that 2'-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2'-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2'-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2'-O-methylation of mRNA suggests that RNA modifications such as 2'-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

Isolation, Characterisation and Experimental Evolution of Phage that Infect the Horse Chestnut Tree Pathogen, Pseudomonas syringae pv. aesculi.

Bleeding canker of horse chestnut trees is a bacterial disease, caused by the bacterium Pseudomonas syringae pv. aesculi, estimated to be present in ~ 50% of UK horse chestnut trees. Currently, the disease has no cure and tree removal can be a common method of reducing inoculum and preventing spread. One potential method of control could be achieved using naturally occurring bacteriophages infective to the causative bacterium. Bacteriophages were isolated from symptomatic and asymptomatic horse chestnut trees in three locations in the South East of England. The phages were found to be belonging to both the Myoviridae and Podoviridae families by RAPD PCR and transmission electron microscopy. Experimental coevolution was carried out to understand the dynamics of bacterial resistance and phage infection and to determine whether new infective phage genotypes would emerge. The phages exhibited different coevolution patterns with their bacterial hosts across time. This approach could be used to generate novel phages for use in biocontrol cocktails in an effort to reduce the potential emergence of bacterial resistance.

Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture.

Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A2α (cPLA2α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA2α activity, which produces lysophospholipids (LPLs) by cleaving at the sn-2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA2α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development.IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2α, which releases fatty acid from the sn-2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments in which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication, and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLA2α inhibitor.

Identification and Characterization of a Ribose 2'-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales.

UNLABELLED: The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2'-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2'-O-MTase activity, although we could not detect 2'-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2'-O-MTase, the roniviral 2'-O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2'-O-MTases and can target only the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with the coronavirus protein, roniviral 2'-O-MTase does not require a protein cofactor for stimulation of its activity and differs in its preference for several biochemical parameters, such as reaction temperature and pH. Furthermore, the ronivirus 2'-O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family. IMPORTANCE: Methylation of the 5'-cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as a 2'-O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales In this study, we identified a 2'-O-methyltransferase encoded by ronivirus that shows common and unique features in comparison with that of coronaviruses. Ronivirus 2'-O-MTase does not need a protein cofactor for MTase activity, whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2'-O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional links between roniviruses and coronaviruses.

The proteome of the infectious bronchitis virus Beau-R virion.

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus infectious bronchitis virus (IBV). It was thought that coronavirus virions were composed of three major viral structural proteins until investigations of other coronaviruses showed that the virions also include viral non-structural and genus-specific accessory proteins as well as host-cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs, purified by sucrose-gradient ultracentrifugation and analysed by mass spectrometry. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus 35 additional virion-associated host proteins. The virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, while others were found to be orthologous to proteins identified in severe acute respiratory syndrome coronavirus virions and also virions from a number of other RNA and DNA viruses.

Synthesis and antiviral properties of spirocyclic [1,2,3]-triazolooxazine nucleosides.

An efficient synthesis of spirocyclic triazolooxazine nucleosides is described. This was achieved by the conversion of ß-D-psicofuranose to the corresponding azido-derivative, followed by alkylation of the primary alcohol with a range of propargyl bromides, obtained by Sonogashira chemistry. The products of these reactions underwent 1,3-dipolar addition smoothly to generate the protected spirocyclic adducts. These were easily deprotected to give the corresponding ribose nucleosides. The library of compounds obtained was investigated for its antiviral activity using MHV (mouse hepatitis virus) as a model wherein derivative 3 f showed the most promising activity and tolerability.

A Novel Non-Destructive Rapid Tool for Estimating Amino Acid Composition and Secondary Structures of Proteins in Solution.

Amino-acid protein composition plays an important role in biology, medicine, and nutrition. Here, a groundbreaking protein analysis technique that quickly estimates amino acid composition and secondary structure across various protein sizes, while maintaining their natural states is introduced and validated. This method combines multivariate statistics and the thermostable Raman interaction profiling (TRIP) technique, eliminating the need for complex preparations. In order to validate the approach, the Raman spectra are constructed of seven proteins of varying sizes by utilizing their amino acid frequencies and the Raman spectra of individual amino acids. These constructed spectra exhibit a close resemblance to the actual measured Raman spectra. Specific vibrational modes tied to free amino and carboxyl termini of the amino acids disappear as signals linked to secondary structures emerged under TRIP conditions. Furthermore, the technique is used inversely to successfully estimate amino acid compositions and secondary structures of unknown proteins across a range of sizes, achieving impressive accuracy ranging between 1.47% and 5.77% of root mean square errors (RMSE). These results extend the uses for TRIP beyond interaction profiling, to probe amino acid composition and structure.

Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories.

Introduction: Pathogen leak from a high-containment laboratory seriously threatens human safety, animal welfare, and environmental security. Transportation of pathogens from a higher (BSL4 or BSL3) to a lower (BSL2) containment laboratory for downstream experimentation requires complete pathogen inactivation. Validation of pathogen inactivation is necessary to ensure safety during transportation. This study established a validation strategy for virus inactivation. Methods: SARS-CoV-2 wild type, delta, and omicron variants underwent heat treatment at 95°C for 10 minutes using either a hot water bath or a thermocycler. To validate the inactivation process, heat-treated viruses, and untreated control samples were incubated with A549-hACE2 and Vero E6-TMPRSS2-T2A-ACE2 cells. The cells were monitored for up to 72 hours for any cytopathic effects, visually and under a microscope, and for virus genome replication via RT-qPCR. The quality of post-treated samples was assessed for suitability in downstream molecular testing applications. Results: Heat treatment at 95°C for 10 minutes effectively inactivated SARS-CoV-2 variants. The absence of cytopathic effects, coupled with the inability of virus genome replication, validated the efficacy of the inactivation process. Furthermore, the heat-treated samples proved to be qualified for COVID-19 antigen testing, RT-qPCR, and whole-genome sequencing. Discussion: By ensuring the safety of sample transportation for downstream experimentation, this validation approach enhances biosecurity measures. Considerations for potential limitations, comparisons with existing inactivation methods, and broader implications of the findings are discussed.

Quantum-Enhanced Detection of Viral cDNA via Luminescence Resonance Energy Transfer Using Upconversion and Gold Nanoparticles.

The COVID-19 pandemic has profoundly impacted global economies and healthcare systems, revealing critical vulnerabilities in both. In response, our study introduces a groundbreaking method for the detection of SARS-CoV-2 cDNA, leveraging Luminescence resonance energy transfer (LRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) to achieve an unprecedented detection limit of 242 femtomolar (fM). This innovative sensing platform utilizes UCNPs conjugated with one primer and AuNPs with another, targeting the 5' and 3' ends of the SARS-CoV-2 cDNA, respectively, enabling precise differentiation of mismatched DNA sequences and significantly enhancing detection specificity. Through rigorous experimental analysis, we established a quenching efficiency range from 10.4\% to 73.6\%, with an optimal midpoint of 42\%, thereby demonstrating the superior sensitivity of our method. By comparing the quenching efficiency of mismatched DNAs to the target DNA, we identified an optimal DNA:UCNP:AuNP ratio that ensures accurate detection. Our comparative analysis with existing SARS-CoV-2 detection methods revealed that our approach not only provides a lower detection limit but also offers higher specificity and potential for rapid, on-site testing. This study demonstrates the superior sensitivity and specificity of using UCNPs and AuNPs for SARS-CoV-2 cDNA detection, offering a significant advancement in rapid, accessible diagnostic technologies. Our method, characterized by its low detection limit and high precision, represents a critical step forward in managing current and future viral outbreaks, contributing to the enhancement of global healthcare responsiveness and infectious disease control.

SARS-CoV-2 Main Protease Inhibitors That Leverage Unique Interactions with the Solvent Exposed S3 Site of the Enzyme.

The main protease (MPro) of SARS-CoV-2 is crucial for the virus's replication and pathogenicity. Its active site is characterized by four distinct pockets (S1, S2, S4, and S1-3') and a solvent-exposed S3 site for accommodating a protein substrate. During X-ray crystallographic analyses of MPro bound with dipeptide inhibitors containing a flexible N-terminal group, we often observed an unexpected binding mode. Contrary to the anticipated engagement with the deeper S4 pocket, the N-terminal group frequently assumed a twisted conformation, positioning it for interactions with the S3 site and the inhibitor component bound at the S1 pocket. Capitalizing on this observation, we engineered novel inhibitors to engage both S3 and S4 sites or to adopt a rigid conformation for selective S3 site binding. Several new inhibitors demonstrated high efficacy in MPro inhibition. Our findings underscore the importance of the S3 site's unique interactions in the design of future MPro inhibitors as potential COVID-19 therapeutics.

Expression dynamics of the aplysia abyssovirus.

Two recent studies documented the genome of a novel, extremely large (35.9 kb), nidovirus in RNA sequence databases from the marine neural model Aplysia californica. The goal of the present study was to document the distribution and transcriptional dynamics of this virus, Aplysia abyssovirus 1 (AAbV), in maricultured and wild animals. We confirmed previous findings that AAbV RNA is widespread and reaches extraordinary levels in apparently healthy animals. Transmission electron microscopy identified viral replication factories in ciliated gill epithelial cells but not in neurons where viral RNA is most highly expressed. Viral transcripts do not exhibit evidence of discontinuous RNA synthesis as in coronaviruses but are consistent with production of a single leaderless subgenomic RNA, as in the Gill-associated virus of Penaeus monodon. Splicing patterns in chronically infected adults suggested high levels of defective genomes, possibly explaining the lack of obvious disease signs in high viral load animals.

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Among them, MPD2 was demonstrated to effectively reduce MPro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of MPro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors.

The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (M Pro ) is a highly conserved and essential protease that plays key roles in viral replication and pathogenesis among various CoVs, representing one of the most attractive drug targets for antiviral drug development. Traditional antiviral drug development strategies focus on the pursuit of high-affinity binding inhibitors against M Pro . However, this approach often suffers from issues such as toxicity, drug resistance, and a lack of broad-spectrum efficacy. Targeted protein degradation represents a promising strategy for developing next-generation antiviral drugs to combat infectious diseases. Here we leverage the proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 M Pro . Our previously developed M Pro inhibitors MPI8 and MPI29 were used as M Pro ligands to conjugate a CRBN E3 ligand, leading to compounds that can both inhibit and degrade SARS-CoV-2 M Pro . Among them, MDP2 was demonstrated to effectively reduce M Pro protein levels in 293T cells (DC 50 = 296 nM), relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing M Pro protein levels in SARS-CoV-2-infected A549-ACE2 cells, concurrently demonstrating potent anti-SARS-CoV-2 activity (EC 50 = 492 nM). This proof-of-concept study highlights the potential of PROTAC-mediated targeted protein degradation of M Pro as an innovative and promising approach for COVID-19 drug discovery.

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease.

Main protease (M Pro ) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 Cα atom in an M Pro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the M Pro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent M Pro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC 50 value in inhibiting SARS-CoV-2 from infecting ACE2 + A549 cells and a selectivity index of 875. The crystallography analyses of M Pro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent M Pro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 M Pro as therapeutics for COVID-19.

Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants.

Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.