In silico evaluation of the impact of Omicron variant of concern sublineage BA.4 and BA.5 on the sensitivity of RT-qPCR assays for SARS-CoV-2 detection using whole genome sequencing.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VoC) Omicron (B.1.1.529) has rapidly spread around the world, presenting a new threat to global public human health. Due to the large number of mutations accumulated by SARS-CoV-2 Omicron, concerns have emerged over potentially reduced diagnostic accuracy of reverse-transcription polymerase chain reaction (RT-qPCR), the gold standard diagnostic test for diagnosing coronavirus disease 2019 (COVID-19). Thus, we aimed to assess the impact of the currently endemic Omicron sublineages BA.4 and BA.5 on the integrity and sensitivity of RT-qPCR assays used for coronavirus disease 2019 (COVID-19) diagnosis via in silico analysis. We employed whole genome sequencing data and evaluated the potential for false negatives or test failure due to mismatches between primers/probes and the Omicron VoC viral genome. METHODS: In silico sensitivity of 12 RT-qPCR tests (containing 30 primers and probe sets) developed for detection of SARS-CoV-2 reported by the World Health Organization (WHO) or available in the literature, was assessed for specifically detecting SARS-CoV-2 Omicron BA.4 and BA.5 sublineages, obtained after removing redundancy from publicly available genomes from National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Influenza Data (GISAID) databases. Mismatches between amplicon regions of SARS-CoV-2 Omicron VoC and primers and probe sets were evaluated, and clustering analysis of corresponding amplicon sequences was carried out. RESULTS: From the 1164 representative SARS-CoV-2 Omicron VoC BA.4 sublineage genomes analyzed, a substitution in the first five nucleotides (C to T) of the amplicon's 3'-end was observed in all samples resulting in 0% sensitivity for assays HKUnivRdRp/Hel (mismatch in reverse primer) and CoremCharite N (mismatch in both forward and reverse primers). Due to a mismatch in the forward primer's 5'-end (3-nucleotide substitution, GGG to AAC), the sensitivity of the ChinaCDC N assay was at 0.69%. The 10 nucleotide mismatches in the reverse primer resulted in 0.09% sensitivity for Omicron sublineage BA.4 for Thai N assay. Of the 1926 BA.5 sublineage genomes, HKUnivRdRp/Hel assay also had 0% sensitivity. A sensitivity of 3.06% was observed for the ChinaCDC N assay because of a mismatch in the forward primer's 5'-end (3-nucleotide substitution, GGG to AAC). Similarly, due to the 10 nucleotide mismatches in the reverse primer, the Thai N assay's sensitivity was low at 0.21% for sublineage BA.5. Further, eight assays for BA.4 sublineage retained high sensitivity (more than 97%) and 9 assays for BA.5 sublineage retained more than 99% sensitivity. CONCLUSION: We observed four assays (HKUnivRdRp/Hel, ChinaCDC N, Thai N, CoremCharite N) that could potentially result in false negative results for SARS-CoV-2 Omicron VoCs BA.4 and BA.5 sublineages. Interestingly, CoremCharite N had 0% sensitivity for Omicron Voc BA.4 but 99.53% sensitivity for BA.5. In addition, 66.67% of the assays for BA.4 sublineage and 75% of the assays for BA.5 sublineage retained high sensitivity. Further, amplicon clustering and additional substitution analysis along with sensitivity analysis could be used for the modification and development of RT-qPCR assays for detecting SARS-CoV-2 Omicron VoC sublineages.

Comparative Analysis of SARS-CoV-2 Variants of Concern, Including Omicron, Highlights Their Common and Distinctive Amino Acid Substitution Patterns, Especially at the Spike ORF.

In order to gain a deeper understanding of the recently emerged and highly divergent Omicron variant of concern (VoC), a study of amino acid substitution (AAS) patterns was performed and compared with those of the other four successful variants of concern (Alpha, Beta, Gamma, Delta) and one closely related variant of interest (VoI-Lambda). The Spike ORF consistently emerges as an AAS hotspot in all six lineages, but in Omicron this enrichment is significantly higher. The progenitors of each of these VoC/VoI lineages underwent positive selection in the Spike ORF. However, once they were established, their Spike ORFs have been undergoing purifying selection, despite the application of global vaccination schemes from 2021 onwards. Our analyses reject the hypothesis that the heavily mutated receptor binding domain (RBD) of the Omicron Spike was introduced via recombination from another closely related Sarbecovirus. Thus, successive point mutations appear as the most parsimonious scenario. Intriguingly, in each of the six lineages, we observed a significant number of AAS wherein the new residue is not present at any homologous site among the other known Sarbecoviruses. Such AAS should be further investigated as potential adaptations to the human host. By studying the phylogenetic distribution of AAS shared between the six lineages, we observed that the Omicron (BA.1) lineage had the highest number (8/10) of recurrent mutations.

Insighting the optoelectronic, charge transfer and biological potential of benzo-thiadiazole and its derivatives.

The current investigation applies the dual approach containing quantum chemical and molecular docking techniques to explore the potential of benzothiadiazole (BTz) and its derivatives as efficient electronic and bioactive materials. The charge transport, electronic and optical properties of BTz derivatives are explored by quantum chemical techniques. The density functional theory (DFT) and time dependent DFT (TD-DFT) at B3LYP/6-31G** level of theory utilized to optimize BTz and newly designed ligands at the ground and first excited states, respectively. The heteroatoms substitution effects on different properties of 4,7-bis(4-methylthiophene-2yl) benzo[c] [1,2,5]thiadiazole (BTz2T) as initial compound are studied at molecular level. Additionally, we also study the possible inhibition potential of COVID-19 from benzothiadiazole (BTz) containing derivatives by implementing the grid based molecular docking methods. All the newly designed ligands docked with the main protease (MPRO:PDB ID 6LU7) protein of COVID-19 through molecular docking methods. The studied compounds showed strong binding affinities with the binding site of MPRO ranging from -6.9 to -7.4 kcal/mol. Furthermore, the pharmacokinetic properties of the ligands are also studied. The analysis of these results indicates that the studied ligands might be promising drug candidates as well as suitable for photovoltaic applications.

Using an Unsupervised Clustering Model to Detect the Early Spread of SARS-CoV-2 Worldwide.

Deciphering the population structure of SARS-CoV-2 is critical to inform public health management and reduce the risk of future dissemination. With the continuous accruing of SARS-CoV-2 genomes worldwide, discovering an effective way to group these genomes is critical for organizing the landscape of the population structure of the virus. Taking advantage of recently published state-of-the-art machine learning algorithms, we used an unsupervised deep learning clustering algorithm to group a total of 16,873 SARS-CoV-2 genomes. Using single nucleotide polymorphisms as input features, we identified six major subtypes of SARS-CoV-2. The proportions of the clusters across the continents revealed distinct geographical distributions. Comprehensive analysis indicated that both genetic factors and human migration factors shaped the specific geographical distribution of the population structure. This study provides a different approach using clustering methods to study the population structure of a never-seen-before and fast-growing species such as SARS-CoV-2. Moreover, clustering techniques can be used for further studies of local population structures of the proliferating virus.

Temporal Patterns in the Evolutionary Genetic Distance of SARS-CoV-2 during the COVID-19 Pandemic.

During coronavirus disease 2019 (COVID-19) pandemic, the genetic mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred frequently. Some mutations in the spike protein are considered to promote transmissibility of the virus, while the mutation patterns in other proteins are less studied and may also be important in understanding the characteristics of SARS-CoV-2. We used the sequencing data of SARS-CoV-2 strains in California to investigate the time-varying patterns of the evolutionary genetic distance. The accumulative genetic distances were quantified across different time periods and in different viral proteins. The increasing trends of genetic distance were observed in spike protein (S protein), the RNA-dependent RNA polymerase (RdRp) region and nonstructural protein 3 (nsp3) of open reading frame 1 (ORF1), and nucleocapsid protein (N protein). The genetic distances in ORF3a, ORF8, and nsp2 of ORF1 started to diverge from their original variants after September 2020. By contrast, mutations in other proteins appeared transiently, and no evident increasing trend was observed in the genetic distance to the original variants. This study presents distinct patterns of the SARS-CoV-2 mutations across multiple proteins from the aspect of genetic distance. Future investigation shall be conducted to study the effects of accumulative mutations on epidemics characteristics.

Genomic Variations in the Structural Proteins of SARS-CoV-2 and Their Deleterious Impact on Pathogenesis: A Comparative Genomics Approach.

A continual rise in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection causing coronavirus disease (COVID-19) has become a global threat. The main problem comes when SARS-CoV-2 gets mutated with the rising infection and becomes more lethal for humankind than ever. Mutations in the structural proteins of SARS-CoV-2, i.e., the spike surface glycoprotein (S), envelope (E), membrane (M) and nucleocapsid (N), and replication machinery enzymes, i.e., main protease (Mpro) and RNA-dependent RNA polymerase (RdRp) creating more complexities towards pathogenesis and the available COVID-19 therapeutic strategies. This study analyzes how a minimal variation in these enzymes, especially in S protein at the genomic/proteomic level, affects pathogenesis. The structural variations are discussed in light of the failure of small molecule development in COVID-19 therapeutic strategies. We have performed in-depth sequence- and structure-based analyses of these proteins to get deeper insights into the mechanism of pathogenesis, structure-function relationships, and development of modern therapeutic approaches. Structural and functional consequences of the selected mutations on these proteins and their association with SARS-CoV-2 virulency and human health are discussed in detail in the light of our comparative genomics analysis.

The substitution spectra of coronavirus genomes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has triggered an unprecedented international effort to sequence complete viral genomes. We leveraged this wealth of information to characterize the substitution spectrum of SARS-CoV-2 and to compare it with those of other human and animal coronaviruses. We show that, once nucleotide composition is taken into account, human and most animal coronaviruses display a mutation spectrum dominated by C to U and G to U substitutions, a feature that is not shared by other positive-sense RNA viruses. However, the proportions of C to U and G to U substitutions tend to decrease as divergence increases, suggesting that, whatever their origin, a proportion of these changes is subsequently eliminated by purifying selection. Analysis of the sequence context of C to U substitutions showed little evidence of apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-mediated editing and such contexts were similar for SARS-CoV-2 and Middle East respiratory syndrome coronavirus sampled from different hosts, despite different repertoires of APOBEC3 proteins in distinct species. Conversely, we found evidence that C to U and G to U changes affect CpG dinucleotides at a frequency higher than expected. Whereas this suggests ongoing selective reduction of CpGs, this effect alone cannot account for the substitution spectra. Finally, we show that, during the first months of SARS-CoV-2 pandemic spread, the frequency of both G to U and C to U substitutions increased. Our data suggest that the substitution spectrum of SARS-CoV-2 is determined by an interplay of factors, including intrinsic biases of the replication process, avoidance of CpG dinucleotides and other constraints exerted by the new host.

Characterizing genomic variants and mutations in SARS-CoV-2 proteins from Indian isolates.

SARS-CoV-2 is mutating and creating divergent variants by altering the composition of essential constituent proteins. Pharmacologically, it is crucial to understand the diverse mechanism of mutations for stable vaccine or anti-viral drug design. Our current study concentrates on all the constituent proteins of 469 SARS-CoV-2 genome samples, derived from Indian patients. However, the study may easily be extended to the samples across the globe. We perform clustering analysis towards identifying unique variants in each of the SARS-CoV-2 proteins. A total of 536 mutated positions within the coding regions of SARS-CoV-2 proteins are detected among the identified variants from Indian isolates. We quantify mutations by focusing on the unique variants of each SARS-CoV-2 protein. We report the average number of mutation per variant, percentage of mutated positions, synonymous and non-synonymous mutations, mutations occurring in three codon positions and so on. Our study reveals the most susceptible six (06) proteins, which are ORF1ab, Spike (S), Nucleocapsid (N), ORF3a, ORF7a, and ORF8. Several non-synonymous substitutions are observed to be unique in different SARS-CoV-2 proteins. A total of 57 possible deleterious amino acid substitutions are predicted, which may impact on the protein functions. Several mutations show a large decrease in protein stability and are observed in putative functional domains of the proteins that might have some role in disease pathogenesis. We observe a good number of physicochemical property change during above deleterious substitutions.

Comparative insight into the genomic landscape of SARS-CoV-2 and identification of mutations associated with the origin of infection and diversity.

The analyses of 2325 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes revealed 107, 162, and 65 nucleotide substitutions in the coding region of SARS-CoV-2 from the three continents America, Europe, and Asia, respectively. Of these nucleotide substitutions 58, 94, and 37 were nonsynonymous types mostly present in the Nsp2, Nsp3, Spike, and ORF9. A continent-specific phylogram analyses clustered the SARS-CoV-2 in the different group based on the frequency of nucleotide substitutions. Detailed analyses about the continent-specific amino acid changes and their effectiveness by SNAP2 software was investigated. We found 11 common nonsynonymous mutations; among them, two novel effective mutations were identified in ORF9 (S194L and S202N). Intriguingly, ORF9 encodes nucleocapsid phosphoprotein possessing many effective mutations across continents and could be a potential candidate after the spike protein for studying the role of mutation in viral assembly and pathogenesis. Among the two forms of certain frequent mutation, one form is more prevalent in Europe continents (Nsp12:L314, Nsp13:P504, Nsp13:Y541, Spike:G614, and ORF8:L84) while other forms are more prevalent in American (Nsp12:P314, Nsp13:L504, Nsp13:C541, Spike:D614, and ORF8:L84) and Asian continents (Spike:D614), indicating the spatial and temporal dynamics of SARS-CoV-2. We identified highly conserved 38 regions and among these regions, 11 siRNAs were predicted on stringent criteria that can be used to suppress the expression of viral genes and the corresponding reduction of human viral infections. The present investigation provides information on different mutations and will pave the way for differentiating strains based on virulence and their use in the development of better antiviral therapy.

A study on non-synonymous mutational patterns in structural proteins of SARS-CoV-2.

SARS-CoV-2 is mutating and creating divergent variants across the world. An in-depth investigation of the amino acid substitutions in the genomic signature of SARS-CoV-2 proteins is highly essential for understanding its host adaptation and infection biology. A total of 9587 SARS-CoV-2 structural protein sequences collected from 49 different countries are used to characterize protein-wise variants, substitution patterns (type and location), and major substitution changes. The majority of the substitutions are distinct, mostly in a particular location, and lead to a change in an amino acid's biochemical properties. In terms of mutational changes, envelope (E) and membrane (M) proteins are relatively more stable than nucleocapsid (N) and spike (S) proteins. Several co-occurrence substitutions are observed, particularly in S and N proteins. Substitution specific to active sub-domains reveals that heptapeptide repeat, fusion peptides, transmembrane in S protein, and N-terminal and C-terminal domains in the N protein are remarkably mutated. We also observe a few deleterious mutations in the above domains. The overall study on non-synonymous mutation in structural proteins of SARS-CoV-2 at the start of the pandemic indicates a diversity amongst virus sequences.

A comparative phylogenomic analysis of SARS-CoV-2 strains reported from non-human mammalian species and environmental samples.

Coronaviruses (CoVs) infect a wide range of domestic and wild mammals. These viruses have a potential and tendency to cross-species barriers and infect humans. Novel human coronavirus 2019-nCoV (hCoV-19) emerged from Wuhan, China, and has caused a global pandemic. Genomic features of SARS-CoV-2 may attribute inter-species transmission and adaptation to a novel host, and therefore is imperative to explicate the evolutionary dynamics of the viral genome and its propensity for differential host selection. We conducted an in silico analysis of all the coding gene sequences of SARS-CoV-2 strains (n = 39) originating from a range of non-human mammalian species, including pangolin, bat, dog, cat, tiger, mink, mouse, and the environmental samples such as wastewater, air and surface samples from the door handle and seafood market. Compared to the reference SARS-CoV-2 strain (MN908947; Wuhan-Hu-1), phylogenetic and comparative residue analysis revealed the circulation of three variants, including hCoV-19 virus from humans and two hCoV-19-related precursors from bats and pangolins. A lack of obvious differences as well as a maximum genetic homology among dog-, cat-, tiger-, mink-, mouse-, bat- and pangolin-derived SARS-CoV-2 sequences suggested a likely evolution of these strains from a common ancestor. Several residue substitutions were observed in the receptor-binding domain (RBD) of the spike protein, concluding a promiscuous nature of the virus for host species where genomic alternations may be required for the adaptation to novel host/s. However, such speculation needs in vitro investigations to unleash the influence of substitutions towards species-jump and disease pathogenesis.

Substitutions in Spike and Nucleocapsid proteins of SARS-CoV-2 circulating in South America.

SARS-CoV-2 is a new member of the genus Betacoronavirus, responsible for the COVID-19 pandemic. The virus crossed the species barrier and established in the human population taking advantage of the spike protein high affinity for the ACE receptor to infect the lower respiratory tract. The Nucleocapsid (N) and Spike (S) are highly immunogenic structural proteins and most commercial COVID-19 diagnostic assays target these proteins. In an unpredictable epidemic, it is essential to know about their genetic variability. The objective of this study was to describe the substitution frequency of the S and N proteins of SARS-CoV-2 in South America. A total of 504 amino acid and nucleotide sequences of the S and N proteins of SARS-CoV-2 from seven South American countries (Argentina, Brazil, Chile, Ecuador, Peru, Uruguay, and Colombia), reported as of June 3, and corresponding to samples collected between March and April 2020, were compared through substitution matrices using the Muscle algorithm. Forty-three sequences from 13 Colombian departments were obtained in this study using the Oxford Nanopore and Illumina MiSeq technologies, following the amplicon-based ARTIC network protocol. The substitutions D614G in S and R203K/G204R in N were the most frequent in South America, observed in 83% and 34% of the sequences respectively. Strikingly, genomes with the conserved position D614 were almost completely replaced by genomes with the G614 substitution between March to April 2020. A similar replacement pattern was observed with R203K/G204R although more marked in Chile, Argentina and Brazil, suggesting similar introduction history and/or control strategies of SARS-CoV-2 in these countries. It is necessary to continue with the genomic surveillance of S and N proteins during the SARS-CoV-2 pandemic as this information can be useful for developing vaccines, therapeutics and diagnostic tests.

Evolving sequence mutations in the Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) has continued to cause sporadic outbreaks of severe respiratory tract infection over the last 8 years. METHODS: Complete genome sequencing using next-generation sequencing was performed for MERS-CoV isolates from cases that occurred in Riyadh between 2015 and 2019. Phylogenetic analysis and molecular mutational analysis were carried out to investigate disease severity. RESULTS: A total of eight MERS-CoV isolates were subjected to complete genome sequencing. Phylogenetic analysis resulted in the assembly of 7/8 sequences within lineage 3 and one sequence within lineage 4 showing complex genomic recombination. The isolates contained a variety of unique amino acid substitutions in ORF1ab (41), the N protein (10), the S protein (9) and ORF4b (5). CONCLUSION: Our study shows that MERS-CoV is evolving. The emergence of new variants carries the potential for increased virulence and could impose a challenge to the global health system. We recommend the sequencing every new MERS-CoV isolate to observe the changes in the virus and relate them to clinical outcomes.

Genetic sequence changes related to the attenuation of avian infectious bronchitis virus strain TW2575/98.

The attenuated avian infectious bronchitis virus (IBV), derived from a wild strain (TW2575/98w) in chicken embryos after 75 passages, is designed as a commercial vaccine strain (TW2575/98vac) to control the disease in Taiwan. The differences in viral infectivity, replication efficiency, and genome sequences between TW2575/98w and TW2575/98vac were determined and compared. TW2575/98vac caused earlier death of chicken embryos and had higher viral replication efficiency. Thirty amino acid substitutions resulting from 44 mutated nucleotides in the viral genome were found in TW2575/98vac. All of the molecular variations lead to attenuation, found in TW2575/98, were not observed consistently in the other IBVs (TW2296/95, Ark/Ark-DPI/81, the Massachusetts strain, GA98/CWL0470/98, and CK/CH/LDL/97I) and vice versa. After further comparisons and evaluations from three aspects: (1) longitudinal analysis on the timing of variations appeared in specific homologous strain passages, (2) horizontal evaluations with the amino acid changes between wild and vaccine strains among the other 5 IBVs, and (3) inspection on alterations in the chemical characteristics of substituted amino acid residues in viral proteins, four amino acid substitutions [V342D in p87, S1493P and P2025S in HD1, as well as F2308Y in HD1(P41)] were selected as highly possible candidates for successful TW2575/98w attenuation. Our findings imply that molecular variations, which contribute to the successful attenuation of different IBVs, are diverse and not restricted to a fixed pattern or specific amino acid substitutions in viral proteins. In addition, four amino acid changes within the replicase gene-encoded proteins might be associated with TW2575/98 virus virulence.