Evolution of the SARS-CoV-2 Mutational Spectrum.

SARS-CoV-2 evolves rapidly in part because of its high mutation rate. Here, we examine whether this mutational process itself has changed during viral evolution. To do this, we quantify the relative rates of different types of single-nucleotide mutations at 4-fold degenerate sites in the viral genome across millions of human SARS-CoV-2 sequences. We find clear shifts in the relative rates of several types of mutations during SARS-CoV-2 evolution. The most striking trend is a roughly 2-fold decrease in the relative rate of G→T mutations in Omicron versus early clades, as was recently noted by Ruis et al. (2022. Mutational spectra distinguish SARS-CoV-2 replication niches. bioRxiv, doi:10.1101/2022.09.27.509649). There is also a decrease in the relative rate of C→T mutations in Delta, and other subtle changes in the mutation spectrum along the phylogeny. We speculate that these changes in the mutation spectrum could arise from viral mutations that affect genome replication, packaging, and antagonization of host innate-immune factors, although environmental factors could also play a role. Interestingly, the mutation spectrum of Omicron is more similar than that of earlier SARS-CoV-2 clades to the spectrum that shaped the long-term evolution of sarbecoviruses. Overall, our work shows that the mutation process is itself a dynamic variable during SARS-CoV-2 evolution and suggests that human SARS-CoV-2 may be trending toward a mutation spectrum more similar to that of other animal sarbecoviruses.

A systematic review on impact of SARS-CoV-2 infection.

Innumerable pathogens including RNA viruses have catastrophic pandemic propensity, in turn, SARS-CoV-2 infection is highly contagious. Emergence of SARS-CoV-2 variants with high mutation rate additionally codifies infectious ability of virus and arisen clinical imputations to human health. Although, our knowledge of mechanism of virus infection and its impact on host system has been substantially demystified, uncertainties about the emergence of virus are still not fully understood. To date, there are no potentially curative drugs are identified against the viral infection. Even though, drugs are repurposed in the initial period of infection, many are significantly negative in clinical trials. Moreover, the infection is dependent on organ status, co-morbid conditions, variant of virus and geographic region. This review article aims to comprehensively describe the SARS-CoV-2 infection and the impacts in the host cellular system. This review also briefly provides an overview of genome, proteome and metabolome associated risk to infection and the advancement of therapeutics in SARS-CoV-2 infection management.

Ribonucleic acid genome mutations induced by the Casimir effect.

In this paper, we investigate the Casimir effect within a virus RNA, particularizing the study to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Then, we discuss the possibility of occurring damage or mutation in its genome due to the presence of quantum vacuum fluctuations inside and around the RNA ribbon. For this, we consider the geometry and the nontrivial topology of the viral RNA as having a simple helical structure. We initially compute the non-thermal Casimir energy associated to that geometry, considering boundary conditions that constrain the zero point oscillations of a massless scalar field to the cylindrical cavity containing a helix pitch of RNA ribbon. Then we extend the obtained result to the electromagnetic field and, following, we calculate the probability of occurring damage or mutation in RNA by using the normalized inverse exponential distribution, which suppresses very low energies, and consider cutoff (threshold) energies corresponding to UV-A and UV-C rays, surely responsible by mutations. Then, by taking into account UV-A, we arrive at a mutation rate per base per infection cycle, which in the case of the SARS-CoV-2 is non-negligible. We find a maximum value of this mutation rate for an RNA ribbon radius, applying it for SARS-CoV-2, in particular. We also calculate a characteristic longitudinal oscillation frequency for the helix pitch value corresponding to the local minimum of the Casimir energy. Finally, we consider thermal fluctuations of classical and quantum nature and show that the corresponding probability of mutation is completely negligible for that virus. Therefore, we conclude that only the nontrivial topology and the geometric attributes of the RNA molecule contribute to the possible mutations caused by quantum vacuum fluctuations in the viral genome.

Global landscape of SARS-CoV-2 mutations and conserved regions.

BACKGROUND: At the end of December 2019, a novel strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been identified in Wuhan, a central city in China, and then spread to every corner of the globe. As of October 8, 2022, the total number of COVID-19 cases had reached over 621 million worldwide, with more than 6.56 million confirmed deaths. Since SARS-CoV-2 genome sequences change due to mutation and recombination, it is pivotal to surveil emerging variants and monitor changes for improving pandemic management. METHODS: 10,287,271 SARS-CoV-2 genome sequence samples were downloaded in FASTA format from the GISAID databases from February 24, 2020, to April 2022. Python programming language (version 3.8.0) software was utilized to process FASTA files to identify variants and sequence conservation. The NCBI RefSeq SARS-CoV-2 genome (accession no. NC_045512.2) was considered as the reference sequence. RESULTS: Six mutations had more than 50% frequency in global SARS-CoV-2. These mutations include the P323L (99.3%) in NSP12, D614G (97.6) in S, the T492I (70.4) in NSP4, R203M (62.8%) in N, T60A (61.4%) in Orf9b, and P1228L (50.0%) in NSP3. In the SARS-CoV-2 genome, no mutation was observed in more than 90% of nsp11, nsp7, nsp10, nsp9, nsp8, and nsp16 regions. On the other hand, N, nsp3, S, nsp4, nsp12, and M had the maximum rate of mutations. In the S protein, the highest mutation frequency was observed in aa 508-635(0.77%) and aa 381-508 (0.43%). The highest frequency of mutation was observed in aa 66-88 (2.19%), aa 7-14, and aa 164-246 (2.92%) in M, E, and N proteins, respectively. CONCLUSION: Therefore, monitoring SARS-CoV-2 proteomic changes and detecting hot spots mutations and conserved regions could be applied to improve the SARS-CoV-2 diagnostic efficiency and design safe and effective vaccines against emerging variants.

SARS-CoV-2 Variants of Concern and Variations within Their Genome Architecture: Does Nucleotide Distribution and Mutation Rate Alter the Functionality and Evolution of the Virus?

SARS-CoV-2 virus pathogenicity and transmissibility are correlated with the mutations acquired over time, giving rise to variants of concern (VOCs). Mutations can significantly influence the genetic make-up of the virus. Herein, we analyzed the SARS-CoV-2 genomes and sub-genomic nucleotide composition in relation to the mutation rate. Nucleotide percentage distributions of 1397 in-house-sequenced SARS-CoV-2 genomes were enumerated, and comparative analyses (i) within the VOCs and of (ii) recovered and mortality patients were performed. Fisher's test was carried out to highlight the significant mutations, followed by RNA secondary structure prediction and protein modeling for their functional impacts. Subsequently, a uniform dinucleotide composition of AT and GC was found across study cohorts. Notably, the N gene was observed to have a high GC percentage coupled with a relatively higher mutation rate. Functional analysis demonstrated the N gene mutations, C29144T and G29332T, to induce structural changes at the RNA level. Protein secondary structure prediction with N gene missense mutations revealed a differential composition of alpha helices, beta sheets, and coils, whereas the tertiary structure displayed no significant changes. Additionally, the N gene CTD region displayed no mutations. The analysis highlighted the importance of N protein in viral evolution with CTD as a possible target for antiviral drugs.

Rampant C-to-U deamination accounts for the intrinsically high mutation rate in SARS-CoV-2 spike gene.

The high mutation rate of SARS-CoV-2 largely complicates our control of the pandemic. In particular, it is currently unclear why the spike (S) gene has an extraordinarily high mutation rate among all SARS-CoV-2 genes. By analyzing the occurrence of fixed synonymous mutations between SARS-CoV-2 and RaTG13, and profiling the DAF (derived allele frequency) of polymorphic synonymous sites among millions of worldwide SARS-CoV-2 strains, we found that both fixed and polymorphic mutations show higher mutation rates in the S gene than other genes. The majority of mutations are C-to-T, representing the APOBEC-mediated C-to-U deamination instead of the previously proposed A-to-I deamination. Both in silico and in vivo evidence indicated that the S gene is more likely to be single-stranded compared to other SARS-CoV-2 genes, agreeing with the APOBEC preference of ssRNA. We conclude that the single-stranded property of the S gene makes it a favorable target for C-to-U deamination, leading to its excessively high mutation rate compared to other non-S genes. In conclusion, APOBEC, rather than ADAR, is the "editor-in-chief" of SARS-CoV-2 RNAs. This work helps us to understand the molecular mechanism underlying the mutation and evolution of SARS-CoV-2, and we believe it will contribute to the control of the pandemic.

Containing the spread of COVID-19 virus facing to its high mutation rate: approach to intervention using a nonspecific way of blocking its entry into the cells.

Viruses have multiple mutation rates that are higher than any other member of the kingdom of life. This gives them the ability to evolve, even within the course of a single infection, and to evade multiple host defenses, thereby impacting pathogenesis. Additionally, there are also interplays between mutation and recombination and the high multiplicity of infection (MOI) that enhance viral adaptability and increase levels of recombination leading to complex and conflicting effects on genome selection, and the net results is difficult to predict. Recently, the outbreak of COVID-19 virus represents a pandemic threat that has been declared a public health emergency of international concern. Up to present, however, due to the high mutation rate of COVID-19 virus, there are no effective procedures to contain the spread of this virus across the globe. For such a purpose, there is then an urgent need to explore new approaches. As an opinion, the present approach emphasizes on (a) the use of a nonspecific way of blocking the entry of COVID-19 virus as well as its variants into the cells via a therapeutic biocompatible compound (ideally, "in a pill") targeting its spike (S) glycoprotein; and (b) the construction of expression vectors via the glycosyl-phosphatidylinositol, GPI, anchor for studying intermolecular interactions between the spike S of COVID-19 virus as well as its variants and the angiotensin-converting enzyme 2 (ACE2) of its host receptor for checking the efficacy of any therapeutic biocompatible compound of the nonspecific way of blocking. Such antiviral drug would be safer than the ACE1 and ACE2 inhibitors/angiotensin receptor blockers, and recombinant human ACE2 as well as nucleoside analogs or protease inhibitors used for fighting the spread of the virus inside the cells, and it would also be used as a universal one for any eventual future pandemic related to viruses, especially the RNA viruses with high mutation rates.

High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination.

For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants.

Early introduction, dispersal and evolution of Delta SARS-CoV-2 in Southern Brazil, late predominance of AY.99.2 and AY.101 related lineages.

The emergence of Variants of Concern (VOC) presenting an unusual number of new mutations is one of the most remarkable features of SARS-CoV-2. The Delta variant, since its appearance, replaced the VOC Gamma, which was responsible for the major COVID-19 wave in Brazil. In this study, we performed a Delta whole-genome sequencing of 183 samples as part of a major genomic surveillance study performed since the beginning of the pandemic. Here, we showed an emergence, widespread dispersion and consolidation of the Delta variant in Rio Grande do Sul State, completely replacing the Gamma variant in a four to five months period. Performing the phylogenetic and phylodynamic analysis, the majority of the sequences generated herein were classified as AY.99.2, AY.99.2-like and AY.101. AY.99.2 Delta-related lineage has been widely reported in Brazil and in the Americas as well. Altogether, our findings provided a mutational profile of the sequences and presented high substitutions per site in the root-to-tip phylogenetic tree, corroborating studies that show the high mutational rate of SARS-CoV-2 over time.

Enhanced High Mutation Rate and Natural Selection to Produce Attenuated Viral Vaccine with CRISPR Toolkit in RNA Viruses especially SARS-CoV-2.

The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.

The variants question: What is the problem?

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan, China in early December 2019 has rapidly widespread worldwide. Over the course of the pandemic, due to the advance of whole-genome sequencing technologies, an unprecedented number of genomes have been generated, providing both invaluable insights into the ongoing evolution and epidemiology of the virus and allowing the identification of hundreds of circulating genetic variants during the pandemic. In recent months variants of SARS-CoV-2 that have an increased number of mutations on the Spike protein have brought concern all over the world. These have been called "variants of concerns" (VOCs), and/or "variants of interests" (VOIs) as it has been suggested that their genome mutations might impact transmission, immune control, and virulence. Tracking the spread of emerging SARS-CoV-2 variants is crucial to inform public health efforts and control the ongoing pandemic. In this review, a concise characterization of the SARS-CoV-2 mutational patterns of the main VOCs and VOIs circulating and cocirculating worldwide has been presented to determine the magnitude of the SARS-CoV-2 threat to better understand the virus genetic diversity and its potential impact on vaccination strategy.

An entropy-based study on mutational trajectory of SARS-CoV-2 in India.

The pandemic of COVID-19 has been haunting us for almost the past two years. Although, the vaccination drive is in full swing throughout the world, different mutations of the SARS-CoV-2 virus are making it very difficult to put an end to the pandemic. The second wave in India, one of the worst sufferers of this pandemic, can be mainly attributed to the Delta variant i.e. B.1.617.2. Thus, it is very important to analyse and understand the mutational trajectory of SARS-CoV-2 through the study of the 26 virus proteins. In this regard, more than 17,000 protein sequences of Indian SARS-CoV-2 genomes are analysed using entropy-based approach in order to find the monthly mutational trajectory. Furthermore, Hellinger distance is also used to show the difference of the mutation events between the consecutive months for each of the 26 SARS-CoV-2 protein. The results show that the mutation rates and the mutation events of the viral proteins though changing in the initial months, start stabilizing later on for mainly the four structural proteins while the non-structural proteins mostly exhibit a more constant trend. As a consequence, it can be inferred that the evolution of the new mutative configurations will eventually reduce.

Identification and characterization of SARS-CoV-2 clusters in the EU/EEA in the first pandemic wave: additional elements to trace the route of the virus.

A high-quality dataset of 3289 complete SARS-CoV-2 genomes collected in Europe and European Economic Area (EAA) in the early phase of the first wave of the pandemic was analyzed. Among all single nucleotide mutations, 41 had a frequency ≥ 1%, and the phylogenetic analysis showed at least 6 clusters with a specific mutational profile. These clusters were differentially distributed in the EU/EEA, showing a statistically significant association with the geographic origin. The analysis highlighted that the mutations C14408T and C14805T played an important role in clusters selection and further virus spread. Moreover, the molecular analysis suggests that the SARS-CoV-2 strain responsible for the first Italian confirmed COVID-19 case was already circulating outside the country.

Assessment of intercontinents mutation hotspots and conserved domains within SARS-CoV-2 genome.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 pathogen, has led to waves of global pandemic claiming lives and posing a serious threat to public health and social cum physical interactions. To evaluate the mutational landscape and conserved regions in the genome of the causative pathogen, we analysed 7213 complete SARS-CoV-2 protein sequences mined from the Global Initiative on Sharing All Influenza Data (GISAID) repository from infected patients across all regions on the EpiCov web interface. Regions of origin and the corresponding number of sequences mined are as follows: Asia - 2487; Oceania - 2027; Europe - 1240; Africa - 717; South America - 391; and North America - 351. High recurrent mutations, namely: T265I in non-structural protein 2 (nsp2), L3606F in nsp6, P4715L in RNA-dependent RNA polymerase (RdRp), D614G in spike glycoprotein, R203K and G204R in nucleocapsid phosphoprotein and Q57H in ORF3a with well-conserved envelope and membrane proteins, 3CLpro and spike S2 domains across regions were observed. Comparative analyses of the viral sequences reveal the prevalence P4715L and D614G mutations as the most recurrent and concurrent in Africa (97.20%), Europe (89.83%) and moderately in Asia (61.60%). Mutation rates are central to viral transmissibility, evolution and virulence, which help them to invade host immunity and develop drug resistance. Based on the foregoing, it is important to understand the mutational spectra of SARS-CoV-2 genome across regions. This will help in identifying specific genomic sites as potential targets for drug design and vaccine development, monitoring the spread of the virus and unraveling its evolution, virulence and transmissibility.

Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates.

Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.6% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 5,472 distinct variants were found in total. The majority of the observed variants were recurrent, but only 21 and 14 recurrent variants were found in at least 1% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 2.6% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition. These findings demonstrate the continuous importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies.

Phylogenetic reconstruction of the initial stages of the spread of the SARS-CoV-2 virus in the Eurasian and American continents by analyzing genomic data.

Samples from complete genomes of SARS-CoV-2 isolated during the first wave (December 2019-July 2020) of the global COVID-19 pandemic from 21 countries (Asia, Europe, Middle East and America) around the world, were analyzed using the phylogenetic method with molecular clock dating. Results showed that the first cases of COVID-19 in the human population appeared in the period between July and November 2019 in China. The spread of the virus into other countries of the world began in the autumn of 2019. In mid-February 2020, the virus appeared in all the countries we analyzed. During this time, the global population of SARS-CoV-2 was characterized by low levels of the genetic polymorphism, making it difficult to accurately assess the pathways of infection. The rate of evolution of the coding region of the SARS-CoV-2 genome equal to 7.3 × 10-4 (5.95 × 10-4-8.68 × 10-4) nucleotide substitutions per site per year is comparable to those of other human RNA viruses (Measles morbillivirus, Rubella virus, Enterovirus C). SARS-CoV-2 was separated from its known close relative, the bat coronavirus RaTG13 of the genus Betacoronavirus, approximately 15-43 years ago (the end of the 20th century).

Mutation Rates, Mutation Frequencies, and Proofreading-Repair Activities in RNA Virus Genetics.

The error rate displayed during template copying to produce viral RNA progeny is a biologically relevant parameter of the replication complexes of viruses. It has consequences for virus-host interactions, and it represents the first step in the diversification of viruses in nature. Measurements during infections and with purified viral polymerases indicate that mutation rates for RNA viruses are in the range of 10-3 to 10-6 copying errors per nucleotide incorporated into the nascent RNA product. Although viruses are thought to exploit high error rates for adaptation to changing environments, some of them possess misincorporation correcting activities. One of them is a proofreading-repair 3' to 5' exonuclease present in coronaviruses that may decrease the error rate during replication. Here we review experimental evidence and models of information maintenance that explain why elevated mutation rates have been preserved during the evolution of RNA (and some DNA) viruses. The models also offer an interpretation of why error correction mechanisms have evolved to maintain the stability of genetic information carried out by large viral RNA genomes such as the coronaviruses.