COVID-19 mutations: An overview

Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the genus Beta coronavirus and the family of Coronaviridae. It is a positive-sense, non-segmented single-strand RNA virus. Four common types of human coronaviruses circulate globally, particularly in the fall and winter seasons. They are responsible for 10%-30% of all mild upper respiratory tract infections in adults. These are 229E, NL63 of the Alfacoronaviridae family, OC43, and HKU1 of the Betacoronaviridae family. However, there are three highly pathogenic human coronaviruses: SARS-CoV-2, Middle East respiratory syndrome coronavirus, and the latest pandemic caused by the SARS-CoV-2 infection. All viruses, including SARS-CoV-2, have the inherent tendency to evolve. SARS-CoV-2 is still evolving in humans. Additionally, due to the development of herd immunity, prior infection, use of medication, vaccination, and antibodies, the viruses are facing immune pressure. During the replication process and due to immune pressure, the virus may undergo mutations. Several SARS-CoV-2 variants, including the variants of concern (VOCs), such as B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617/B.1.617.2 (Delta), P.1 (Gamma), and B.1.1.529 (Omicron) have been reported from various parts of the world. These VOCs contain several important mutations; some of them are on the spike proteins. These mutations may lead to enhanced infectivity, transmissibility, and decreased neutralization efficacy by monoclonal antibodies, convalescent sera, or vaccines. Mutations may also lead to a failure of detection by molecular diagnostic tests, leading to a delayed diagnosis, increased community spread, and delayed treatment. We searched PubMed, EMBASE, Covariant, the Stanford variant Database, and the CINAHL from December 2019 to February 2023 using the following search terms: VOC, SARS-CoV-2, Omicron, mutations in SARS-CoV-2, etc. This review discusses the various mutations and their impact on infectivity, transmissibility, and neutralization efficacy.

Key Words: Variant of concern, SARS-CoV-2, Omicron, N501Y mutation, E484K mutation

Core Tip: The severe acute respiratory syndrome coronavirus-2 virus is constantly evolving because to natural immunity and vaccine-induced immunity which exert continual immunological pressure, resulting in the generation of newer variants and numerous new mutations. This study detailed the many variants of concern (VOCs), including their transmissibility, severity, and immune-evasion capacities. We have also discussed several key mutations and their consequences. The tables summarized the major points of the paper and provided a full discussion of the important mutations found in these VOCs. Readers will benefit from our article's concise overview of these areas.

INTRODUCTION

Variants are coronaviruses that have the same inherited set of very distinctive mutations[1]. It is worth noting that RNA viruses tend to have higher mutation rates than DNA viruses, and single-stranded viruses mutate quicker than double-stranded viruses[2]. However, the rate of mutations among the coronaviruses is lower than that of most RNA viruses. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) acquires 2-3 single-nucleotide (nt) mutations in its genome per month. This is half the rate of influenza (4 nt/month) and one-fourth the rate of human immunodeficiency virus (8 nt/month) mutations[3,4]. The slow rate of mutation may be explained by the existence of a novel 3′-to-5′ exoribonuclease (ExoN) in nsp14, which serves as a proofreader and corrects some replication errors[5]. Genetic inactivation of this ExoN function causes a 15- to 20-fold rise in mutation rates[6]. When variants with various mutations infect the same host, they accumulate mutations and create diversity via recombination[7-9]. Human hosts may also contribute to the diversity via host-mediated RNA editing[10,11]. It is important to know that not every mutation will have a long-lasting effect on the virus. Typically, synonymous mutations are neutral, whereas non-synonymous mutations are persistent[12]. Wang et al[13] observed that in SARS-CoV-2, non-synonymous mutations are 14 times less likely to persist as weak deleterious mutations are slowly eliminated[13]. Mutations may increase infectivity, transmissibility, disease severity, and decreased neutralization efficacy by monoclonal antibodies (mAbs) and convalescent sera, as well as lower vaccine efficacy (VE). Moreover, a few variants may be responsible for negative results on the diagnostic tests. For example, with the Alpha and Omicron variants, there could be “S” gene target failure (SGTF)[14-16]. Delay in diagnosis increases the likelihood of viral transmission. The surface transmembrane spike (S) protein consists of S1 and S2 subunits. The S1 subunit has the N-terminal domain (NTD) and a receptor-binding domain (RBD) and it mediates the critical step of viral entry into the cell via interacting with the angiotensin-converting enzyme 2 (ACE2) receptor of the host cell[17]. The S1 subunit is also the primary target of neutralizing antibodies upon infection and various therapeutic and vaccines[18]. S2 mediates fusion of the viral and cellular membranes[19]. The RBD must be in an upright position to bind with ACE2 receptors and initiate the viral entry process. It is followed by the cleavage of S1/S2, which helps in membrane fusion (Figure 1A). The SARS-CoV-2 also enters in the cell by endosomal/lysosomal pathway. Inside the cell, the virus undergoes replication, translation, assembly, and exocytosis followed by viral release (Figure 1B). The most common site of mutation in SARS-CoV-2 is the spike protein, but it may involve other proteins as well. The diagnostic assays for SARS-CoV-2 are based on the two most abundant and immunogenic viral proteins, such as spike or nucleocapsid (N) proteins[20]. The spike protein contains sequences that are unique to the SARS-CoV-2 virus, thereby reducing the risk of cross-reactivity. However, spike protein has the highest potential to undergo mutation, and it has the potential to cause false-negative tests on immunoassays that are based only on detecting spike protein. Since N protein is less susceptible to mutations, it is the best target for developing diagnostic assays[20]. Recently, several variants of concern (VOCs) have been de-escalated. It will provide updated knowledge on the de-escalated status of various VOCs, subcategories, and VE. A detailed evaluation of the existing mutations and emerging mutations has been of immense help in studying their impact on transmissibility, severity, neutralization potency, and VE. This narrative review will provide an updated version of SARS-CoV-2 VOCs, associated mutations, the effect of mutations, and the present status of the VOCs in detail. Understanding the biological characteristics of the mutations will guide us in the surveillance, prevention, and control of coronavirus disease 2019 (COVID-19) infection.

Figure 1 Showing the S protein mediated binding, fusion and various steps of severe acute respiratory syndrome coronavirus-2 infection cycle in the humans. A: Showing the S protein mediated binding and fusion with human cell; B: Showing the various steps in the severe acute respiratory syndrome coronavirus-2 infection cycle in human. It includes viral cellular entry, transcription, replication and packaging, translation, assembly within cellular organelle. SARS-CoV-2: Severe acute respiratory syndrome coronavirus-2; ER: Endoplasmic reticulum; ERCIC: ER-to-Golgi intermediate compartment; RdRP: RNA-dependent RNA polymerase; 3CLpro: 3C-like proteinase; Hel: Helicase.

SARSCOV2 VARIANT’S NOMENCLATURE SYSTEM

The SARS-CoV-2 interagency group (SIG) of the United States Department of Health and Human Services has classified the newly developing variations of SARS-CoV-2 into many groups. The SIG is responsible for better coordination among various United States departments and agencies, rapid characterization of emerging variants, and actively monitoring their potential impact on vaccines, therapeutics, and diagnostics[21]. Due to the lack of a standardized system, various nomenclature systems are in use. The Global Initiative on Sharing Avian Influenza Data, Nextstrain, and Phylogenetic Assignment of Named Global Outbreak (PANGO) are the most commonly used nomenclature systems by the scientific community worldwide[22]. The PANGO lineage system contains an alphabetical prefix and a suffix containing up to three numbers separated by periods indicating sub-lineages (such as B.1.1.7). Starting from high impact to low impact, SARS-CoV-2 variants may be classified as variants of high consequence, VOCs, variants of interest, and alerts for further monitoring. The VOC is characterized by increased transmissibility, a severe disease associated with an increased rate of hospitalizations or deaths, a significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures[23].

TRANSLATIONAL SCIENCE

The COVID-19 pandemic has demonstrated the importance of translational research in pandemic control. Translational research encompasses a wide range of disciplines, including diagnostics, newer drug development, pathogenesis, epidemiology, and vaccine development. The COVID-19 pandemic has changed the traditional approach to clinical research[24].

NATURAL SELECTION AND IMMUNE IMPRINTING

Two phenomena play a significant role in the natural history of SARS-CoV-2 virus evolution. These include natural selection and immune imprinting. Animal experiments have confirmed the existence of natural selection. Lei et al[25] investigated the composition and codon use of the SARS-CoV-2 virus in infected humans and animals[25]. They reported the maximum mutations in mink. SARS-CoV-2 in mink showed that substitutions of cytidine contributed to approximately 50% of substitutions. The corresponding figure for other animals was 30%. However, the incidence of adenine transversion in SARS-CoV-2 in other animals is three times higher than in mink. They also found lower adaptability than humans in all other animals except for mink. Furthermore, a binding affinity analysis revealed that the spike protein of the SARS-CoV-2 variant in mink had a greater preference for binding with the mink receptor ACE2 than the human receptor, particularly with the mutations Y453F and F486L in mink SARS-CoV-2, which improved the binding affinity for the mink receptor. This study demonstrates that SARS-CoV-2's natural history in mink includes both natural selection and host adaptation. Similarly, Fu et al[26] showed that natural selection had a stronger influence on some SARS-CoV-2 sequences than mutational pressure[26]. The Y453F and N501T mutations in mink SARS-CoV-2 increased viral spike binding to the mink receptor. It confirmed the role of these mutations in natural selection and viral fitness. Natural selection favors the strains with beneficial mutations and reduces the number of strains with deleterious mutations[27]. However, it is still unclear whether natural selection occurred first in an animal host before zoonotic transfer or whether natural selection occurred in humans after zoonotic transfer[28]. Rubio-Casillas et al[29] coined the term “intermittent virulence”, which is basically an evolutionary equilibrium between transmissibility and virulence[29]. They considered this phenomenon to be due to natural selection. Habib et al[30] scanned the RBD of the Omicron spike protein for adaptive evolution based on a public database in Bangladesh[30]. It was reported that the adaptive mutations in the RBD domain were characterized by a non-synonymous to synonymous nt substitution rate of more than one. This indicates a positive selection. Some of the adaptive sites mediate increased viral fitness. Immune imprinting is the mechanism by which memory B lymphocytes induced by an initial viral infection prevent the development of B cells in response to a subsequent infection with a novel but related virus[31]. Chemaitelly et al[32] conducted a retrospective cohort study in Qatar to compare the incidence of SARS-CoV-2 reinfection in persons who had received primary-series (two-dose) vaccination, no vaccination, or booster (three-dose) vaccinations[32]. They found that a history of primary-series immunization enhanced immune protection against omicron reinfection, whereas a history of booster vaccination compromised protection against omicron reinfection. In the future, a study elucidating the pathogenetic mechanism behind the phenomenon of immune imprinting may provide useful insights for creating a more effective vaccine against the SARS-CoV-2 virus. In addition, we should not forget the short-term public health benefits of vaccination.

VOC

Several VOCs have been identified, and they differ from one another in terms of infectivity, transmissibility, severity, therapeutic efficacy, and neutralization efficacy by mAbs, convalescent sera, or vaccines. These are B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617/B.1.617.2 (Delta), P.1 (Gamma), and B.1.1.529 (Omicron). The European Center for Disease Prevention and Control declared a new category in July 2021 as de-escalated variants. These VOCs have been de-escalated because they are either not circulating or, if they are, have no epidemiological impact. Moreover, they are not associated with any concerning properties[33].

Alpha (B.1.1.7 lineage) VOC

The B.1.1.7 variant was the first VOC to be detected in southeast England in September 2020. It eventually became the dominant variant in the United Kingdom and many other countries due to its increased transmissibility[33]. The B.1.1.7 variant was found to be 50%-75% more transmissible than the origin lineage, with a R0 value that was 1.75 times higher[34]. Another feature is the increased disease severity compared to the pre-existing SARS-CoV-2 variants of that time. Davies et al[35] had shown that the hazard of death with SGTF was 55% (95%CI: 39%–72%) higher than that in cases without SGTF after adjustment for age, sex, ethnicity, deprivation, care home residence, local authority of residence, and test date[35]. The B.1.1.7 variant's higher transmissibility is due to the presence of the N501Y mutation and Deletion69/Deletion70, which increase binding affinity to ACE2[36,37]. Other characteristics include SGTF due to mutations in the S gene and no change in susceptibility to monoclonal antibody therapy such as Bamlanivimab-etesevimab, casirivimab-imdevimab, and sotrovimab[38-40]. However, E484K and/or other NTD mutations (especially deletions) may result in a considerable reduction in neutralizing efficacy[41]. The ChAdOx1 nCoV-19 (AstraZeneca) vaccine showed an efficacy of 70.4%[42]. The first and second doses of the BNT162b2 vaccine (Pfizer–BioNTech) reported 48.7% and 93.7% effectiveness, respectively[43]. The reported efficacy of two doses of the mRNA-1273 (Moderna) vaccine was 98.4%[44].This variant has been de-escalated[33].

Beta (B.1.351) VOC

Tegally et al[45] detected this variant, also known as 501Y.V2, in late 2020 in the Eastern Cape, South Africa[45]. The beta variants also show increased transmissibility, similar to the B.1.1.7 variants. In comparison to the alpha and gamma versions, this variant increased the likelihood of hospitalization, intensive care unit (ICU) admission, and mortality. However, it causes less severe disease than the delta variant[46]. These variants also show immune-evasion properties. There was a 45-fold decrease in susceptibility to Bamlanivimab-etesevimab therapy; however, casirivimab-imdevimab and sotrovimab retained susceptibility[38-40]. Furthermore, beta versions demonstrate lower neutralization by convalescent and post-vaccination sera[47]. In a systematic review and meta-analysis, Zeng et al[48] assessed 11 COVID-19 vaccines (BNT162b2, mRNA-1273, ChAdOx1, Ad26.COV2.S, BBV152, CoronaVac, NVX-CoV2373, BBIBP-CorV, CVnCoV, SCB-2019, and HB02) and reported full vaccination efficacy against Alpha, Beta, Gamma, Delta, and Omicron variants of 88.0% (95%CI: 83.0%–91.5%), 73.0% (95%CI: 64.3%–79.5%), 63.0% (95%CI: 47.9%–73.7%), 77.8% (95%CI: 72.7%–82.0%), and 55.9% (95%CI: 40.9%–67.0%), respectively[48]. The efficacy of booster vaccination was higher against Delta and Omicron variants, 95.5% (95%CI: 94.2%–96.5%) and 80.8% (95%CI: 58.6%–91.1%), respectively. They also reported a higher efficacy of mRNA vaccines (mRNA-1273/BNT162b2) against VOC over others.

Gamma (P.1; GR/501Y.V3)

The P.1 variant was first reported from Japan on January 6, 2021, by four people who had arrived in Tokyo after visiting Amazonas, Brazil[49]. Faria et al[50] further published the genomic and epidemiological analysis of this Brazilian variant from Manaus[50]. They reported 17 mutations in the P.1 variants, including three in the spike protein RBDs (K417T, E484K, and N501Y). These mutations caused enhanced binding to the human ACE2 receptor. The P.1 variant is 1.7 to 2.4 times more transmissible than the previous (non-P.1) infection. Infection with P.1 is also 1.2 to 1.9 times more likely to cause mortality than previous lineages[50]. This variant also possesses immune evasion properties. Although the P.1 variant retained susceptibility to Casirivimab-imdevimab and Sotrovimab, there was a 511-fold decrease in susceptibility to Bamlanivimab-etesevimab[38-40]. This variant has been deescalated. Full vaccination efficacy against the gamma variants was 63.0%[48].

Delta (G/478 K.V1; B.1.617.2)

The B.1.617.2 variant was first identified in India in October 2020 and quickly became the dominant variant in India and globally until the emergence of the Omicron variant. This variant was 40%-60% more transmissible than the B.1.1.7 variant and almost twice as transmissible as the original Wuhan strain[51]. The B.1.617 variant has three sublineages: B.1.617.1, B.1.617.2, and B.1.617.3. The B.1.617.2 variants show increased transmissibility and replication advantages. This variant shows 1260-fold higher viral loads than those for the 2020 infections with clade 19A/19B viruses. It makes the person more infectious[52]. Compared to the B.1.1.7 cases, the B.1.617 variant is associated with an increased severity of the disease[53,54] and an increased risk of hospitalization[54,55]. The common signature mutations located in the spike protein are D111D, G142D, L452R, E484Q, D614G, and P681R. The L452R, E484Q, and P681R mutations contribute to increased transmissibility, and the E484Q and P681R mutations influence antibody binding. Neutralization by mAbs is affected minimally. Although there is a moderate reduction in VE against symptomatic COVID-19 infection, efficacy against severe disease and hospitalization showed no significant impact[47]. The 2-dose mRNA-1273 vaccine showed 86.7% (95%CI: 84.3%- 88.7%) efficacy against infection and 97.5% (95%CI: 92.7%-99.2%) efficacy against hospital admission with the B.1.617.2 variant[44]. However, VE decreased from 94.1% at 14-60 d after immunization to 80.0% at 151-180 d following vaccination. The efficacy of 2-doses of BNT162b2 and ChAdOx1 nCoV-19 vaccines was 88.0% (95%CI: 85.3%-90.1%) and 67.0% (95%CI: 61.3%-71.8%) among those with the delta variant, respectively[43]. Effectiveness after one dose of vaccine with both BNT162b2 and ChAdOx1 nCoV-19 vaccine was notably lower among persons with the delta variant (30.7%; 95%CI: 25.2%- 35.7%).

Omicron (B.1.1.529 lineage) variant

The Omicron variant was initially reported from Botswana and then from South Africa. Very soon, it became the dominant variant globally. The World Health Organization classified it as VOC on November 26 and named it Omicron[56]. Subsequently; several sublineages of the SARS-CoV-2 variant were identified. These are BA.1, BA.2, BA.4/BA.5, BA.4.6 (BA.4), BA.2.75.2 (BA.2), BQ.1/BQ.1.1 (BA.5), and XBB/XBB.1/XBB.1.5 (BA.2.10.1 and BA.2.75 recombinant) sublineages[47]. TheBA.4 and BA.5 sublineages have identical spike proteins similar to BA.2 except for the addition of 69-70 deletions, L452R, F486V, and the wild-type amino acid at Q493[57]. However, other sublineages differ from the others by at least one spike protein mutation[58]. Typical features of Omicron variants are high transmissibility, increased risk of reinfection or breakthrough infection, less severe disease compared to delta variants, and reduced or absent neutralization efficacy by vaccines and monoclonal antibody therapies. The Omicron variant is heavily mutated, as it contains up to 59 mutations in its genome, including 36 occurring within the spike protein and more than 30 involving the RBD[58,59]. The Omicron variant has a replication advantage over the B.1.617.2 variant, with the basic reproduction number (R₀) exceeding 3[60,61]. The high R0 is the result of both higher transmissibility and immunological evasion. Compared to the earlier surge, hospitalized patients with the B.1.1.529 variant in Tshwane, Gauteng Province, South Africa, showed lower rates of ICU admissions (1% vs 4.3%, P < 0.00001), in-hospital death (4.5% vs 21.3%, P < 0.00001), and length of hospital stay (4.0 d vs 8.8 d)[62]. Omicron sublineages show immune evasion properties. Yue et al[62] reported that Omicron subvariant XBB.1.5 was more transmissible than other XBB sublineages[62]. This subvariant XBB.1.5 has an additional Ser486Pro substitution. The authors demonstrated a higher ACE2-receptor binding affinity and significant immune evasion in convalescent plasma. Moreover, Bebtelovimab showed no neutralization effect against the XBB.1/XBB.1.5 subvariant[63]. Similarly, Sotrovimab, Tixagevimab-cilgavimab, and Casirivimab-imdevimab remain inactive against the XBB/XBB.1/XBB.1.5 sublineages[47]. The mAbs resistant in B.1.1.529 variants may be explained by the presence of the following mutations: K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, S371L, and Y505H, which are located within or close to the epitopes targeted by these antibodies. The Omicron variety (B.1.1.529 lineage) also has SGTF, which causes a delay in diagnosis and increases the risk of infection transmission. However, the BA.2 lineage does not show SGTF due to a lack of deletions in positions 69-70[64]. The above-mentioned monoclonal antibody cocktails should not be used against the B.1.1.529 variants[65]. Mass vaccination is a crucial public health intervention that lowers COVID-19-related hospitalization and mortality. However, the duration of protection wanes over time. Wu et al[66] in a meta-analysis, studied the long-term efficacy of COVID-19 vaccinations against infection, hospitalization, and mortality up to 307 d after completion of the primary vaccination series and 139 d after a first booster vaccination[66]. They reported a vaccine effectiveness of 83% against infection, 92% against hospitalization, and 91% against mortality after the primary COVID-19 vaccination. However, the efficacy decreased over time. The VE against the omicron sublineages was 61% and 71% against infection and hospitalization, respectively. However, a booster dose increased the vaccine effectiveness against the omicron variant to 67% against infection and 89% against hospitalization. Andrews et al[67] reported a decreased efficacy of the vaccine against the omicron variant compared to the delta variant[67]. The two doses of ChAdOx1 nCoV-19 (AstraZeneca) showed no efficacy against the symptomatic disease caused by the Omicron. The VE of 2-dose BNT162b2 doses and the mRNA-1273 vaccine were 65.5% (95%CI: 63.9%-67.0%) and 75.1% (95%CI: 70.8%-78.7%), respectively. However, efficacy decreases over time. Among patients who received ChAdOx1 nCoV-19 as the primary vaccine, a BNT162b2 and mRNA-1273 booster dose increased the efficacy to 62.4% (95%CI: 61.8%-63.0%) and 70.1% (95%CI: 69.5%-70.7%), respectively. Booster doses are required to mount a more appropriate immune response against omicron infection[46]. Among patients who received BNT162b2 as primary vaccine, a BNT162b2 booster dose increased the efficacy to 67.2% (95%CI: 66.5%-67.8%). The BNT162b2 vaccine showed an efficacy of 70% against hospitalization[68]. Table 1 shows the characteristic features of various VOCs.

Table 1 Showing characteristic features of various variants of concerns.

VOCs	Transmissibility	Severity	Effect on neutralization by mABs		SGTF	Present status	Vaccine efficacy
Alpha	Increased transmissibility (50%-100%) with R0 1.75-fold higher compared to original lineage[32]	Increased severity. Hazard of death of 55% (95%CI: 39%–72%) higher than in cases without SGTF after adjustment[33]	No impact on neutralization by mABs, and minimal impact by convalescent and/or post-vaccination sera[18]. E484K and/or various NTD mutations cause a significant fall in neutralization efficacy[39]		Presence	De-escalated	The ChAdOx1 nCoV-19 vaccine showed an efficacy of 70.4%[40]. The first and second dose of BNT162b2 vaccine (Pfizer–BioNTech) reported 48.7% and 93.7% effectiveness, respectively[41]. The reported efficacy of 2-doses of mRNA-1273 vaccine 98.4%[42]
Beta	Increased transmissibility	Increased risk of hospitalization, ICU admission, and mortality in comparison to Alpha and Gamma variants, but less severe disease compared to Delta[44]	45-fold decreased susceptibility to Bamlanivimab-etesevimab therapy. Casirivimab-imdevimab and sotrovimab retained susceptibility[38-40]. Moderate reduction in neutralization by convalescent and post-vaccination sera[45]		Absent	De-escalated	Full vaccination efficacy 73.0% (95%CI: 64.3%–79.5%)[46]
Gamma	1.7 to 2.4-fold higher transmissible than previous (non-P.1) infection[48]. Increased risk of reinfection	1.2 to 1.9 times more likely to result in mortality compared with previous lineages[48]	> 511 fold decreased susceptibility to Bamlanivimab-etesevimab but no change in susceptibility with Casirivimab-imdevimab and Sotrovimab[38-40]. Reduced neutralization to convalescent and post-vaccination sera		Absent	De-escalated	Full vaccination efficacy against Gamma variants 63.0% (95%CI: 47.9%–73.7%)[46]
Delta	40%-60% more transmissible than Alpha variant[49]	Increased severity of the disease[51,52] and increased risk of hospitalization[52,53].A shorter time interval between disease onset to hospitalization in comparison to the wild-type variant[44]	Neutralization is affected minimally		Absent	De-escalated	Moderate reduction in vaccine efficacy against symptomatic infection but retained efficacy against severe disease and hospitalization[45]. The 2-dose mRNA-1273 vaccine: 86.7% (95%CI: 84.3%-88.7%) efficacy against infection and 97.5% (92.7%-99.2%) efficacy against hospital admission. The 2-doses of BNT162b2 and ChAdOx1 nCoV-19 vaccine 88.0% (95%CI: 85.3%-90.1%) and 67.0% (95%CI: 61.3%-71.8%), respectively[41]
Omicron	Increased risk of transmissibility, reinfection/breakthrough infection	Severity less compared to Delta variant	Reduced or absent neutralization efficacy by vaccines and mABs[56]		SGTF except BA.2 lineage[64]	Few sublineages de-escalated (BA.1, BA.2, BA.3, BA.4, BA.5 etc.)	Booster doses are needed to mount a more appropriate immune response against symptomatic or non-symptomatic infections, transmission, and serious manifestations[44]

VOCs: Variants of concerns; SGTF: “S” gene target failure; ICU: Intensive care unit; mAbs: Monoclonal antibodies; NTD: N-terminal domain.

COMMON MUTATIONS AND THEIR IMPACTS

D614G mutation

Genomic surveillance of SARS-CoV-2 during the first year of the COVID-19 pandemic revealed that the D614G mutation in the spike glycoprotein (Spike protein) was the predominant mutation in February 2020[69,70]. Later on, it spreads globally. The D614G mutation is caused by an aspartic acid-to-glycine substitution at position 614 of the spike glycoprotein. The D614G change is also associated with three other mutations: A, C-to-T mutation in the 5′ untranslated region (5’-UTR), a silent C-to-T mutation at position 3037, and a C-to-T mutation at position 14408, which causes an amino acid change in RNA-dependent RNA polymerase (RdRp)[70]. The D614G-mutated variants almost always carry these three mutations. Remdesivir targets the RdRp enzyme. Plante et al[71] examined the replication kinetics of the D614G variants in human lung epithelial cells (Calu-3 cells) and primary human airway tissues[71]. They found 2.4-fold more infectious virus at 36 hpi, indicating that the D614G mutation enhanced viral replication. Similarly, the golden Syrian hamster model infected with the D614G mutation produced higher infectious viral titers in the nasal washes and trachea but not in the lungs[71,72]. As a result, the D614G mutation may enhance viral loads in COVID-19 patients' upper respiratory tracts, increasing transmission. Korber et al[70] reported a lower real-time reverse transcription–polymerase chain reaction assay cycle threshold, which suggests higher viral loads and high infectivity[70]. However, the mechanism underlying improved replication fitness is unclear. Few studies have reported that increased cleavage efficiency of the spike protein into S1/S2 influences the SARS-CoV-2 infection[73,74]. However, Plante et al[71] observed no substantial differences in spike cleavage between the D614 and G614 virions, indicating that the enhanced infectivity is unlikely due to a D614G-mediated spike cleavage difference[71]. Another mechanism could be the disruption of the interprotomer latch between S1 and S2. Normally, the carboxyl groups in D614 form a hydrogen bond with the hydroxyl group in Thr859 across the S1/S2 interface[70]. The cryo-EM studies had shown that D614G disrupts the interprotomer latch between D614 in S1 and T859 in S2 and promotes the RDB domain to an “up” or open conformation and a higher chance of binding with the human ACE2 receptor. The ratio of closed and open conformation in D614 and G614 is 82% and 18%, and 42% and 58%, respectively[75]. Kannan et al[76] suggested that D614G alone would not be able to explain the high infectivity of the SARS-CoV-2 virus, and other coexistence mutations such as P323L (nsp12) and C241U (5’-UTR) and nsp mutations may also contribute to the infectivity[76]. D614G, by increasing the number of spike proteins per virion, may also be responsible for the increased infectivity[77,78]. However, since the 614 position lies outside the RBD, this mutation does not alter the affinity of spike protein to ACE2. Zhang et al[79] hypothesized that increased stability of the S-trimer in the presence of the D614G mutation may explain the enhanced infectivity as the S1 subunit dissociates more readily from the virus with an aspartic acid residue at position 614 than the virus having glycine at position 614[79]. The D614G mutation has been detected in B.1.1.7, B.1.351, P.1, and B.1.617.2 and B.1.1.529 lineages, indicating a transmission advantage of this mutation. However, it does not cause immune escape. Garcia-Beltran et al[80] had shown that the sera from convalescent individuals showed effective cross-neutralization of both wild type and D614G variants[80].

N501Y mutation

It includes replacing the amino acid asparagine (N) with tyrosine (Y) at position 501. The N501Y mutation has been identified in the B.1.1.7, B.1.351, P.1, and B.1.1.529 lineages. This mutation can alternatively be represented as S: N501Y, indicating that it occurs in the spike protein. The N501Y mutation is responsible for higher binding affinity to human ACE2 receptors, but has no impact on immune escape mechanisms[81]. Luan et al[82] in an in-silico study, had similarly shown that the N501Y mutation can increase the spike protein’s receptor binding affinity with the human ACE2 receptor[82]. The N501Y mutation on RBD may produce an aromatic ring-ring contact and an extra hydrogen bond with ACE2 receptors, increasing binding affinity by tenfold over the wild strain[83]. Moreover, the N501Y mutation decreases the polarity of critical residues located in RBD, thereby increasing the affinity between RBD and ACE2 receptors[84,85]. Zhu et al[86] reported that a higher number of ACE2 receptors bind with N501Y spikes as compared to N501[86]. Furthermore, using cryo-electron microscopy, the N501Y was placed into a cavity at the binding contact at Y41 of ACE2.This provides a structural basis for the N501Y mutant's higher ACE2 affinity, which is likely related to its greater infectivity. Teruel et al[87] in a modeling analysis demonstrated that D614G and N501Y mutations allow the RBD to remain in open conformation for a longer period of time[87]. However, large structural changes in the antibody-binding epitopes do not occur as the N501 is located outside the major neutralizing epitopes on the RBD[88]. Therefore, the N501Y mutation causes only minimal changes in the sensitivity to neutralizing antibodies. The N501Y mutation co-occurs with several other mutations, such as P681H and deletion of the amino acid at the 69^th and 70^th residues (Deletion69/Deletion70) on the spike protein. Leung et al[89] reported that the N501Y lineage with amino acid deletion Deletion69/Deletion70, detected among the United Kingdom strain, was 75% (70%–80%) more transmissible than the N501 lineage[89]. However, the N501Y mutation does not impact the binding and neutralization of most mAbs[90-95]. Similarly, it rarely shows reduced susceptibility to convalescent plasma[37,92-94].

E484K mutations

The E484K mutation is situated in the RBD and is critical for ACE2 receptor binding and antibody recognition. The E484K mutation has been detected in the B.1.1.7, B.1.351, P.1, and B.1.617.2 variants[53,95]. It involves the replacement of the amino acid glutamic acid (E) with lysine (K) at position 484 of the spike protein. The E484K mutation is an escape mutation, which permits the virus to slip past the body's immunological defenses[95]. Collier et al[96] observed that the B.1.1.7 variant carrying the E484K mutation increased the amount of serum antibody needed to prevent infection of cells substantially[96]. The E484K mutations reduce neutralization by antibodies and may cause breakthrough infections[41,95]. The E484 mutation with amino acid changes to K, Q, or P reduces neutralization by convalescent sera by more than an order of magnitude. Greaney et al[97] reported that the E484 mutation with K, Q, or P reduces the neutralization titer of the convalescent plasma collected from the subject on day 32 by 35 to 115-fold[97]. They also found that each of the four discovered mutations (E484A, E484D, E484G, and E484K) conferred resistance to all four convalescent sera tested. The E484 mutation is notable for causing the most significant decreases in neutralization titers. On the other hand, the K444E, G446V, L452R, and F490S mutations escaped three of the four sera tested. The G446V mutation caused approximately a 30-fold decrease in the neutralization titer. By co-incubating the pseudovirus with SARS-CoV-2 spike proteins and mAbs, Liu et al[98] demonstrated that that the E484 mutations resulted in considerably lower neutralization efficacy by both mAbs and convalescent sera[98]. Nelson et al[99] in a molecular dynamic simulation study, reported that the combination of E484K, K417N, and N501Y mutations resulted in the highest degree of conformational alterations of the RBD domain when bound to ACE2, compared to either E484K or N501Y alone[99]. These mutations favor ACE2 receptor binding. Zahradník et al[100] used an in vitro evolution model and found that S477N, E484K, and N501Y mutations were among the first to be selected[100]. Moreover, the E484K and N501Y mutations are the tightest binding mutations emerging from the B3 library. Wang et al[101] reported that E484K, N501Y individually, or K417N/E484K/N501Y mutations together showed a small but significant reduction in neutralization efficacy with Moderna and Pfizer-BioNTech vaccines[101].

L452R and E484Q mutations

Due to the presence of these two prominent mutations at the same location, it was initially called a “double mutant”. The L452R and E484Q are also the key mutations in the B.1.617.2 variants. The L452R and E484Q double mutants are the two-spike protein RBD mutations and have been detected in 15% to 20% of positive cases in the Maharashtra state of India on March 24, 2021, by the Indian SARSCoV-2 Consortium on Genomics[102]. The L452R and E484Q mutations are responsible for the overall stability of virus-host interaction[103]. They are also responsible for resistance to neutralization by monoclonal and polyclonal antibodies. In the pseudovirus-based study, the L452R mutation caused more cellular entry compared with that of the D614G mutation alone, but it was lower than the N501Y mutation[104]. The L452R mutation raised spike protein expression (0.32 times) and improved binding affinity to ACE2 receptors. It increases the virus's infectivity[84]. The L452R mutation also allows immune escape from human leukocyte antigen (HLA)-restricted cellular immunity[105].

P681R mutation

The furin cleavage site is located at the spike S1/S2 junction. The cleavage of this region is the key to host cell entry. This mutation is responsible for efficient furin cleavage, subsequent internalization, and better transmissibility. A unique feature of the B.1.617.2 variant is the P681R mutation in the spike protein, where proline is substituted by arginine. The P681R mutation is located adjacent to the furin cleavage site[106]. The P681R mutation makes the sequence less acidic and causes furin to function more effectively[51]. Increased furin cleavage will make more spike proteins primed to enter human cells. In the Delta variant, more than 75% of the spikes are primed to infect a human cell, whereas the values in the Alpha variant and original strain were 50% and 10%, respectively[107]. The P681R mutation is highly conserved in the B.1.617.2 variant and is responsible for the higher pathogenicity of the B.1.617.2 variant[108]. P681R mutation in the B.1.617.2 variant enhances SARS-CoV-2 fitness. In an experimental study, Liu et al[109] reported that the B.1.617.2 variants outnumbered other variants based on a replication competition assay done on human lung epithelial cells and primary human airway tissues[109]. The mechanism of increased infectivity was explained by the accumulation of the P681R mutation in the B.1.617.2 variant, which causes furin cleavage of the S1/S2 protein, leading to increased infectivity. Moreover, reverting the P681R mutation to wild-type P681 significantly reduced replication.

P681H mutation

The P681H mutation involves the substitution of proline (P) with histidine (H) at position 681. The P681H mutation is also near the S1/S2 furin cleavage site that is responsible for efficient SARS-CoV-2 transmission and infection[85,110]. The P681H mutation may also reduce class 3 antibody recognition[111].

T478K mutation

The T478K mutation is found within the critical receptor binding motif of S gene[112]. It alters the virus's affinity for human cells, increasing viral infectivity. The T478K mutation is a shift in amino acid from polar, uncharged threonine (T) to basic, charged lysine. It may raise the electrostatic potential of spike protein, resulting in a more positive surface in an area that directly contacts ACE2. Furthermore, the longer side chain of lysine is expected to exacerbate the mutant's steric hindrance, perhaps altering the spike/ACE2 interaction[113]. The T478K mutation is frequently co-occurring with three other spike mutations located outside the canonical ACE2 interaction regions, such as D614G (99.83% co-occurrence), P681H, and T732A, with 93.8% and 88.7% co-occurrence with T478K, respectively[114].

N439K mutations

This mutation was identified in March 2020 in Scotland from lineage B.1 on the background of D614G. It has also appeared independently in multiple lineages. As of January 6, 2021, it was reported in 34 countries and was the second most commonly observed RBD mutation worldwide[115]. N439K enhances the binding affinity for the ACE2 receptor and is also responsible for immune evasion. The N439K mutation confers resistance against several neutralizing monoclonal and polyclonal antibodies[116]. The N439K mutation located in the RBD region creates a strong salt bridge with ACE2 receptors, which may enhance the electrostatic complementarity and binding affinity of spike proteins to ACE2[117].

Y453F mutations

The Y453F mutation is located on the RBD and has been detected in human and mink infections. The bidirectional transmission has been reported in the Netherlands[118,119]. Initially, in Denmark, one new lineage was identified and was known as “Cluster 5” and contained mutations in the spike protein[120]. Later on, the mutation was identified as a Y453F mutation located in the RBD domain[121]. The Y453F mutation enhances binding to ACE2. The Y453F mutations involve a tyrosine-to-phenylalanine substitution at amino acid 453 (Y453F). Y453F mutation significantly lowers susceptibility to casirivimab (74-fold), but not to other Food and Drug Administration/Emergency Use Authorisation approved mAbs[122,123]. The Y453F mutation is also found to escape from HLA-restricted cellular immunity[105].

N440K mutations

This mutation was detected in various parts of India in March and April 2021. The N440K mutation is also associated with the P323L substitution in the RdRP gene. The N440K variant can generate significantly higher viral loads within a short period, leading to its rapid spread. This variant has shown localized spread in the following four states: Karnataka, Maharashtra, Telangana, and Chhattisgarh. They together contributed to about 50% of these samples submitted for analysis[124]. The N440K mutation has also been reported to cause reinfection[125]. The frequency of the N440K variant was 2.1% in India and was particularly high in the state of Andhra Pradesh (33.8% of 272 genomes)[126]. The N440K variant is responsible for immune escape as it has shown resistance to class 3 mAbs and an enhanced binding affinity to the human ACE2 receptor[127].

AMINO-TERMINAL DOMAIN OR NTD MUTATIONS

NTD mutations in spike protein are often the neglected area in the SARS-CoV-2 genomic study. However, NTD mutations have been reported among the B.1.1.7 and B.1.351 lineages[128]. A significant transmission of a six-nt deletion in the S gene has been reported by Gupta et al[128] leading to a loss of two amino acids: H69 and V70[128].

Here we report recurrent emergence and significant onward transmission of a six-nt deletion in the S gene, which results in loss of two amino acids: H69 and V70. The H69/V70 variant also co-occurs with N501Y, N439K, and Y453F mutations on the RBD. The H69/V70 deletion increases infectivity twofold, and the effect on viral fitness is independent of the RBD changes. The H69/V70 mutations may also boost SARS-CoV-2’s ability to generate new variants, such as vaccine escape variants[129].

K417N/T mutations

The K417N/T mutation has been reported in the B.1.351 (as K417N), P.1 (as K417T), and B.1.1.529 variants (as K417N). Interestingly, the K417N/T mutation usually occurs in presence of other RBM mutations as these mutations may decrease the binding to ACE2 receptors[84,115]. The K417N/T mutation may cause immune evasion as well. The K417N mutation confers reduced susceptibility to etesevimab[130] and casirivimab[92] but retains susceptibility to bamlanivimab, imdevimab, and sotrovimab[124]. It also retains susceptibility to convalescent plasma or sera from patients vaccinated with the mRNA vaccine[92,123]. The K417N, E484K, or N501Y mutations showed a reduced or abolished neutralization by 14 of the 17 most potent mAbs tested[101]. Li et al[131] in a pseudovirus model, showed that the K417N mutation increases viral sensitivity to neutralization. Normally, the K417 variant allows a closed conformation, leading to reduced binding to ACE2 and reduced access to neutralization antibodies. The K417N mutation helps in an open conformation, resulting in the exposure of more epitopes to neutralizing antibodies and subsequently increased virus neutralization. Table 2 shows the five VOCs and their mutations.

Table 2 Showing the five variants of concerns and their mutations.

WHO label	Pango lineage	GISAID clade	Nextstrain clade	Spike protein substitutions	First detected	WHO date of designation
Alpha	B.1.1.7	GRY	201(V1)	Deletion 69-70, Deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H	United Kingdom	18th December 2020
Beta	B.1.351	GH/501Y.V2	20H(V2)	D80A, D215G, DeletionL242, DeletionA243, DeletionL244, K417N, E484K, N501Y, D614G, A701V	South Africa	18th December 2020
Delta	B.1.617.2	G/478K.V1	21A	T19R, T95I, G142D, Deletion156, Deletion157, R158G, L452R, T478K, D614G, P681R and D950N	India	VOI: 4th April, 2021 VOC: 11th May, 2021
Gamma	P.1	GR/501Y.V3	20J(V3)	L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I	Brazil	11th January 2021
Omicron	B.1.1.529 lineage	GR/484A	21K	A76V, T95I, Y145del, G339D, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, L212I, S371L, S373P, S375F, K417N. ORF1a: K856R, ORF1a: L2084I, ORF1a: A2710T, ORF1a: T3255I, ORF1a: P3395H, ORF1a: I3758V, ORF1b: P314L, ORF1b: I1566V, and ORF9b: P10S	Botswana and South Africa	26th November 2021

WHO: World health organization; GISAID: Global initiative on sharing all influenza data; VOI: Variants of interest; VOC: Variants of concern.

CONCLUSION

The emergence of SARS-CoV-2 variants is an important phenomenon in the natural history of SARS-CoV-2 infection because it poses a considerable public health risk. Currently, we have five VOCs. These variants are more transmissible than the Wuhan strain. Various mutations identified in these VOCs are located on the spike protein, especially in the RBD. These mutations influence virus-host cell interaction, binding affinity, furin cleavage, and neutralizing efficacy by antibodies and vaccines. The most recent VOC detected is the omicron variant; however, this will not be the last variant we encounter. We will also see newer variants in the future, too. Characterization of the genomic character of the VOCs will help in identifying newer mutations quickly and in exploring phenotypic effects on the virus. In this article, we looked at the characteristics of the five VOCs, as well as the associated mutations, and how they affect SARS-CoV-2 virus’s infectivity, transmissibility, and immune evasion. The best way to prevent the development of new variants is to vaccinate as many people as possible, closely adhere to infection prevention and control measures, and eliminate vaccine inequalities that limit future human transmission and acquisition.