**3. Emergence of SARS-CoV-2 variants – genotype to phenotype analysis and global public health effects**

A series of measures have been taken globally for optimal management of the emergence of SARS-CoV-2 variants. A global system has been established to detect SARS-CoV-2 lineages and to assess the potential risk for the circulating viral variants. For effective surveillance and viral characterization worldwide, it was essential to better describe the recently emerging variants and to establish a joint nomenclature.

Considering the large amount of collected data generated by sequencing, WHO experts after consultation with the Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE) establish the current nomenclature in use when referring to SARS-CoV-2 variants [54]. Thus, for each identified lineage, it was assigned a Greek alphabet letter (e.g., Alpha, Beta, Delta, or Omicron) and when choosing the working terminology for SARS-CoV-2 variants, it was considered the current terminology adopted by well-known open-source databases that performed phylogenetic analysis. Such examples are **PANGOLIN** – The Phylogenetic Assignment of Named Global Outbreak Lineages [55]; **Nextstrain** [56] or **GISAID** – The Global Initiative on Sharing All Influenza Data [57]. At the same time, the term "index virus" was established when referring to the SARS-CoV-2 genome characterized at the beginning of the pandemic (December 2019) in the situation of the first cases reported [58]. Considering the rapid evolution of the virus from a mutational point of view, the present nomenclature may undergo changes.

Specific viral variants that pose a risk to public health have been named, are considered a priority in monitoring, and are categorized using the following working terms:


**Variant of Interest (VOI) -** a SARS-CoV-2 variant presenting a certain genetic constellation that is predicted to cause changes in virus properties impacting its transmissibility, virulence, the diagnostic and treatment methods, the severity of the disease, and immune system escape. It is also acknowledged to be responsible for high community spread, prevalence in multiple clusters or many countries, or hint of an emerging risk to global public health. At present, there are no circulating VOIs [58].

Among the circulating VOIs reported in the past [58, 59] (presented chronologically with spike mutation of interest and using the WHO and Pango lineage terminology) are:


According, to WHO a **Variant of Concern (VOC)** is defined as a viral variant that meets the criteria for a VOI and in addition correlates with a higher degree of virulence and transmission or a change in COVID-19 epidemiology or clinical presentation, or a decrease in the effectiveness of currently available public health measures or of diagnostics, therapeutics, or vaccines [58].

Current VOC is **Omicron lineage (B.1.1.529/VOC: 26-Nov-2021)** including sublineages **BA.1, BA.2, BA.3, BA.4, BA.5,** and **XE** (**BA.1/BA.2** circulating recombinant) that WHO recommend to be monitored by public health authorities as distinct lineages.

The mutational profiles in spike sequence in Omicron sub-lineages are listed below:

• **BA.1** - A67V, Δ69–70, T95I, G142D, Δ143–145, N211I, Δ212, ins215EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F;


From the collected data, a higher transmission is observed also accompanied by a lower severity for the Omicron BA.1 and BA.2 variants with BA.2 displaying a dominant transmission in the EU/EEA countries. On the other hand, for the BA.4 and BA.5 there is not enough evidence regarding the impact of these variants on transmission rate or severity [59].

VOCs circulating in the past include:


**VOC lineages under monitoring (VOC-LUM)** are characterized as variants that, from a phylogenetic point of view, belong to VOCs, which are currently circulating, nevertheless compared to the circulating VOCs exhibit some genetic alterations that show better transmission and also present amino acids changes that might explain the epidemiologic modifications compared to circulating variants [58].

Currently, all **VOC-LUMs** are Omicron sublineages being under surveillance. Thus, the **BA.4 and BA.5** sublineages from a phylogenetic point of view belong to the same circulating VOC, presenting the same mutations profile for S (**BA.2 like** + del69/70, L452R, F486V, Q493) and also displaying two Nsp4 reversions: L438 and ORF6:D61. On the other hand, these two VOC-LUMs vary from each other through mutations outside spike, thus BA.4 shows the following additional mutations: ORF7b:L11F, N: P151S while BA.5 exhibits D3N mutation in M gene. **BA.2.12.1** is another current VOC-LUM belonging to BA.2 sublineage and which shares the same S mutational pattern plus L452Q and S704F. **BA.2.9.1** and **BA.2.13** come from BA.2 sublineage with which they share S mutational profile and also L452M additional mutation, while **BA.2.9.1** displays other genomic mutations outside of S, respectively, in N gene (P67S, S412I) and in ORF3a:H78Y [58].

It is very important to keep these lineages and their modifications under close observation in key sites or hotspots, in order to detect in real-time a divergence from the generating VOC and a possible new risk to global public health.

**Variants under monitoring (VUM)** are defined as variants that are suspected to pose future risk but with unclear evidence of phenotypic or epidemiological impact which requires enhanced monitoring. Currently, there is no variant under monitoring.

In the generation of SARS-CoV-2 variants, two processes, replication and recombination, have major implications. The SARS-CoV-2 replication process requires an RNA-dependent RNA polymerase (RdRp) that is prone to an error leading to the replication-associated changes. Due to the proofreading process assured by the virus-encoded 3′ exonuclease, nsp14, the mutation rate remains in the low range if we consider the magnitude of the SARS-CoV-2 extension [60].

The factors that may impact SARS-CoV-2 replication, error rate accumulation, and the variants selection include wildlife reservoirs (permissive host species and species-specific adaptation in different hosts) [61], biochemical characteristics of different infected cell types generating a heterogeneous mix of viral proteins [62], and population-level immunity driving selection of these variants [63]. Recombination can occur in a cell coinfected with more than one virus variant through discontinuous transcription of SARS-CoV-2 genomes via 'strand switching' by the viral RdRp or through breakage and rejoining of genomes based on homology regions to form chimeric genomes [63, 64].

Careful monitoring of new SARS-CoV-2 variants must reduce transmission rate, pathogenicity, and resistance to immune responses. This refers especially to molecules involved in cell entry and those that provide antigenicity.

The characteristics of SARS-CoV-2 variants are determined by the structural spike (S) protein cleaved in infected cells by a cellular protease, furin, yielding two subunits, S1 and S2. The S1 subunit binds the receptor ACE2 and the S2 subunit anchors the S protein to the membrane and mediates membrane fusion [65, 66].

In order to identify SARS-CoV-2 genome hotspots, a phylogenetic analysis of the virus is required. This analysis enables detection of the occurrence of variants that may present concern [67]. Until now numerous SARS-CoV-2 genome hotspots have been recognized. Being considered an important SARS-CoV-2 hotspot, spike genomic sequence is frequently altered (various substitutions/deletions) causing modifications in the protein sequence that ultimately may have a significant impact on virus evolution and can cause major difficulties in pandemic management. Consequently, certain mutations in spike protein can determine an increase in infectivity and also a more severe disease, while on the other hand can affect the therapeutic effectiveness [68].

During the early evolution of SARS-CoV-2, the **D614G** substitution in the receptor-binding domain (RBD) on Spike is considered to be one of the earliest S hotspots being detected in almost all previously circulating VOCs (Alpha, Beta, Delta, Gamma) as well in the current circulated VOC Omicron [69]. The mutation, originated from genetic drift and obtained a selective advantage [70, 71], being detected first in Europe in January 2020 and accounting for 98% of the SARS-CoV-2 spread in September 2021 [72]. In these variants, replacing the aspartic acid with glycine at position 614 of the spike protein induced structural change that increased binding affinity to ACE2 and virus entry [73], associated with increases in transmissibility (*in vivo* infectivity) but without affecting the severity of disease [71, 74].

Another recognized S hotspot is considered to be N501Y substitution also in RBD region, shared by Alpha, Beta, and Gamma lineages from previous VOCs. N501Y is known to promote viral replication through increasing affinity between the receptorbinding domain (RBD) and ACE2 and to facilitate the antibody escape [75]. P681H augments viral infectivity being located in the furin cleavage site [75], and delH69/ V70 detected in Alpha and Omicron VOCs, was associated with disease severity and long-term infection [76].

Interfering also with antibody escape and thus being associated with vaccination failure were another two hotspots found in RBD region namely E484K and K417N/T

#### *SARS-CoV-2 Variant Surveillance in Genomic Medicine Era DOI: http://dx.doi.org/10.5772/intechopen.107137*

substitutions. E484K is found in Beta, Gamma, Delta, and Omicron VOCs while K417N/T is only in Beta and Gamma [77]. Hoter and Naim analyzed the biosynthetic forms and glycosylation of intracellular and secreted forms of double mutants L452R and E484Q (Indian B.1.617 variant) in comparison with E484K and N501Y (B.1.351 and P.1 variant) and observed that the double mutants L452R and E484Q were comparatively highly secreted, associated with a strong interaction with ACE2 in the human lung Calu3 cells [78]. L452R and T478K hotspots, identified in Delta variant, were associated with an increased ACE2 binding and also with antibody escape, which led to an increase in virulence [79, 80].

Compared with wild-type Wuhan-1 bearing D614G, the **Delta (B.1.617.2)** was six-fold less sensitive to serum neutralizing antibodies from previously infected individuals, and eight-fold less sensitive to vaccine-elicited antibodies, lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees [81]. The B.1.617.2 variant proved to be highly fusogenic and notably more pathogenic than its parental virus due to the highly conserved P681R mutation in the spike protein facilitating cleavage of the spike protein and enhancing viral fusogenicity [82].

It is crucially important to determine spike mutations that affect antigenic profiles and the level of cross-protection provided by prior infection with other viruses. The immunogenic regions of the spike refer especially to the spike receptor-binding domain (RBD) because ~90% of the serum neutralizing antibodies from SARS-CoV-2 infected individuals target this region [83–85]. But, also, the N-terminal domain (NTD) of the S protein is targeted [86–89].
