key: cord-0976946-09tdk4tq
authors: Antony, Priya; Vijayan, Ranjit
title: Role of SARS-CoV-2 and ACE2 variations in COVID-19
date: 2021-04-21
journal: Biomed J
DOI: 10.1016/j.bj.2021.04.006
sha: a820027c55204a59ec37894e46369c840305415f
doc_id: 976946
cord_uid: 09tdk4tq

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is one of the worst medical emergencies that has hit the world in almost a century. The virus has now spread to a large number of countries/territories and has caused over two million deaths. Evidently, the virus has been mutating and adapting during this period. Significant effort has been spent on identifying these variations and their impact on transmission, virulence and pathogenicity of SARS-CoV-2. Binding of the SARS-CoV-2 spike protein to the angiotensin converting enzyme 2 (ACE2) promotes cellular entry. Therefore, human ACE2 variations could also influence susceptibility or resistance to the virus. A deeper understanding of the evolution and genetic variations in SARS-CoV-2 as well as ACE2 could contribute to the development of effective treatment and preventive measures. Here, we review the literature on SARS-CoV-2 and ACE2 variations and their role in COVID-19.

In late December 2019, hospitals in Wuhan City, Hubei province of China observed a string of respiratory infections of unknown etiology. Most of the infected patients had a history of exposure to a seafood and wet animal market in Wuhan [1, 2] . By January 7, 2020 a previously unrecognized virus was isolated from the throat swab of a patient by the Chinese Center for Disease Control and Prevention (CDC). Subsequently, its genome was elucidated by using next-generation sequencing [3, 4] . The virus was initially named as the 2019 novel coronavirus (2019-nCoV) by the World Health Organization (WHO). On February 12, 2020 the virus was formally named as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses and the disease associated with it as the coronavirus disease 2019 . The naming was based on the phylogenetic relationship of the new virus with the severe acute respiratory syndrome coronavirus (SARS-CoV). Due to the alarming spread and severity of COVID-19, on January 30, 2020, the WHO declared the SARS-CoV-2 outbreak as a Public Health Emergency of International Concern. Since the first reported case, the morbidity and mortality increased exponentially around the globe. This led to the declaration of COVID-19 as a global pandemic by the WHO on March 11, 2020 . Indisputably, COVID-19 is one the worst medical emergencies that has hit the world in almost a century. Recently, a number of vaccines based on inactivated viruses, viral vectors, nucleic acids and viral proteins have been approved around the world for emergency use and more than 50 vaccine candidates are in clinical trials [5] . The major phenotype of this disease could include some or all of fever, cough, shortness of breath and respiratory disorders. A vast number of COVID-19 patients are either asymptomatic or mildly symptomatic but, in severe cases, infection can cause pneumonia, acute respiratory syndrome, kidney failure and possibly death [6] . COVID-19 has now spread to nearly two hundred countries or regions including Antarctica. In a span of just a year, the virus has caused more than two million deaths and confirmed cases have exceeded 98 million globally [7] .

Coronaviruses (CoVs) are the largest group of viruses that belong to the Coronaviridea family. They cause respiratory and intestinal infections in animals and humans. Due to genomic nucleotide substitutions and recombination, CoVs are one of the most rapidly evolving viruses [8] . Until the outbreak of SARS in 2002 in China, these viruses were not considered to be highly pathogenic [9] . CoVs are relatively large, enveloped, single-stranded, positive-sense RNA viruses. They get their name from the crown-like viral protein that projects out of their envelope. Based on serological and genomic characteristics, CoVs are classified into four generaalphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus [10] . They are highly contagious and cause disease in a variety of domestic and wild animals. Alphacoronaviruses and betacoronaviruses primarily infect mammals while gammacoronaviruses and deltacoronaviruses mainly infect birds [11] .

Currently seven species of coronaviruses are known to infect humans. Before the outbreak of SARS, only two human CoVs (HCoVs) -the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 were known to cause human disease such as mild common colds [12] . SARS was caused by a betacoronavirus eventually named as SARS-CoV. It affected over 8000 individuals and caused nearly 800 deaths [13] . Subsequent to the SARS epidemic, two more HCoVsthe alphacoronavirus HCoV-NL63 and the betacoronavirus HCoV-HKU1 -were reported that were capable of causing mild to serious lower respiratory tract infections in infants, children and adults [14] . In 2012, another novel betacoronavirus was identified and reported in the Middle East that could cause severe respiratory illness. The virus was named the Middle East respiratory syndrome coronavirus (MERS-CoV) [15] . The most recent novel betacoronavirus is the SARS-CoV-2 that causes COVID-19.

Coronaviruses carry relatively large RNA genomes ranging from 27 to 32 kilobases (kb). Like other coronaviruses, SARS-CoV-2 is an enveloped virus with a linear single-stranded positive-sense RNA of ~30 kb [16, 17] . The SARS-CoV-2 genome consists of six major open reading frames (ORFs) and a number of accessory genes [18] . Roughly, two third of the viral RNA encodes two large overlapping ORFs namely ORF1a and ORF1b, or ORF1ab together, that produces non-structural proteins (nsps). ORF1a is translated to produce polyprotein 1a (pp1a) while ORF1ab yields polyprotein 1ab (pp1ab). These polypeptides are further cleaved by viral encoded proteases into 16 nsps (nsp1-16) that are involved in the viral replication and transcription. During the time of their infection in host cells, SARS CoV-2 replicates its genomic RNA using nsp12 that harbors RNA-dependent RNA polymerase (RdRP). The remaining one third of the genome encodes four structural proteins namely spike (S), envelope (E), membrane (M) and nucleocapsid (N) [17] . Another interesting feature of CoVs is the synthesis of a nested set of subgenomic RNAs (sgRNAs) from genomic RNA by discontinuous transcription. These sgRNAs are translated into structural proteins (S, E, M, N) and several accessory proteins. Each viral sgRNA contains a common leader sequence derived from the 5′ end of the genomic RNA. The leader sequence plays a critical role in gene expression and the length of this sequence varies between different CoV species [17, 19] . The S protein, located on the virion surface, is a trimeric class I fusion protein. It is one of the most variable and probably one of the most important part of the CoV genome. It mediates binding of the virus to the host receptor as well as membrane fusion for invading the host cell [20] . The M glycoprotein could be regarded as the central organiser since it interacts with several other structural proteins. It is the most abundant structural protein and spans the membrane bilayer three times [21] . It also plays a significant role in the budding process as well as the sorting of viral components into virions. Among the structural proteins, the E protein is a smallest and this membrane protein is essential for assembly and budding of virions. Even though they are abundantly expressed during replication, only a small number of these E proteins are incorporated into the virion envelope. The primary function of the N protein is to make up the nucleocapsid. Additionally, it is also assists with viral transcription and replication [22] .

Initial analysis of the sequenced genome revealed that SARS-CoV-2 shared 80% sequence identity with SARS-CoV and 96.2% with the bat coronavirus (BatCoV) RaTG13, obtained from the bat Rhinolophus affinis [18, 23] . As SARS-CoV-2 genome was highly similar to the BatCoV genome, it was suggested that bats may act as the primary and natural reservoir of these viruses [18] . Like SARS-CoV and MERS-CoV, this zoonotic disease was also believed to have been transmitted from an intermediate host. In both cases, bats acted as the natural reservoir while masked palm civet and the dromedary camel acted as the intermediate host [9, 24] . Another interesting observation was subsequently made from coronaviruses isolated from Malayan pangolins (Manis javanica). Genomic and evolutionary analysis revealed that PangolinCoV is 91.02% and 90.55% identical to SARS-CoV-2 and BatCoV RaTG13, respectively. This makes PangolinCoV the second closest relative of SARS-CoV-2 behind RaTG13. This suggests that pangolin could also be considered as possible hosts of SARS-CoV-2 [25, 26] .

The entry of SARS-CoV-2 into the host cell is mediated by the transmembrane S glycoprotein protruding from the viral surface [27] . The S protein is functionally divided into two subunits S1 and S2 that mediate attachment and membrane fusion respectively. The S1 subunit is further divided into two independent domains -J o u r n a l P r e -p r o o f the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD). Depending on the type of CoV, receptor binding domain (RBD) could be in either NTD or CTD [28] . In SARS-CoV-2, the RBD is located in the S1 subunit and composed of a core and an extended insertion. Five antiparallel β-strands along with short connecting helices and loops constitute the core. It is also responsible for formation of the S trimer. The extended insertion, that hosts the receptor binding motif (RBM), contains most of the residues that allow the SARS-CoV-2 to bind to the host receptor [29] .

In the mature virus, three S1/S2 subunits assemble to form a spike-like trimer structure that protrudes from the viral envelope. The entry of virus into host cell is a complex process involving a series of steps that include the binding of the S protein to the host receptor and proteolytic cleavage of the protein to facilitate virus cell fusion. The two functional subunits (S1 and S2) of the S protein remain non-covalently bound in the prefusion state stabilized by the RBD of the S1 subunit [30] [31] [32] . Binding of S1 to the host receptor results in substantial structural rearrangement that destabilizes the trimer [33] . These conformational changes are crucial for membrane fusion since this exposes the S1/S2 cleavage site to host proteases [34, 35] . Sequence analysis of SARS-CoV-2 S protein reveals an insertion of four amino acids ('PRRA') at the boundary of S1/S2 subunits. Interestingly, this insertion is not present in SARS-CoV and other SARS-related CoVs but was observed in human CoVs HCoV-OC43, HCoV-HKU1 and MERS-CoV [36, 37] . The insertion introduces a "PRRARS/V" sequence, which is a furin recognition site, in SARS-CoV-2 that aids effective cleavage of S1/S2 by furin and other proteases [38] . It has been suggested that the presence of multiple arginines (multibasic) in the cleavage site could activate ubiquitously expressed proprotein convertases, like furin, that could assist the systematic spread of the virus [39] . It has been suggested that SARS-CoV-2 S protein behaves in a manner similar to that of MERS-CoV. Initially, the S protein of both viruses are cleaved by furin at the S1/S2 junction followed by plasma membrane-associated type II transmembrane serine protease (TMPRSS2) mediated membrane fusion. [36] . TMPRSS2 expressing cell lines are highly susceptible to SARS-CoV-2 infection when compared to nonexpressing cell lines, supporting the role of TMPRSS2 in viral infection [40] Analysis of amino acid variation patterns of S1 proteins from SARS-CoV-2, SARS-CoV, RaTG13 and Pangolin-CoV revealed that the S1 subunit of Pangolin-CoV is more closely related to SARS-CoV-2 than RaTG13. It was also observed that RBD of both SARS-CoV-2 and Pangolin-CoV are highly conserved with just a single amino acid change (glutamine at position 500 in SARS-CoV-2 S1 and histidine in Pangolin-CoV) suggesting a similar mechanism of infection in Pangolin CoV and SARS-CoV-2 [25] .

Depending on the species, CoVs adopt different target receptor recognition patterns for binding to the host. For instance, MERS-CoV identifies and binds to the transmembrane dipeptidyl peptidase 4 (DPP4), which is also known as cluster of differentiation (CD26) [41, 42] . The SARS-CoV, and other related viruses, targets angiotensin converting enzyme 2 (ACE2) for their entry into human cells [43, 44] . SARS-CoV-2 has also been shown to target ACE2 for host cell entry [18, 36, 45] . ACE2 is an 805 amino acid long trans-membrane type I glycoprotein with two functional domainsthe N terminal peptidase domain and a C terminal domain homologous to glycoprotein collectrin. The peptidase domain of ACE2 has two lobes that harbors the active site [46] . The primary physiological role of ACE2 is the maturation of the peptide hormone angiotensin that controls blood pressure and vasoconstriction [47] . Additionally, it also cleaves a number of biologically active peptides like kinetensin, neurotensin, casomorphins and apelins [48] . ACE2 is expressed in type I and II alveolar epithelial lung cells, intestine, kidney, heart, adipose tissues and blood vessels, which can be correlated with high frequency of pneumonia and bronchitis in COVID-19 patients [49] . A high expression of ACE2 in the oral cavity, specifically on the tongue than buccal and gingival tissues, has also been reported. This could potentially increase the risk of SARS-CoV-2 infection as it grants easy access to the host [50] . During infection, the S1 subunit directly binds to the peptidase domain of ACE2. ACE2 is subsequently cleaved by either disintegrin and metallopeptidase domain 17 (ADAM17)/tumor necrosis factor α-converting enzyme (TACE) or TMPRSS2. It has been shown that processing by TMPRSS2 promotes viral entry [51, 52] .

The association of SARS-CoV-2 and ACE2 has been reported to be stronger than SARS-CoV due to mutations in the S protein RBD. The RBD of SARS-CoV-2 S protein has evolved structural changes in the ACE2 binding ridge when compared to SARS-CoV. In SARS-CoV-2, the more compact RBD structure produced by residues between 482-485 (Gly-Val-Glu-Gly) forms better contacts with the N terminal helix of ACE2. It has been suggested that the replacement of Leu472 in SARS-CoV with Phe486 in SARS-CoV-2 could produce stronger van der Waals interaction with ACE2. Similarly change from Val404 to Lys417 increases the association of SARS-CoV-2 RBD and ACE2 due to the formation of a salt bridge between Lys417 of S protein J o u r n a l P r e -p r o o f and Asp30 of ACE2 [53, 54] . These structural features enhance the binding affinity of SARS-CoV-2 to ACE2 when compared to SARS-CoV. In addition, S2 subunit of SARS-CoV-2 exhibits superior plasma membrane fusion capability than SARS-CoV [55] . Apart from these, crystallographic studies and biophysical assays suggest that SARS-CoV-2 S protein binds to ACE2 with 10 to 20-fold higher affinity than SARS-CoV, which could account for the higher infectivity and transmissibility [56] . In fact, it has been suggested that two S protein trimers of SARS-CoV-2 could independently bind to an ACE2 homodimer [54] .

As SARS-CoV-2 targets multiple organs efficiently, work has been done to identify potential targets other ACE2. Cluster of differentiation 147 (CD147), also known as basigin (BSG) or extracellular matrix metalloproteinase inducer (EMMPRIN), is one such receptor that has been reported to enable SARS-CoV-2 entry. Anti-CD147 humanized antibody meplazumab, currently undergoing phase II clinical trials, has been reported to prevent SARS-CoV-2 infection by blocking CD147 [57] . Data from two recent preprints suggest that the S1 subunit of SARS-CoV-2 could directly bind to two forms of neuropilin, a cell surface receptor, namely NRP1 and NRP2 [58, 59] . Analysis of lung tissue of patients who died from COVID-19 showed an increased expression of NRP1 and NRP2 suggesting an upregulation of these genes at the site of inflammation [60] . NRP1 and NRP2 are type 1 transmembrane proteins involved in physiological processes like axon guidance, angiogenesis and control of cellular signal cascades. The extracellular region of these proteins are composed of five structured domains including two CUB (for complement C1r/C1s, Uegf, Bmp1) domains (a1 and a2), two coagulation factor domains (b1 and b2), and a MAM (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu) domain [61] . These studies have demonstrated that furin cleaved substrates of S protein, mainly the S1 subunit, is able to bind to the b1 domain of NRP1. Thus, these observations suggest that, apart from ACE2, other host cell proteins could also be targeted in a SARS-CoV-2 infection.

Mortality due to SARS-CoV-2 infection depends on several factors including age, gender, race, quality of health care, restriction in movement, quarantine, difference in genetics and immune system response. Apart from these, genetic variations in the virus also alters its pathogenicity and virulence. The role played by these mutations and emergence of new viral strains cannot be ignored due to the rapid spread and mortality rate of the virus. The mutation rate of RNA viruses is dramatically high as error rates in RdRP are generally high due to the lack of a proofreading mechanism [62] . Additionally, viruses could also exploit genetic variations to improve their virulence and evolve to evade the immune response [63] . Soon after the emergence of SARS-CoV-2 in late December 2019, the viral genome was sequenced and made publicly accessible [64] .The reference genome of SARS CoV-2 is available from the Global Initiative on Sharing All Influenza Data (GISAID; https://www.gisaid.org; hCoV-19/Wuhan-Hu-1/2019, EPI_ISL_402125) as well as NCBI (Accession: NC_045512). Since then, extensive sequencing of viral genomes was done in a number of countries and made available to the public through the GISAID initiative and NCBI.

A number of studies have now looked at the genomic data to identify new variants and their association with disease severity. An analysis of 95 SARS-CoV-2 whole genome sequences identified 116 unique variants, most of which were missense and synonymous variations. However, this study did not consider the association of these variations with severity of disease [65] . Another analysis of 86 genomic sequences obtained from GISAID detected 3 deletions and 93 variations in genomes of viral isolates from Japan, USA and Australia. Most of the variations were observed in the ORF1ab and RBD of S protein [66] . As S protein plays a critical role in binding to the host receptor, it was suggested that some of these variations could induce conformational changes that alter the virulence of the virus. Genome diversity analysis of 7666 publicly available genome assemblies identified 198 recurrent mutations in the SARS-CoV-2 genome of which nearly 80% were missense variations. Most of the recurrent mutations were observed in the ORF1ab region coding for nsp6, nsp11, and nsp13, and the S protein. Since majority of these variations are in protein coding regions, it was suggested that the SARS CoV-2 is possibly adapting [67] . Another analysis of mutation data from four geographic areas -Asia, Oceania, Europe, North America -using 220 SARS-CoV-2 genomes identified 8 novel mutation hot spots: 5 of these were predominantly observed in European samples while 3 were exclusively present in North American samples. Mutations at positions 3036, 14408 and 23403 (nsp3, RdRP and S protein) were recurrent in the European region. The mutation at position 14408 causes an amino acid substitution at position 323 (Pro323Leu) in the RdRP protein, a key component of the replication and transcription machinery. As RdRP works with several other viral cofactors involved in proofreading activity (ExoN, nsp7 and nsp8) this mutation could have J o u r n a l P r e -p r o o f altered the binding capability or impaired the proofreading. It was proposed that this could have led to an increase in mutations in the European region since February 2020 [68] . To further understand the effects of RdRP mutations on viral transmission, their association with mutations identified in the M and E glycoproteins was also investigated. Ten mutations were frequently observed in the RdRP region and mutations found at positions 14408, 14805, 15324, and 13730 were observed to increase the risk of mutations in M/E glycoproteins. Among these, the 14408C>T mutation was found to be associated with and increased likelihood of M/E mutations. Thus, these findings suggest that RdRP significantly contributes to the evolution of SARS-CoV-2 [69] (Table 1) .

A phylogenetic network analysis using 160 complete viral genomes attempted to reconstruct the evolutionary path of SARS-CoV-2 from Wuhan to Europe and North America. Based on the analysis, three distinct strains of the virus were identified and clustered into three related lineages designated as Type A, Type B, and Type C. Type A represents the root of the outbreak; Type B was derived from A separated by two mutations (T8782C and C28144T) and Type C is derived from B separated by a single mutation (G26144T). Foster and colleagues found that Type A closely resemble the virus that was found in bats and pangolins. Initially, Type A was found in patients from China and later in patients from the United States and Australia. Even though Type A was found in Wuhan, it was not the predominant strain found in the city. Type B was the major viral strain found in Wuhan and East Asian regions and this strain didn't travel much beyond these regions. This suggests an environmental or immunological adaptation of the viral strain that limited it to the Asian region. Type C was observed across much of Europe, most notably among the early patients from France, Italy, Sweden, and the United Kingdom. This network provides a detailed picture of evolutionary paths of COVID-19 during the early stages of the pandemic [70] . SARS-CoV-2 genes exhibits varying levels of selective pressure. While M and E integral proteins tend to evolve slowly, proteins that are involved in viral replication, transmission and pathogenesis, such as viral replicase, S and N proteins, are more prone to mutations due to host adaptation [71] . As the pandemic spread, several mutations specific to spike proteins have emerged. These variations alter viral infectivity and transmission as well as the efficiency of neutralizing antibodies that play an important role in the host defense mechanism.

Phylogenetic analysis of the S gene from 144 SARS-CoV-2 genomes from several countries confirmed the presence of a novel missense mutation 23403A> G. This non-conservative mutation causes a change in amino acid from an aspartate to a glycine (Asp614Gly; D614G). This change, from a negatively charged aspartate to a achiral glycine without a side chain and greater flexibility, is located near the furin recognition site at the S1-S2 junction. Asp614 was the predominant form during the early days of the epidemic. In the course of its expansion to Europe, the mutant form Gly614 gained foothold before spreading to other regions. Furthermore, it was observed that both forms (Asp614 and Gly614) were circulating in most sampled countries. By the end of March 2020, the mutated Gly614 was the major variant in most reported samples. The Asp614Gly mutation could have altered the infectivity of virus by improving receptor binding, fusion activation or antibody dependent enhancement elicitation [72] . Zhang et al. reported that D614G mutation increases viral infectivity and virion spike density. This mutation may also provide a fitness advantage to the virus by providing greater flexibility and stability to the S protein for efficient transmission (Figure 1 ). The mutated variant was reported to infect ACE2 expressing cells more efficiently thereby promoting viral infection and transmission [73] . However, this contradicts another study that suggested that Asp614Gly mutation is not associated with increased viral transmissibility. In this analysis 15000 SARS-CoV-2 viral genomes from 75 countries were assessed for the transmissibility of recurrent mutations. Results suggest that the observed mutations do not increase the transmissibility of the virus and most of the mutations are either detrimental or neutral to the virus. Additionally, most of the recurrent mutations, including Asp614Gly, was suggested to occur due to the interaction with the human immune system [74] .An analysis of 2147 viral genomes from 50 countries identified 23-point mutations in the RBD of the S protein. Among these, 9 mutations exhibited increased affinity and binding to ACE2 while 14 mutations showed lower affinity and stability. Additionally, an analysis of 1150 S protein variants reported by CDC found 76 missense variants in the RBD region (Thr333-Cys525). Out of these, five variants had enhanced binding affinity while three increased the stability of virus-receptor complex [75] . [80] [81] [82] Emerging viral variants that can surpass human immune system could pose a challenge to researchers. Immune escape will make people more susceptible to reinfection and less responsive to vaccines. The human immune system fights against viral invasion by producing neutralizing antibodies against RBD region to disrupt the viral binding to the ACE2 receptor. However, in an effort to survive, the virus could mutate to evade the host immune system. Several mutations have been reported in the spike region and one of the most dominant mutation is the D614G. This mutation, combined with others, could make the virus more infectious. Another important mutation reported in the RBD region of SARS-CoV-2 is Asn439Lys (N439K) (Figure 1 ). This mutation results in a stronger association with human ACE2 (hACE2) due to the formation of a new salt bridge in the RBD-hACE2 interface. Apart from this, additional hydrogen bonds and van der Waals interactions were also observed in the mutated virus-hACE2 complex. Consequently, the stronger binding of this mutated strain may be the reason for it being more infectious than the original strain [76] . Furthermore, this strain has been reported to be resistant to neutralizing antibodies and capable of immune escape from a panel of neutralizing monoclonal antibodies [83] . Thus, this variant has raised concerns about reduced antibody production and vaccine efficacy.

In addition to this, in early November 2020, Denmark reported several COVID-19 cases caused by SARS-CoV-2 variants from farmed minks. Viral variants identified from these infections were grouped into five closely related clusters. Initial observations and clinical data suggest that the severity and transmission of these variants are closely related to other SARS-CoV-2 strains except for the cluster 5 variant. This variant had four substitutions and two deletions in the spike protein region including Y453F, I692V, M1229I, S1147L and 69-70Δ HV. In early experiments the cluster 5 variant showed moderately decreased sensitivity towards neutralizing antibodies suggesting that this variant could be less responsive to vaccines or antibodies. By taking proper preventive measures and mass culling, Danish authorities have declared that the variant is under control [77] . Figure 1 . Rendering of the SARS-CoV-2 spike glycoprotein trimer based on an X-ray crystal structure (Protein Database ID: 6XLU) [84] . For clarity, only one subunit is shown in color while the other two are shown in translucent white cartoon representation. In the trimer, the S1 subunit is shown in orange and the RBD harbored within the S1 subunit is shown in pink. The S2 subunit is shown in yellow. Locations with mutations reported in the literature are shown in van der Waal's representation. The image was rendered using Visual Molecular Dynamics 1.9.3 [85] .

A rapidly spreading SARS-CoV-2 variant (SARS-CoV-2 VUI 202012/01) was first reported in the United Kingdom (UK) in December 2020 as the country faced rapid increase in COVID-19 cases. Genomic sequencing of the variant identified multiple spike protein mutations (N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, Δ 69-70, Δ144) and other genomic variations. Preliminary data indicated that the newly reported strain was more transmissible than previously reported variants with up to 70% higher transmissibility rate [78] . Among the mutations in the spike region, N501Y is one of the six key amino acids involved in the RBD-hACE2 complex formation. This novel mutation could therefore facilitate a stronger association between the spike protein and hACE2. Additionally, this strain also harbors the D614G mutation which help the virus to spread rapidly. In early January 2021, the National Institute of Infectious Diseases (NIID) of Japan reported a J o u r n a l P r e -p r o o f new variant isolate of SARS-CoV-2. The new variant had previously reported mutations in the spike protein including N501Y and E484K. The E484K mutation has been identified as a neutralizing antibody escape mutation, which suggest a change in antigenicity [86] . On December 18, 2020, the South African government announced the emergence of a new SARS-CoV-2 strain (501Y.V2) harboring eight mutations in the spike protein. In addition to D614G, three mutations were found in key residues in the RBD (N501Y, E484K and K417N), three at the N terminal domain (L18F, D80A and D215G) and one in the loop 2 region (A701V). Initial reports suggested that the new variant was highly transmissible and exhibited complete immune escape from neutralizing antibodies [80, 81] . Furthermore, recent genomic studies reported the emergence of two B.1.1.28 sub-clades designated as P.1 (B.1.1.28.1) and P.2 (B.1.1.28.2) in Brazil. These emerging variants harbours several mutations in the spike protein including N501Y, E484K and K417N (Figure 1 ) [82] . These rapidly spreading strains, with reduced antibody neutralization, were found in the United Kingdom, South Africa and Brazil and has now been classified as Variants of Concern (VOC). [79] .

Thus, it is evident that over the course of time, this virus is expected to mutate and evolve resulting in the emergence of novel viral variants. Persistent monitoring is imperative during this its evolution for efficient vaccine development. Studies have also reported that introduction of vaccines could cause selection pressure in S protein which may change the T cell and B cell epitopes with immune escape property. These strategic mutations could hamper the ability of host immune system in recognizing and combating the virus [87, 88] .

Genome-wide association studies have also been performed to identify whether genetic variations are linked to the severity of COVID-19. In one such study, 152 complete viral genomes were analyzed from symptomatic or asymptomatic patients. Variations at the position 11083, located in the coding region of nsp6, were found to be associated with disease severity. Two variations were observed at the site, a thymine variant (11083T) was often found in asymptomatic patients and a guanine variant (11803G) in symptomatic infections. It was suggested that interaction between viral RNA and host microRNAs play a crucial role in the development of viral infections. Three microRNAs -miR-485-3p, miR-539-3p, and miR-3149were found to target the two variants differentially. It was suggested that targeting the 11083G variant by miRNAs could be a way to alter the severity of the disease [89] .

As SARS-CoV-2 primarily depends on ACE2 for fusion and entry, specific genetic variants of ACE2 could potentially alter the binding affinity and susceptibility to infection. Several studies have been reported the association between ACE2 variants and diseases like cardiovascular disorders, hypertension and diabetes [90, 91] . Structural studies have documented the key residues that are involved in the binding interface of ACE2 [27, 53, 54, 56] . Crucial modifications occurred in several CoV S protein RBDs, including Bat CoVs and other suspected intermediate host CoVs like pangolin and palm civet, that facilitated SARS-CoV and SARS-CoV-2 infection of humans [53, 54] . It has been demonstrated, by mutagenesis in rat ACE2, that variations in ACE2 alter the binding affinity of S protein of SARS-CoV. Mutating Lys353, a residue involved in S protein binding, to histidine partially inhibited the S protein mediated viral entry. Similarly, by modifying glycosylation site (Asp90) in rat ACE2 altered the ACE2 conformation which in turn improved S protein mediated SARS-CoV infection [92] .Thus, it is likely that genetic variations in ACE2 could affect the expression level as well as protein conformation and stability. This could, in turn, also alter SARS-CoV-2 S protein affinity making individuals more resistant or susceptible to an infection. However, more work is required to ascertain this.

An analysis of ACE2 expression profiles in normal lung cells showed that Asian males have a higher expression of ACE2 compared to white and African populations suggesting that they could be more susceptible to a viral infection [93] . Mortality data, suggests that men succumbed to COVID-19 more often than women. Multiple reasons have been proposed, including pregnancy, sex chromosomes, estrogen signalling, and ACE2 receptor levels [94] . However, contradicting this, in another study no significant difference was observed in ACE2 expression among Asians and Caucasians, among different age groups (>60 vs <60) and between males and females. This study also compared ACE2 expression in smokers and non-smokers and it was found that smokers were more prone to COVID-19 [95] . The differing conclusions in these studies may have occurred due to a difference in sample size or purity of ACE2 expressing cells.

Recent studies have also looked at the relationship between ACE2 variants and susceptibility to SARS-CoV-2 infection. Thirty-two ACE2 variants that included 7 hotspot variants (Lys26Arg, Ile468Val, Ala627Val, Asn638Ser, Ser692Pro, Asn720Asp, and Leu731Ile/Leu731Phe) have been identified in data from the 1000 genomes project and the China Metabolic Analytics Project. Thus, genetic variants among different populations could also affect overall ACE2 function [96] . A similar result was reported in another study involving Europeans, Africans, Asians and Americans that identified 31 variants of ACE2. Among these ACE2 variants, 13 were found to bind more efficiently to SARS-CoV-2 while 18 variants were characterized as interaction inhibitors. Among the 13 that improved binding, Ser19Pro, Ile21Thr, Lys26Arg, Thr27Ala, Asn64Lys, and His378Arg were found abundantly in all population groups [97] . Lending further support, a comprehensive analysis of 290000 genomic samples representing more than 400 population groups identified multiple ACE2 variants. The data suggested that ACE2 variants Ser19Pro, Ile21Val, Glu23Lys, Lys26Arg, Thr27Ala, Asn64Lys, Thr92Ile, Gln102Pro and His378Arg found in the binding region increased disease susceptibility whereas the variants Lys31Arg, Asn33Ile, His34Arg, Glu35Lys, Glu37Lys, Asp38Val, Tyr50Phe, Asn51Ser, Met62Val, Lys68Glu, Phe72Val, Tyr83His, Gly326Glu, Gly352Val, Asp355Asn, Gln388Leu and Asp509Tyr exhibited lower binding to SARS-CoV-2 S protein [98] . Analysis of the whole exome sequence of 6930 Italians also identified 30 ACE2 variants. Of these, three commonly found missense variants (Lys26Arg, Gly211Arg, and Asn720Asp) were reported to affect protein structure and stability. Lys26 is located near the N terminal region and forms a hydrogen bond with Asn90. The variant Lys26Arg is likely to cause a destabilization in secondary structure of ACE2 protein. Gly211 is located in the turning point of a loop and its neighbouring Val212 forms strong hydrophobic interaction with Leu91 that stabilizes ACE2 structure. A change from an achiral and flexible glycine to charged and polar arginine is not favorable for the loop and it also introduces a hydrophilic group that weakens the Val212-Leu91 interaction and possibly destabilizing the ACE2 structure. The Asn720Asp variant is located close to the TMPRSS2 cleavage sequence and could affect virion intake [99] . ACE2 variants from Genome Aggregation Database identified 12 deleterious missense variants -Ser19Pro, Asp206Gly, Gly211Arg, Arg219His, Arg219Cys, Lys341Arg, His378Arg, Ser547Cys, Ile468Val, Arg697Gly, Ser692Pro, Leu731Phe. Based on structural analysis, nine of these variants could disrupt protein structure or its interaction with the RBD of SARS CoV-2 [100] . Since the impact of a CoV infection varies between countries, age and race, it is possible that an individual's ACE2 could also have an effect on susceptibility or resistance to the virus. Identifying the frequently found variants will aid in developing preventive measures against COVID-19. However, much more work needs to be done to establish a strong association between ACE2 variants and COVID-19.

The rapid spread and mortality rate of COVID-19 has undoubtedly grabbed global attention and has been a significant threat to public health systems. In this review, we have focused on the current state of knowledge of genetic adaptations that may have occurred in SARS-CoV-2 genome as well as its host receptor ACE2. Adaptive mutations in SARS-CoV-2 have the potential to empower it to attain increased virulence, high transmissibility and immune escape. Similarly, natural genetic variants of host receptors could also confer increased susceptibility or resistance against evolving pathogenic SARS-CoV-2. Gaining a deeper understanding of these variations is crucial for developing effective measures to develop solutions and to manage future outbreaks of SARS-CoV-2.

Novel Wuhan (2019-nCoV) Coronavirus

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

A novel coronavirus outbreak of global health concern

A systematic review of SARS-CoV-2 vaccine candidates

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Human Coronaviruses: A Review of Virus-Host Interactions

Origin and evolution of pathogenic coronaviruses

Coronaviruses: an overview of their replication and pathogenesis

Bat Coronaviruses in China

Identification of new human coronaviruses

Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection

Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia

A decade after SARS: strategies for controlling emerging coronaviruses

RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak

The Architecture of SARS-CoV

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Subgenomic messenger RNA amplification in coronaviruses

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Genotype and phenotype of COVID-19: Their roles in pathogenesis

Coronavirus envelope protein: current knowledge

The proximal origin of SARS-CoV-2

Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China

Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

Pre-fusion structure of a human coronavirus spike protein

Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Structure of MERS-CoV spike receptorbinding domain complexed with human receptor DPP4

Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26

Structure of SARS coronavirus spike receptor-binding domain complexed with receptor

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis

A novel angiotensinconverting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues

High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa

Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2

TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein

Structural basis of receptor recognition by SARS-CoV-2

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

CD147 as a Target for COVID-19 Treatment: Suggested Effects of Azithromycin and Stem Cell Engagement

Neuropilin-1 is a host factor for SARS-CoV-2 infection

Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system

Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

Neuropilin Functions as an Essential Cell Surface Receptor

Quasispecies theory and the behavior of RNA viruses

RNA virus mutations and fitness for survival

A new coronavirus associated with human respiratory disease in China

Genomic characterization of a novel SARS-CoV-2

Genetic diversity and evolution of SARS-CoV-2

Emergence of genomic diversity and recurrent mutations in SARS-CoV-2

Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant

RdRp mutations are associated with SARS-CoV-2 genome evolution

Phylogenetic network analysis of SARS-CoV-2 genomes

Codon Usage and Phenotypic Divergences of SARS-CoV-2 Genes

Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2

Binding Ability Prediction between Spike Protein and Human ACE2 Reveals the Adaptive Strategy of SARS-CoV-2 in Humans

N439K variant in spike protein may alter the infection efficiency and antigenicity of SARS-CoV-2 based on molecular dynamics simulation

Corona's new coat: SARS-CoV-2 in Danish minks and implications for travel medicine

Emergence of a new SARS-CoV-2 variant in the UK

Centre for Disease Prevention and Control. Risk related to spread of new SARS-CoV-2 variants of concern in the EU/EEA, first update -21

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa

Genomic characterisation of an emergent SARS-CoV-2 lineage in Manaus: preliminary findings

The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity

Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains

VMD: Visual molecular dynamics

Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies

The influence of major S protein mutations of SARS-CoV-2 on the potential B cell epitopes

SARS-CoV-2 escapes CD8 T cell surveillance via mutations in MHC-I restricted epitopes

SARS-CoV-2 genetic variations associated with COVID-19 severity

Association of ACE2 genetic polymorphisms withhypertension-related target organ damages in south Xinjiang

Association of polymorphisms of angiotensin I converting enzyme 2 with retinopathy in type 2 diabetes mellitus among Chinese individuals

Receptor and viral determinants of SARScoronavirus adaptation to human ACE2

Single-Cell RNA Expression Profiling of ACE2, the Receptor of SARS-CoV-2

Estrogen regulates the expression of SARS-CoV-2 receptor ACE2 in differentiated airway epithelial cells

Tobacco-use disparity in gene expression of ACE2, the receptor of 2019-nCov

Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations

The Expression and Polymorphism of Entry Machinery for COVID-19 in Human: Juxtaposing Population Groups, Gender, and Different Tissues

Human ACE2 receptor polymorphisms predict SARS-CoV-2 susceptibility

ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population

Investigation of the genetic variation in ACE2 on the structural recognition by the novel coronavirus (SARS-CoV-2)