Since the emergence of SARS-CoV-2, it has been evolving. Its high transmissibility, persistence in the community, viral shedding through various bodily secretions including nasal and throat secretions (sputum and saliva), and stool has made it more adaptable. Moreover, the rapid adaptation to human-to-human transmission with increasing morbidity and mortality in patients with underlying comorbidities has generated a public health concern.

Disease behavior has been highly varied and unpredictable among various population groups. While reports from European countries initially demonstrated the highest mortality per million population, the US reported the highest number of cases during the first wave. Asian countries on the other hand have witnessed the lowest mortality rate per million. The difference in disease behavior could be attributed to differences in the host population or circulating viral strain. The highly variable ‘S’ glycoprotein of the SARS-CoV-2 which facilitates viral entry into the host cell plays a pivotal role in determining the transmissibility and infectivity of the virus, therefore studied to understand any adaptive evolutionary changes. The presence of a positively selected site (D614G) in the Asian and European groups points to diversifying selection in the European and Asian groups. The presence of diversifying selection in the S1 domain of the S protein could be immunologically driven or could imply viral adaptation to the new human host. Positively selected sites (diversifying selection) were also observed in the initial phase of the SARS epidemic in 2002 when the virus crossed a species barrier to adapt to the human host.

Numerous comparisons have been made between the present pandemic and the previous H1N1 and SARS outbreaks. Both infections were caused by novel RNA viruses that crossed the species barrier to infect the respiratory tract causing respiratory symptoms in humans. The HA and spike proteins of the H1N1 and SARS viruses respectively plays a part in viral attachment, which is homologous to the function of the S protein in the SARS-CoV-2. Adaptive evolutionary studies investigating Inf A (H1N1) viruses from the early and post-pandemic period reported positively selected residues located on the conserved HA stem region and HA globular head respectively, possibly due to adaptation to the new host and/or may be attributed to host immune response. Positions of positively selected sites on mushroom-shaped HA proteins in Inf A (H1N1) was markedly different between the pandemic and post-pandemic periods.²

In contrast, the location of positively selected sites in the variable head portion of the SARS-CoV-2 S protein during the present phase of the pandemic could suggest a diversifying selection that could be driven by the host immune response. Other studies investigating selection pressure have analyzed orf1ab genes and S proteins from a dataset of 91 and 39 SARS-CoV-2 sequences respectively. Most sites for both genes demonstrated ω values of <1, indicating purifying selection, with 7 sites under negative selection (215, 474, 541, 809, 820, 921, and 1044), and none under positive selection.

In the SARS epidemic of 2002, the S protein played a key role in the virus crossing the species barrier and was therefore under high selective pressure. Selection pressure studies during the SARS epidemic in the early and late epidemic phases have reported several positively selected sites in the early and mid-phases of the pandemic implying viral adaptation to the new human host with the majority of positively selected sites located in the S1 domain of the S protein responsible for binding to human angiotensin-converting enzyme-2 (ACE2) receptors. The late phase of the epidemic, however, provided evidence of purifying selection with no positively selected sites reported.

Genomic analysis of 103 SARS-CoV-2 genome sequences suggested 96.2% similarity to bats infected with the SARS-related coronavirus (SARSr-CoV; RaTG13) as reported in earlier studies from China. Variability among SARS-CoV-2 and SARSr-CoV; RaTG13 in genomic nucleotides (4%) and at neutral sites (17%), suggests a degree of divergence between them.³ Additionally, the L lineage (more prevalent, [approximately 70%]) and S lineage (approximately 30%) of SARS-CoV-2 were also found. Tang et al.,⁴ highlighted the role of natural selection leading to new variations in the receptor-binding domain (RBD) of the S protein of SARS-CoV-2 and pangolin SARSr-CoVs.

Zhang et al.,⁵ identified positively selected amino acid sites on the surface of the S protein (239, 311, 479, 609, 743, 778, 1148, and 1163), thereby suggesting its crucial role in SARS-CoV transmission and survival. The variation in positive selective sites and pressures have in large part been identified in the RBD and HR1–HR2 regions of the S protein and tend to be changeable in different epidemics before stabilization. By influencing receptor recognition and/or membrane fusion, such variations help in understanding the molecular adaptation of the S protein for interspecies transmission from animals to humans.

The S protein is an important determinant of the pathogenicity of SARS-CoV-2 and is highly susceptible to a large number of mutations.⁶ Laamarti et al.,⁷ analyzed the genetic variations in 3067 whole genomes of SARS-CoV-2 collected from 55 countries and found 512 variant sites with a non-synonymous effect in addition to 10 hotspot mutations distributed in six SARS-CoV-2 genes. Among genomes from the US isolates, V483A mutation in the S receptor was also identified, suggesting the specificity of certain genotypes to the geographical location. Consistent with the present study, Ou et al.⁸ identified eight stains from China, the US, and France harboring the V367F mutation, which was previously reported to enhance affinity to the ACE2 receptor.

On comparison, selection pressure analysis of the SARS-CoV-2 S protein revealed a single positively selected residue located in the S1 domain; the head region of the S protein, suggesting the role of diversifying selection during the current stage of the pandemic.

The presence of 29 negatively selected sites in only the European group of strains may also have a role in the viral fitness in that group. To comment on whether the evidence of purifying selection observed only in the European strains could influence viral fitness sufficient to be correlated with increased disease severity in Europe would be premature as such, a larger number of sequences is needed to substantiate this claim. Nevertheless, we identified several (n=29) negatively selected sites in the S protein, limited only to the European strains. Mutational analysis of the study strains revealed a D614G mutation in approximately 60% of the sequences. Geographical mapping of these variants revealed a maximum proportion of variants in Europe (63/101). The D614G S protein variant has been correlated with fatal outcomes in the European population in the first quarter of the year. Analysis of 86 SARS-CoV-2 genomes⁹ revealed 14 nucleotide and 8 missense mutations (F32I), (H49Y), (S247R), (N354D), (D364Y), (V367F), (D614G), (P1143L) located in the RBD region of the S glycoprotein. Such mutations may induce conformational changes (open status) and expose the cleavage domain of the spike protein to FURIN, or TMPRSS2 thereby enhancing its cleavage. Variability of SARS-CoV-2 mutations, may contribute to the emergence of different phylogenetic clades which may explain the disparity in death rate. However, both the host and viral genetic factors, along with the geographical distribution have been associated with the high and low COVID-19 related fatalities.¹⁰

Findings of our study are supported by those from Eaaswarkhanth et al.,¹¹, who reported the D614G variant in European populations from Spain, Italy, France, Switzerland, Belgium, and the Netherlands resulting in a high mortality rate. However, this evidence is circumstantial and may vary across different geographical populations. Among different European countries, high case fatality rate and G614 mutated viral strains have been strongly correlated. During the first quarter of this year, the D614G S protein variant was correlated with fatal outcomes in European populations and countries including Italy, France, Belgium, and Spain. Surprisingly, investigators identified 12 positive selective pressure sites in 2002 SARS-CoV outbreak in China, including 613 in domain 1 of the S protein, which is a shift of one codon from SARS-CoV-2. We observed D614G variants under high selective pressure in Asian and European strains; however, the difference was not statistically significant in the US strains. In addition, the presence of 29 negatively selected codon sites under low selection pressure in the European group may imply viral fitness compared with circulating strains in other continents. Thus, it appears that selective pressure-driven fitness with high substitution rates in the S protein may facilitate SARS-CoV-2 adaptive evolution and its widespread transmission as well as its persistence in the community.