Comparative analysis of SARS-CoV-2 and its receptor ACE2 with evolutionarily related coronaviruses

To the Editor,

The current pandemic COVID-19 is rapidly spreading worldwide. This disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; previously called 2019-nCoV), seriously threatening the human health [1]. Since december 12, 2019, when the first patient was confirmed [2], more than 20 million cases have been confirmed, with over 740,000 deaths globally. Due to the rapidly increasing numbers of confirmed cases and deaths of COVID-19, the WHO has raised the risk of spread and impact of this disease to a very high level [1, 2].

Coronaviruses, first described from the common cold patients in 1966, are enveloped positive single-stranded RNA nuclear viruses, which can infect a large variety of host species including humans [3, 4]. SARS-CoV-2 is a member of the Coronavirus family, Betacoronavirus genus and Sarbecovirus subgenus, with a 30 kb genome [5, 6]. Currently the bat coronavirus RaTG13 (GenBank No.: MN996532) is shown to be the most closely related with SARS-CoV-2 by whole genome comparisons [7, 8], and pangolin, mink, snake and turtle are deemed to be the intermediate hosts of this virus [1, 9, 10]. However, to date the origin and the intermediate hosts of SARS-CoV-2 remain unclear.

Here, we analyzed the complete genome sequences of 200 SARS-CoV-2 strains, including 176 from America (USA), 17 from China (CHN), 2 from Spain (ESP), 2 from Hungary (HUN), 1 from Peru (PER), 1 from Colombia (COL) and 1 from Pakistan (PAK), using the MEGA-X software [11]. As shown in Figure 1, the SARS-CoV-2 strains could be grouped into 3 clades, C I, CII and CIII. The viral genomes showed regional aggregation. The SARS-CoV-2 strains from China belong to the C III clade in the same branch of the evolutionary tree (GenBank accession numbers: MT259226, MT259230, MT259231, MT259227, MT259228, MT259229) or to the C I clade, also closely together in a same branch of the evolutionary tree (GenBank accession numbers: MT253704, MT253696, MT253697, MT253698, MT253699, MT253701, MT253702, MT253703, MT253705).

In order to elucidate the relationships between SARS-CoV-2 and the common coronaviruses that also infect humans, we chose genome sequences of six SARS-CoV-2 strains, i.e., MT263395 (furthest), MT263421 (nearest); MT251973 (furthest), MT263420 (nearest); MT259229 (furthest), MT263389 (nearest), which were in the clades C I, C II and C III, respectively, and were the furthest or nearest from the root of the evolutionary tree. We then combined the six SARS-CoV-2 strains with 293 common coronavirus strains that infect humans in the comparative sequence analysis. As shown in Figure 2, the 293 common coronaviruses that infect humans were divided into 3 clades, and there were 12 common coronaviruses that were particularly close to the SARS-CoV-2 strains in evolution (Figure 2 and Table 1). Very interestingly, the disease caused by the 12 common coronaviruses was exclusively respiratory syndrome (Table 1); these common coronaviruses were identified in 2013, 2014 and 2015 (Table 1).

So far, the bat, pangolin, mink, snake and turtle have been assumed to be the intermediate hosts of the SARS-CoV-2 virus [1, 7–10]. Researchers have also found many coronaviruses in other organisms [1, 9, 10]. In order to identify the intermediate hosts of SARS-CoV-2, we chose genome sequences of the six SARS-CoV-2 strains and made comparisons with those of 53 common coronaviruses that infect other organisms. As shown in Figure 3, the common coronaviruses were divided into 3 clades, with six common coronaviruses being particularly close to the SARS-CoV-2 strains in evolution (Figure 3 and Table 2). The diseases caused by the six common coronaviruses were respiratory syndrome and epizootic catarrhal gastroenteritis (Table 2). The hosts of the common coronaviruses closest to SARS-CoV-2 were Apodemus chevrieri (a rodent), Delphinapterus leucas (beluga whale), Hypsugo savii (bat), Camelus bactrianus (camel) and Mustela vison (Mink) (Table 2). Those common coronaviruses were identified in 1998, 2006, 2011 and 2015 (Table 2).

The Angiotensin-Converting Enzyme-2 (ACE2) gene encodes the ACE2 protein, which is the receptor of SARS-coronavirus (SARS-CoV), human respiratory coronavirus NL63 and SARS-CoV-2 [8, 12]. To understand whether different features of ACE2 might be correlated with the infection of SARS-CoV, NL63 or SARS-CoV-2 [13–15], we compared the genome sequences of the ACE2 genes from 29 organisms, including man, chimpanzee, rat, bat, camel, mink, bovine, and Beluga Whale. As shown in Figure 4, the 29 ACE2 gene sequences from different organisms were divided into 3 clades. The ACE2 gene sequence from Nannospalax galili (Upper Galilee mountains blind mole rat, MW008344634) was the closest to humans in evolution, followed by the sequences from Phyllostomus discolor (pale spear-nosed bat, NC040911), Mus musculus (house mouse, NC000086), Delphinapterus leucas (beluga whale, NW022098033) and Catharus ustulatus (Swainson's thrush, NC046222).

In summary, in this work, we found 1, the SARS-CoV-2 strains analyzed could be divided into 3 clades with regional aggregation; 2, the common coronaviruses that infect humans or other organisms causing respiratory syndrome and epizootic catarrhal gastroenteritis were particularly similar to COVID-19 and could be divided into 3 clades, with SARS-CoV-2 being clearly separated from the common coronaviruses in evolution; 3, the hosts of the common coronaviruses closest to SARS-CoV-2 were Apodemus chevrieri (a rodent), Delphinapterus leucas (beluga whale), Hypsugo savii (bat), Camelus bactrianus (camel) and Mustela vison (mink); and 4, the gene sequences of the receptor ACE2 from different hosts could be divided into 3 clades. The ACE2 gene sequences closest to that of humans in evolution include those from Nannospalax galili (Upper Galilee mountains blind mole rat), Phyllostomus discolor (pale spear-nosed bat), Mus musculus (house mouse), Delphinapterus leucas (beluga whale), and Catharus ustulatus (Swainson's thrush).

Based on these analyses, we conclude that SARS-CoV-2 may have evolved from a relatively distant common ancestor with the other coronaviruses but not a branch of any of them, implying that the prevalent pandemic COVID-19 agent SARS-CoV-2 may have existed in a yet to be identified primary host for a long time.