4.3. α HCoV-NL63, β2 SARS-CoV and β2 SARS-CoV-2 Use hACE2 Receptors

4.3.1. α HCoV-NL63
HCoV-NL63 was first isolated from the culture supernatant of tertiary monkey kidney cells inoculated with a nasopharyngeal aspirate specimen, no. NL63, obtained from a 7-month-old baby girl with bronchiolitis and conjunctivitis in Slotervaart Hospital, Amsterdam, The Netherlands in 2003 and it was reported in 2004 [207]. In 2005, a novel strain, HCoV-New Haven (HCoV-NH), was identified by RT-PCR from RNA respiratory specimens collected from children less than 5 years of age from 2002 to 2003 in Yale-New Haven Hospital, New Haven, Connecticut, US. HCoV sequence comparisons revealed that HCoV-NH is likely to be the same species as HCoV-NL63, suggesting worldwide distribution of respiratory tract disease caused by HCoV-NL63, particularly in children [208].
In 2010, metagenomic analysis of viruses from feces, oral swabs, urine and tissues of 3 North American bat species, including big brown bats (Eptesicus fuscus), tricolored bats (Perimyotis subflavus) and little brown bats (Myotis lucifugus) in the Ridge and Valley physiographic province, by Appalachian Laboratory of the University of Maryland Center revealed that HCoV-NL63-related CoV, named Appalachian Ridge CoV strain 2 (ARCoV.2), existed in feces of tricolored bats in the family Vespertilionidae [209]. The results of molecular clock analysis suggested that HCoV-NL63 shares a common ancestor with ARCoV.2 with a most recent common ancestor (MRCA) of approximately 1190–1449 C.E [210]. In 2017, BtKYNL63-9a was reported to be the most closely related to HCoV-NL63 among three HCoV-NL63-related CoVs that were identified in fecal specimens collected between 2007 and 2010 from African trident bats (Triaenops afer) in the family Hipposideridae in Kenya and to be more closely related to HCoV-NL63 than to ARCoV.2 [127]. However, the genetic distance between HCoV-NL63 and ARCoV.2 or between HCoV-NL63 and BtKYNL63-9a is too large, and both ARCoV.2 and BtKYNL63-9a are therefore classified as not conspecific with HCoV-NL63 [211]. Based on the results of phylogenetic analyses, all proteins of HCoV-NL63 were found to cluster with the Triaenops bat NL63-related group, except for the S protein, which was nested within the Hipposideros bat 229E-related group, suggesting a chimeric genome of HCoV-NL63. Genome comparison indicated that there are two recombination breakpoints in the S gene: (i) near the 5′ end and (ii) at around 200 nucleotides upstream of the 3′ end. These data suggested that the conspecific ancestor of HCoV-NL63 is a recombination virus that emerged through co-infection between the Triaenops bat NL63-related CoV and the Hipposideros bat 229E-related CoV [127]. Thus, the recombinant virus should exist in bats or in an intermediate host (currently unknown for HCoV-NL63, probably a terrestrial mammal) that should be further identified. In addition, investigation of the relationship between Perimyotis ARCoV.2 and Triaenops bat NL63-related CoV may contribute to an understanding of the evolutionary origin prior to emergence of the recombinant ancestor of HCoV-NL63.

4.3.2. β2 SARS-CoV
SARS-CoV caused an epidemic outbreak of severe acute respiratory syndrome (SARS), which was first reported in November 2002 in Foshan, Guangdong in South China and it spread quickly from late February 2003. A global alert was issued by the WHO in March 2003, and it was declared to be contained in July 2003 [212,213]. The cumulative number of confirmed cases of SARS in the global epidemic from 1 November 2002 to 11 July 2003 was 8437 with 813 deaths (case fatality ratio of 9.6%) in 26 countries (29 areas) of 6 WHO regions [6]. In the post-global epidemic of SARS, 15 additional cases with one death from reappearance four times were reported during the period from December 2003 to May 2004. One reappearance with four cases in which only mild symptoms occurred was related to a restaurant serving palm civet meat (three cases) and house rats (one case) in Guangdong, China [214], indicating reintroduction of animal viruses into humans and the importance of effective surveillance for zoonotic diseases [215]. The other three reappearances were related to laboratory accidents in Singapore, Taiwan and China. The viruses from Singapore and Taiwan laboratories were not transmitted to others, while two infected cases from a Beijing laboratory subsequently spread to seven others with close contact, resulting in one death. This evidence indicated the importance of biosafety and biosecurity in a laboratory. Since the announcement by the WHO on 18 May 2004 that the SARS outbreak in China was contained, no SARS case has been reported [216].
Since reemergence of SARS from an animal virus could happen at any time, an understanding of the molecular evolution of the virus causing the past global epidemic may help to control future SARS outbreaks. Most of the human cases at the beginning of the SARS epidemic were caused by exposure to market animals (zoonotic source) [217]. Although more than 10 mammalian species, but no avian species, were discovered to be susceptible to infection with either SARS-CoV or SARS-related CoVs (SARSr-CoVs), both the number of animals traded in Guangdong, China, and the detection rate were higher in Himalayan or masked palm civets (Paguma larvata) than in other animals. Moreover, there were close matches between sequences of civet viruses and sequences of human viruses from each human outbreak, including the 2002–2003 epidemic and the 2003–2004 episode. These findings suggested that palm civets are important intermediate hosts for transmission of the virus to humans [218], probably through direct/indirect contact or inhalation of contaminated materials/droplets. Screening of SARSr-CoVs in palm civets by real-time RT-PCR and nested RT-PCR for detection of the N gene and P gene revealed the presence of the virus in rectal and/or throat swabs [100]. Major genetic variations in the S gene were found by genomic sequence analyses of civet viruses (SARSr-CoVs) and human viruses (SARS-CoVs), indicating that changes in the S gene are likely to be critical for shifting the virus from civet-to-human to human-to-human transmission that caused the 2002–2003 epidemic [214]. Kan et al. [100] analyzed all available SARS S gene sequences and found 27 signature nucleotide variations (SNVs). Based on SNVs and a phylogenetic tree of the SARS S genes from animal and human viruses, the viruses were divided into four groups. (i) Viruses without SNVs in the S gene, called a prototype group, were isolated from raccoon dogs and a palm civet but not from humans, suggesting that they can cause only animal-to-animal transmission. (ii) Viruses with two to seven SNVs generate up to six aa changes at the positions of 147, 228, 240, 479, 821 and 1080. These viruses were isolated from palm civets and from mild symptomatic patients (so-called low-pathogenic group) during the 2003–2004 episode, indicating that the virus in a palm civet can acquire the ability to infect humans. (iii) Viruses with 17 to 22 SNVs cause further eleven aa changes at the positions of 360, 462, 472, 480, 487, 609, 613, 665, 743, 765, and 1163. These viruses were isolated from palm civets and raccoon dogs in 2003 and from patients with severe symptoms (so-called high-pathogenic group) who had close contact with infected animals or patients in the early-phase epidemic (16 November 2002 to 30 January 2003). This evidence indicated the possibility that animal species other than civets may be intermediate hosts transferring the animal virus to humans. The SNVs of viruses in this group indicated that the virus in palm civets and raccoon dogs can evolve not only to infect humans but also to spread from one human to other humans by close contact, indicating the possibility that these animals and humans may share similar receptor structures. (iv) Viruses with 25 to 27 SNVs cause up to four aa further changes at the positions of 227, 244, 344, and 778. These viruses in this group were isolated from patients with severe symptoms in the middle phase (beginning on 31 January 2003: hospital phase) and late phase (beginning on 21 February 2003: hotel phase) of the 2003 epidemic that was responsible for the global outbreak, so-called large epidemic outbreak group [100].
While SARSr-CoVs are not widespread in farmed palm civets [100] and wild palm civets [219], SARSr-CoVs collected from bat anal/fecal swabs show high genetic diversity and are widespread in wild Chinese horseshoe bats in the genus Rhinolophus (Rhinolophus sinicus [95], R. pussilus (seropositive in blood specimens), R. pearsoni, R. macrotis and R. ferrumequinum [220]). Based on these observations, bats were believed to be the natural reservoir of SARS-CoVs. However, phylogenetic relationships suggested that none of the virus isolates from wild bats are direct ancestral viruses of SARS-CoV [130]. It is most likely that SARS-CoV evolved through recombination of bat SARSr-CoVs [128,129,130].

4.3.3. β2 SARS-CoV-2
SARS-CoV-2, which shares about 80% nt sequence identity with its elder cousin SARS-CoV [221], causes an acute respiratory disease that was officially named coronavirus disease 2019 (COVID-19). It is a novel coronavirus (2019-nCoV) that was first recognized in December 2019 in Wuhan, Hubei in Central China [222]. Different from SARS-CoV, with a mortality rate of 9.6% in cases of infection, SARS-CoV-2 generally causes mild to moderate disease, but it can also lead to severe disease and death in some cases [223,224]. Keeping the infected hosts alive enabled SARS-CoV-2 to adapt to humans with efficient human-to-human transmission. It spread rapidly worldwide, finally causing the COVID-19 pandemic, with outbreaks sustained in more than one WHO region [154], on 11 March 2020. As of 8 September 2020, the pandemic is ongoing and has caused 27,205,275 confirmed cases reported from 182 countries and 33 territories and reported from international conveyances throughout six WHO regions, resulting in 890,392 deaths, giving a tentative 3.3% case fatality rate [225].
Analysis of whole-genome sequences showed that SARS-CoV-2 shares about 96.1% identity to a bat SARSr-CoV isolated from Rhinolophus affinis (Ra) in Yunnan, China in 2013 (BatCoV RaTG13) [132,133], 93.3% identity to a bat SARSr-CoV isolated from Rhinolophus malayanus (Rm) in Yunnan (YN), China in 2019 (BetaCoV/Rm/Yunnan/YN02/2019, RmYN02) [133], and 87.8% and 87.6% identity to two bat SARSr-CoVs, BatCoV RpZC45 and BatCoV RpZXC21, respectively, detected in Rhinolophus pusillus (Rp) bats from Zhoushan City (ZXC or ZC), Zhejiang Province, China in 2015 [226]. As was the case for SARS-CoV, SARS-CoV-2 is likely to have originated from Chinese horseshoe bats in the genus Rhinolophus. Although there is high sequence identity between the longest encoding gene region (1ab) of RmYN02 and 1ab of SARS-CoV-2 (nt 97.2%, aa 98.8%), there is low sequence identity between the SARS-CoV-2 S RBD and RaTG13 S RBD (nt 85.3%, aa 89.3%) or RmYN02 S RBD (nt 61.3%, aa 62.4%) [133]. The RBD is a major determinant of host range and it is therefore likely that there is an intermediate host facilitating a bat SARSr-CoV to acquire efficient ability to infect humans.
Although the first outbreak occurred in Wuhan, animal specimens from the Huanan seafood wholesale market in Wuhan, which sells both live and dead animals including bats, civets, snakes, poultry, pigs and dogs, were negative for SARSr-CoVs [226]. Snakes and canids (dogs) were presumed to be intermediate hosts of SARS-CoV-2 based on sequence analysis of the relative synonymous codon usage (RSCU) bias between SARS-CoV-2 and animal host species [227] and based on analysis of zinc finger antiviral protein (ZAP) expression in animal host species and tissues that drive CoV evolution to have a low-CpG (5′-Cytosin-phosphate-Guanine-3′) viral genome [228], respectively. However, SARSr-CoV-2 has not been isolated from snakes and it is unlikely that the viruses can cross the species barrier from bats, warm-blooded mammals, to humans via snakes, cold-blooded reptiles. Although results of RT-PCR and serological tests confirmed SARS-CoV-2 infections in dogs, the infected dogs were with infected owners and thus humans are likely to have transferred the virus to their pets [101]. Cats, tigers and lions that were cared for by infected owners/zookeepers were also reported to have tested seropositive for SARS-CoV-2, suggesting human-to-animal transmission of COVID-19 [101]. Experimental studies showed that pigs, chickens and ducks were not susceptible to SARS-CoV-2, that dogs had little susceptibility and that both ferrets and cats were highly susceptible [229,230]. Experimental studies showed that there is little transmission of SARS-CoV-2 among ferrets but that cats have the potential for airborne transmission of the virus between them [230]. However, no cat-SARSr-CoV-2 has been isolated. Furthermore, there has so far been no evidence of transmission of SARS-CoV-2 or SARSr-CoV-2 from these SARS-CoV-2-susceptible animals, including canines, ferrets and felines, to humans [101,229]. Nonetheless, surveillance of both infection and dissemination of SARS-CoV-2 should be implemented. SARSr-CoV-2 has been isolated from smuggled Malayan pangolins (Manis javanica), but it has not been isolated from Chinese pangolins (Manis pentadactyla) seized in Guangxi (GX) and Guangdong (GD) in southern China [103,226,231]. Whole-genome comparison indicated that pangolin-SARSr-CoVs have a significant sequence difference from SARS-CoV-2 sequences, suggesting that current pangolin-SARSr-CoV isolates are unlikely to be the virus directly transmitted to cause SARS-CoV-2 outbreaks in humans. However, all of the studies [103,133,226,231] showed high aa sequence identities (97.4%) between pangolin SARSr-CoV (pangolin/GD/2019 with a single consensus sequence merged from the GD/P1L and GD/P2S sequences) S RBD and SARS-CoV-2 S RBD. Thus, SARSr-CoV from Malayan pangolins may be able to infect humans or may provide an RBD gene region to a coinfected CoV. Malayan pangolins may serve as a vessel to generate a CoV with human receptor-binding potential due to the high aa sequence similarity between pangolin and human ACE2 receptors (84.8%) [231], suggesting the need for pangolin surveillance for public health. The continued search for a SARS-CoV-2 intermediate host is essential for understanding the emergence of the COVID-19 pandemic and for future prevention and control of zoonotic CoV-related diseases.

4.3.4. Receptor Binding Specificity of α HCoV-NL63, β2 SARS-CoV and β2 SARS-CoV-2
Results obtained by direct biochemical methods and X-ray crystallographic studies showed that α HCoV-NL63 [84,232], β2 SARS-CoV [233] and β2 SARS-CoV-2 [195] use their S1-CTD RBD to bind to a host receptor, angiotensin-converting enzyme 2 (ACE2, a zinc peptidase), that is essential for virus entry into cells. As shown in Figure 4b, ACE2 is a homodimeric type I transmembrane protein having an orientation with the N-terminus outside and the C-terminus inside the cytoplasm [195]. The virus-binding site (VBS) of these three CoVs is not the peptidase active site but the outer surface of the ACE2 N-terminal lobe. Analyses of cocrystal structures between RBDs of HCoV-NL63 (pdb: 3kbh [232]), SARS-CoV (pdb: 2ajf [234]) or SARS-CoV-2 (pdb: 6m0j [235]) and the human ACE2 (hACE2) receptor indicated aa residues covering the CoV–ACE2 interfaces, divided into a common region of hACE2 recognized by all three ACE2-recognizing CoVs (a hotspot region) and unique regions bound by HCoV-NL63, SARS-CoV or SARS-CoV-2 (Figure 4b, middle row). Evidence indicating that HCoV-NL63 and SARS-CoV bind to the same hotspot region on hACE2 and that their binding is important for infection was obtained from infection inhibition studies showing that the SARS-CoV RBD can inhibit lentivirus infections mediated by the S protein of either SARS-CoV or HCoV-NL63 into hACE2-expressing HEK293T cells [236]. Likewise, the use of the HCoV-NL63 RBD as a competitive inhibitor can inhibit infections of murine leukemia viruses (MLVs) mediated by SARS-CoV S protein into hACE2-expressing HEK293T cells [237]. In addition, aa changes in the hotspot region in hACE2, either L353A or D38A substitution, resulted in a significant reduction of binding interactions between the SARS-CoV or HCoV-NL63 RBD and hACE2 and reduction of MLV infections mediated by SARS-CoV or HCoV-NL63 S protein [237]. The results of these studies suggested that the hotspot region on the hACE2 VBS is a potential target for development of drugs against ACE2-binding CoVs. It should be noted that MLN-4760, an ACE2 inhibitor that binds to the ACE2 catalytic center and induces hACE2 conformational changes, did not affect interactions of SARS-CoV S1 with the hACE2 surface and did not affect SARS-CoV S protein-mediated infection. Likewise, binding of SARS-CoV S1 to hACE2 did not affect hACE2 catalytic activity [238].
The aa sequences of hACE2 were aligned with aa sequences of ACE2 orthologues of possible natural reservoirs and possible intermediate hosts. Only the aa sequences corresponding to the hACE2 interface are shown in Figure 4b. Adaptation of each zoonotic virus to interact with aa residues at the hACE2 binding interface is critical for efficient transmission among humans. The hACE2 residues K31, D38, Y41 and K353 are important host determinants of adaptation of civet SARSr-CoV to human SARS-CoV (Figure 4b), and viral S1 RBD residues at the positions of 479 and 487 are important determinants of SARS-CoV binding preference (Figure 8a–c). The K479N mutation from civet to human viral S1 RBD can accommodate K31 on hACE2 [238]. The S487T mutation from civet to human viral S1 RBD can accommodate a hydrophobic pocket between Y41 and K353, neutralized by D38, on the hACE2 receptor for efficient interactions [234]. These findings agree with results obtained by Kan et al. [100] suggesting that viruses with SNVs leading to aa changes at these two positions are able to be transmitted from animals to infect humans and from humans to humans by close contact. However, the roles of the additional 4 aa substitutions at position 344 in the RBD but outside the RBS (Figure 8b) and positions 227, 244 and 778 outside the RBD in viruses isolated from patients during the global epidemic [100] in human-to-human transmission remain unknown.
Crystal structure analysis indicated that the SARS-CoV-2 RBD to which hACE2 binds is almost identical to the SARS-CoV RBD [235]. Later, hACE2 amino acids at or near the RBD/ACE2 interface (Figure 4b) that could affect RBD/ACE2 binding were used for screening the capability of ACE2 of various animals used by SARS-CoV and SARS-CoV-2. ACE2 of possible SARS-CoV and SARS-CoV-2 intermediate hosts, masked palm civet and Malayan pangolin, respectively, and ACE2 of many mammals including cats, dogs, cows, buffalos, goats and sheep, but not rats (Rattus norvegicus), were predicted to be potentially recognized by SARS-CoV and SARS-CoV-2 [239,240], supporting the finding that rat ACE2 has less efficiency for binding to the SARS-CoV S1 domain and is less susceptible to SARS-CoV S protein-mediated infection [241]. However, young female Fischer 344 (F344) rats of 4 weeks of age were shown to be susceptible to infection with SARS-CoV by intranasal inoculation [242]. Western blot analysis showed that ACE2 expression in Sprague Dawley rats decreased with aging without a gender difference [243]. However, it remains unknown whether there is a difference in ACE2 sequence depending on the age of rats and whether there are differences in ACE2 expression and sequence depending on the rat strain. Based on ACE2 residues 31, 35, 38, 82 and 353, Chinese horseshoe bats, which are thought to be a natural reservoir, can be divided into two groups [240]. First, bat ACE2 of SARSr-CoV–RT-PCR-positive R. ferrumequinum (bat Rf) [220] was predicted not to have the ability to bind to either SARS-CoV-2 or SARS-CoV S protein. Second, bat ACE2 of SARSr-CoV–seropositive and –RT-PCR-positive R. pearsonii, R. macrotis [220], and SARSr-CoV–RT-PCR-positive R. sinicus [95] was predicted to be able to bind to both SARS-CoV-2 and SARS-CoV S proteins. Based on residues 20, 31, 41, 68, 83, 353, 355, 357 and 383, R. sinicus ACE2 was confirmed to have the potential to be used by SARS-CoV-2 [239]. These findings indicate the possibility of cross-species transmission of the virus from humans to animals carrying similar host receptor sequences, although other host factors, such as target organ temperature and cellular proteins interacting with the virus, may be involved in host range restriction of the virus. Thus, surveillance of transmission both back and forth between humans and animals is needed. The susceptible host range of HCoV-NL63, which causes mild respiratory disease [244], has not been determined. However, the host range of HCoV-NL63 might be similar to that of SARS-CoV and SARS-CoV-2, and thus a mixed infection of these different viruses to the same host cells may occur.
Although α HCoV-NL63, β2 SARS-CoV and β2 SARS-CoV-2 recognize the same ACE2 receptor and all bind to the hotspot region on ACE2 (Figure 4b, middle), they have aa differences in the RBS at the viral RBD interface (Figure 8a), suggesting that they have undergone convergent evolution for efficient ACE2 binding [232,235]. While SARS-CoV and SARS-CoV-2 have similar RBD structures with a concave surface, α HCoV-NL63 has no structural RBD homology to βCoV RBDs (Figure 8b) [232,235]. Thus, we superimposed hACE2 receptors (green) in complex with HCoV-NL63 (pdb: 3kbh), SARS-CoV (pdb: 2ajf) and SARS-CoV-2 (pdb: 6m0j) as shown in Figure 8b. However, since all three viruses interact with the same hotspot region on the hACE2 receptor, RBS residues of these different viruses occupy similar positions in the hotspot area. For example, S535/T487/N501 of HCoV-NL63/SARS-CoV/SARS-CoV-2 are located near K353 and Y41 of hACE2, while Y498/Y491/Y505 of HCoV-NL63/SARS-CoV/SARS-CoV-2 are located near K353, E37 and D38 of hACE2 (Figure 8c) [235,237]. By using surface plasmon resonance with a Biacore 2000/3000 instrument, equilibrium dissociation constant (Kd, smaller value indicating greater binding affinity) values between HCoV-NL63 RBD and immobilized hACE2 and between SARS-CoV RBD and immobilized hACE2 were determined to be 34.9 and 20.8 nM, respectively [232,237]. It should be noted that NL63-CoV RBD-hACE2 interactions have lower koff and kon values than do SARS-CoV RBD-hACE2 interactions, suggesting that NL63-CoV RBD/hACE2 complex has less electrostatic and more hydrophobic interactions [237]; three hydrogen bonds were observed in HCoV-NL63 RBD-hACE2 complex, but nine hydrogen bonds were observed in SARS-CoV RBD-hACE2 complex (Figure 8c). By surface plasmon resonance with a Biacore T200 instrument, the Kd values of SARS-CoV RBD-immobilized hACE2 and SARS-CoV-2 RBD-immobilized hACE2 were determined to be 31 nM and 4.7 nM, respectively [235]. As mentioned earlier, S487T and K479N substitutions in the civet SARSr-CoV RBS are critical for civet-to-human transmission [238]. It appears that both T487 and N479 are substituted by N501 and Q493, respectively, in the SARS-CoV-2 RBS. N501 in the SARS-CoV RBD and T486, but not T487, in the SARS-CoV RBD (Figure 8c) form a hydrogen bond with hACE2 Y41. It is likely that subtle differences between the SARS-CoV RBD and SARS-CoV-2 RBD in interactions with hACE2 are responsible for the difference in Kd values of the SARS-CoV RBD and SARS-CoV-2 RBD for hACE2 [235]. For example, while N479 in the SARS-CoV RBD does not cause hydrogen bond formation, its substituted Q493 in the SARS-CoV-2 RBD makes two hydrogen bonds with E35 of hACE2. K417 in the SARS-CoV-2 RBD provides a unique interaction with huACE2 D30 and a positive charged patch on the SARS-CoV-2 RBD, which is not found on the SARS-CoV RBD (Figure 8c) [235]. A virus with a great binding affinity, which can trigger infection efficiently, could be a factor of the rapid spread of SARS-CoV-2. Other factors may be involved in driving the rapid spread of the virus. For example, the presence of a polybasic (RRAR) site at the S1/S2 cleavage site found in SARS-CoV-2, but not in other βCoVs in lineage B, which is cleaved by furin pre-activating the viral S proteins during virus exit, reduces dependence of the viral S proteins on target cell proteases for virus entry and thus facilitates the virus infection [245].