9  The pathogenesis of COVID-19
Current understanding of the pathogenesis of HCoVs infection is still limited, especially for SARS-CoV-2. Before 2019, there were six CoVs that could infect humans and cause respiratory disease. HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are sometimes attributed to the “common cold”, but in rare cases can cause severe infections in infants, young children and elderly people. On the other hand, SARS-CoV and MERS-CoV can infect the lower respiratory tract and cause a severe respiratory syndrome in human [3,12]. The new coronavirus SARS-CoV-2 is similar to SARS-CoV and MERS-CoV and can infect lower respiratory tract and cause severe pneumonia.
The origin of SARS-CoV-2 was thought to be wild animals in the Huanan Seafood Market in Wuhan. However, not all cases have an apparent connection with the Wuhan Huanan Seafood Wholesale Market. It is evident now that SARS-CoV-2 is capable of person-person transmission. We list the major pathogenic CoVs in Table 2 for better understanding of the pathogenesis of HCoV [87].
Table 2 Partial list of important pathogenic human coronaviruses [87].
Virus Genus Symptoms
HCoV-229E alpha mild respiratory tract infections
HCoV-NL63 alpha mild respiratory tract infections
HCoV-OC43 beta mild respiratory tract infections
HCoV-HKU1 beta pneumonia
SARS-CoV beta severe acute respiratory syndrome, 11% mortality rate
MERS-CoV beta severe acute respiratory syndrome, 34% mortality rate
SARS-CoV-2 beta severe acute respiratory syndrome, 2.6% mortality rate
The term “cell pyroptosis” was first proposed in 2001 [88]. In recent decades, there has been increasing evidence suggesting that “pyroptosis” is a novel inflammatory form of programmed cell death. In 2019, Chen et al. found that SARS-CoV Viroporin 3a triggered the activation of the NLRP3 inflammasome and the secretion of IL-1β in bone marrow-derived macrophages, suggesting SARS-CoV induced cell pyroptosis [89]. Studies have shown that patients infected with SARS-CoV-2 have increased IL-1β in the serum [26]. As the rise of IL-1β is a downstream indicator of cell pyroptosis, this may suggest that cell pyroptotic activity is likely to be activated and involved in the pathogenesis of COVID-19 patients. Nevertheless, as both classical and non-classical pyroptosis signaling can induce the release of IL-1β, it is unclear which pathway is involved in COVID-19. Based on existing data, SARS-CoV-2 is likely to cause cell pyroptosis, especially in lymphocytes, through the activation of NLRP3 inflammasome. The pathways involved in the activation of the signaling between NLRP3m IL-1β, IL-18 and GSDMD are illustrated in Fig. 6 and are a subject of study in samples from SARS-CoV-2 patients [90].
Fig. 6 A hypothesis of the relationship between SARS-CoV-2 and cell pyroptosis. The COVID-19 may be linked to cell pyroptosis, especially in lymphocytes through the activation of the NLRP3 inflammasome. Morphological changes in lymphocytes and macrophages, nucleic acid and protein levels in classical and non-classical cells, detection of NLRP3 and GSDMD, and the role of inflammatory cytokines IL-1β and IL-18 requires further research.

9.1  The genomic structure of SARS-CoV-2
The rapid sequencing of the nearly 30,000 nucleotide SARS-CoV-2 genome was accomplished in approximately 3 weeks from the time of the first hospitalized patient on the December 12, 2019 by Zhang's group and several others in China [91]. The genomic structure is shown in Fig. 7 and shows greater than 99.9% consistency [19,[91], [92], [93], [94], [95]].
Fig. 7 Schematic diagram of the SARS-CoV-2 genome [95]. The genomic structure of SARS-CoV-2 is 5′-UTR-orf1a-orf1ab-S (Spike)–E (Envelope)-M (Membrane)-N (Nucleocapsid)-3′UTRpoly (A) tail. Accessory genes are interspersed within the structural genes at the 3′ end of genome. The pp1a protein encoded by the orf1a gene and the pp1ab protein encoded by the orf1ab gene contains 10 nsps (nsp1-nsp10). The pp1ab protein also includes nsp12-nsp16.
The SARS-CoV-2 genome was found to possess 14 ORFs encoding 27 proteins. The orf1ab and orf1a genes are located at the 5′-terminus of the genome and encode 15 non-structural proteins (nsps) from nsp1 to nsp10, and from nsp12 to nsp16. The 3′-terminus of the genome contains 4 structural proteins (S, E, M and N) and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14). At the amino acid level, the SARS-CoV-2 is quite similar to that of SARS-CoV, but there are some notable differences. For example, the 8a protein is present in SARS-CoV and absent in SARS-CoV-2; the 3b protein is 154 amino acids in SARS-CoV but shorter in SARS-CoV-2 with only 22 amino acids. Further studies are needed to characterize how these differences affect the functionality and pathogenesis of SARS-CoV-2 [95].
The phylogenetic tree based on whole genomes showed that SARS-CoV-2 is most closely related to bat SARS-like coronavirus bat-SL-CoVZC21 (NCBI accession number MG772934) and bat-SL-CoVZC45 (NCBI accession number MG772933), which share ~89% sequence homology [[91], [92], [93]]. Their genomic organization is typical of a lineage B beta coronavirus. Further phylogenetic analysis has posited that SARS-CoV-2 is a product of recombination with previously identified bat coronaviruses, but a recent report has subsequently identified a bat CoVs sequence, RaTG13, with 92–96% sequence identity with the novel virus, demonstrating that RaTG13 is the closest relative of the SARS-CoV-2 and forms a distinct lineage from other SARS-CoVs. This rejects the hypothesis of emergence as a result of a recombination event [94,96]. Even though there are high similarities between SARS-CoV-2 S and RaTG13 S, there are two distinct differences: one is an “RRAR” furin recognition site formed by an insertion residues in the S1/S2 protease cleavage site in SARS-CoV-2, rather than the single Arginine in SARS-CoV [[97], [98], [99], [100], [101]]; the other difference is the presence of 29 variant residues between SARS-CoV-2 S and RaTG13 S, 17 of which mapped to the receptor binding domain (RBD) [97].
The identities of 5′- and 3′-UTR sequences are more than 83.6% consistent between SARS-CoV-2 and other β-CoVs, such as SARS-CoV [102]. The replicase polyproteins ppla and pp1ab encoded by the largest genes orf1ab are proteolytic and have been reported to function in the replication of CoVs by regulatory elements located within the non-structural proteins [102]. Four structural proteins (S, E, M and N) contribute to virion assembly and infection of CoVs. The spike protein located on the surface of viral particles is made up of homotrimers of S proteins and is the key for the viral attachment to host receptors [103,104].
Spike glycoprotein consists of S1 and S2 subunits. The S1 subunit contains a signal peptide, an N-terminal domain (NTD) and RBD, while the S2 subunit includes the conserved fusion peptide (FP), heptad repeat 1 and 2 (HR1 and HR2), transmembrane domain (TM), and cytoplasmic domain (CP) [102,105]. Furthermore, the S2 subunit of SARS-CoV-2 is highly conserved and shares 99% similarity with those of Bat-SL-CoVZC45, Bat-SL-CoVZC21 and human SARS-CoV [102]. The S2 subunit is therefore targeted when screening broad spectrum antiviral peptides, which is an important piece of information that can be used to develop preventive and treatment measures. Most recently, the 3D structure of S protein was elucidated using Cryo-electron microscopy (Cryo-EM), and the RBD structure of the S protein is closer to the central location of SARS-CoV-2 compared to SARS-CoV [97].
The E protein plays a role in virus assembly and release, and is required for pathogenesis [106,107]. The N protein contains two domains, both of which can bind virus RNA genomes via different mechanisms. It has been reported that the N protein can bind nsp3 protein to help tether the genome to replicase-transcriptase complex (RTC) and package the encapsulated genome into virions [12,108,109]. The N protein is also an antagonist of interferon and viral encoded repressor (VSR) of RNA interference (RNAi), which benefits viral replication [110].

9.2  Entry into host cell
Cell entry is an essential component of cross-species transmission, especially for the β-CoVs. All CoVs encode a surface glycoprotein, spike, which binds to the host receptor and mediates viral entry [111]. For β-CoVs, the RBD of the spike protein mediates the interaction with host receptor. Upon binding the receptor, the spike protein is cleaved by nearby host proteases and releases the signal peptide to facilitate virus entry into host cells [[112], [113], [114], [115]].
Angiotensin converting enzyme 2 (ACE2) and dipeptidyl peptidase 4 (DPP4) are known host receptors for the β-CoVs SARS-CoV and MERS-CoV, respectively [116,117]. In similar fashion to SARS-CoV, SARS-CoV-2 also uses ACE2 to gain entry into host cells. Hoffmann et al. found that the cellular protease MPRSS2 blocks entry by cleaving the spike protein and may constitute a treatment option [118]. Zhou et al. also confirmed that SARS-CoV-2 is able to use all but mouse ACE2 as an entry receptor for ACE2-expressing cells, but not cells without ACE2, indicating that the cell receptor for SARS-CoV-2 could be ACE2, and not other coronavirus receptors such as aminopeptidase N and dipeptidyl peptidase 4 [94]. Huang also showed that the affinity of the SARS-CoV-2 S-RBD binding to ACE2 is less than that of SARS-CoV through Monte Carlo algorithm [119]. However, Wrapp et al. found that SARS-CoV-2 S binding to ACE2 has approximately 10- to 20- fold higher affinity than SARS-CoV S, which can provide one explanation why SARS-CoV-2 has more human-to human spread compared to SARS-CoV [97].
The combination of SARS-CoV-2 S and ACE2 of host cells is similar to the combination of SARS-CoV and ACE2, indicating that they have the same mechanism to entry into host cells [97]. The S protein of metastable prefusion conformation undergoes a series of structural rearrangements to combine with the viral membrane of host cells [111,120]. This process consists of the S1 subunit binding to the host cell receptor, triggering of the prefusion trimer's instability, and shedding of the S1 subunit, resulting in a highly stable post-fusion conformation of the S2 subunit [121]. During the binding of the subunit S1 to its cognate receptor, it is important to note that the S1 subunit exists in 2 different states, a “down” conformation and an “up” conformation state, which corresponds to a receptor-inaccessible state and an unstable receptor-accessible state, respectively [[122], [123], [124], [125]]. Unfortunately, there are significant conformational differences between SARS-CoV and SARS-CoV-2 such that the commercially available monoclonal antibodies against SARS-CoV do not react with SARS-CoV-2 [97].
SARS-CoV-2 may directly bind to ACE2 positive cholangiocytes but not necessarily hepatocytes via specific expression of ACE2 in healthy liver tissues using cell RNA-seq data of two independent cohorts [126]. Though the respiratory systems is a primary target of SARS-CoV-2, bioinformatic analysis of single-cell transcriptomes datasets of lung, esophagus, gastric, ileum and colon reveal that the digestive system is also a potential route of entry for COVID-19, as ACE2 was not only highly expressed in lung AT2 cells, esophagus upper and stratified epithelial cells but also in absorptive enterocytes from the ileum and colon [127].