Pathogenesis of SARS-CoV-2

Viral Entry and Replication
A virus starts its infection by binding viral particles to the host’s surface cellular receptors. The recognition of cellular receptors is the first step towards viral entry into host cells, in addition to determining their tropism. The ability to engage receptors and the affinity of binding can define the efficiency of a virus when infecting an organism, while the amount of these receptors present in cells can indicate the intensity of infection. Viruses that have a high capacity to bind to more conserved receptors are more likely to migrate between different species, which may also reflect the susceptibility of hosts and increase viral pathogenicity (40, 41).
As well as the other β-CoVs, the SARS-CoV-2 genome has a long open reading frame (ORF) 1ab region, followed by regions that encode S, E, M, and N proteins (42). Homotrimers of S proteins are present on the viral surface and are responsible for attaching to host receptors (43). The E protein plays a role in the assembly and release of the virus, in addition to being involved in viral pathogenesis (44). The M protein has three transmembrane domains and shapes the virions, promotes membrane curvature, and binds to the nucleocapsid (45, 46). Lastly, the N protein contains two domains that can bind to the RNA virus and is also an antagonist of interferon (IFN) and a virally-encoded repressor of RNA interference, which appears to benefit viral replication (47, 48).
The S protein of SARS-CoV-2 plays an important role in determining tropism for being able to activate receptors in host cells and induce the invasion process. This protein is cleaved by proteases into the S1 and S2 subunits, which are responsible for receptor recognition and membrane fusion, respectively (39). Several articles have experimentally demonstrated that the RDB in the S protein, especially in the S1 region, binds to the peptidase domain (PD) of the ACE2 receptor, which is part of the renin-angiotensin-aldosterone system, an enzyme present in the plasma membrane mainly of pulmonary, endothelial, cardiac, renal, and intestinal cells (7, 22, 38, 49, 50). The S2 subunit is known to contain the fusion peptide, in which it is inserted into the host cell membrane to trigger the fusogenic reaction (7, 51, 52). The interaction of the S glycoprotein with the CD26 receptor and CD209L (39, 53, 54) is also suggested, however, its role remains unclear.
The binding of the virus to the ACE2 receptor causes stabilization of the RBD in the standing-up state and triggers conformational changes in the S complex, resulting in the release of the S1 subunit and activation of S2 fusogenic activity (55). The S2 subunit contains an N-terminal fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), a transmembrane region (TM), and a cytoplasmic tail (CT). During the fusion process, the FP portion is exposed and inserts into the membrane of the target cell, leading to a modification in S2, then the HR1 and HR2 come together to form a six-helical bundle (6-HR) structure, which allows the fusion between the membranes (55–57).
Therefore, CoVs need to elicit exogenous proteases to perform modifications of their binding receptors necessary for the connection to occur. SARS-CoV-2 has its own furin-like proteases, which play a role in these changes, providing it with an evolutionary advantage in relation to other coronaviruses and improving the process of cell infection and viral dissemination. Concerning exogenous proteins, SARS-CoV-2 can also use host proteins to prepare its S glycoprotein for receptor binding (49). Hoffman et al. (7) demonstrated in vitro that strains of the virus isolated from COVID-19 patients can use both the host protease transmembrane serine protease 2 (TMPRSS2) and cathepsins B/L to prime the S protein.
The entry mechanism of CoVs in host cells depends on the strain and species considered, as well as tissue and cell-type specificities (receptor/protease availability and local microenvironment) (58). After binding to a target host cell via interactions with cellular receptors, viral entry of CoVs can occur in two manners: (i) the endosomal pathway and (ii) the non-endosomal pathway (59, 60). The endosomal pathway is facilitated by low pH and pH-dependent endosomal cysteine protease cathepsins, helping to overcome the energetically unfavorable membrane fusion reaction and facilitating endosomal cell entry of CoVs (61, 62). The non-endosomal pathway is dependent on TMPRSS2, which allows the activation of the S protein for viral entry (63).
Once the viral genome is inside the host cell cytoplasm, translation of viral RNA produces RNA-dependent RNA polymerase (RdRp), which uses viral RNA as a template to generate virus-specific mRNAs (subgenomic mRNAs) from subgenomic negative-strand intermediates (64–66). Translation of subgenomic mRNAs leads to the production of structural and nonstructural viral proteins. Thus, after their formation, structural proteins are inserted into the membrane of the endoplasmic reticulum or Golgi, and viral particles germinate into the endoplasmic reticulum-Golgi intermediate compartment. Finally, the vesicles containing the virus particles fuse with the plasma membrane to release the virus (65, 67, 68).
Another possible mechanism for CoV entry may occur through antibodies. During the binding of the virus-antibody complex, simultaneous binding of viral proteins to antigen-binding fragment (Fab) regions of immunoglobulin G (IgG) and of the fragment crystallizable (Fc) portion of the antibody to Fc gamma receptors (FcγRs) that are expressed by immune cells occurs, promoting viral entry without the use of the ACE2 receptor (69, 70). However, the presence of viral RNA in the endosomes signals via the Toll-like 3 (TLR3), TLR7, or TLR8 receptor, activating the host cell to release pro-inflammatory cytokines that lead to exacerbated tissue damage, a phenomenon called antibody-dependent enhancement (ADE) (71).
Such a mechanism for SARS‐CoV‐2 is not yet fully understood, but previous coronavirus infections or SARS‐CoV‐2 convalescent patients with different SARS‐CoV‐2 strains could promote ADE, as experimentally shown for antibodies against the MERS‐CoV or SARS‐CoV-1 spike S protein (72). Several studies have shown that sera administration induced increased SARS-CoV-1 viral entry into cells that express the Fc receptor, and serum-dependent SARS-CoV-1 entry does not pass through the endosome pathway (73, 74).
This mechanism was characterized by Yip et al. (75) and Wang et al. (76), who revealed that the anti-Spike protein antibodies were in fact responsible for the infection of immune cells, and the enhancement of the infection can be improved by increasing the dilutions of antibodies. In relation to MERS-CoV, a similar mechanism has been demonstrated, since neutralizing monoclonal antibodies (nAb) are able to bind to the spike-S surface protein, allowing conformational changes and being subject to proteolytic activation. Meanwhile, nAb binds to the cell surface IgG Fc receptor, guiding viral entry through canonical pathways dependent on the viral receptor (77). Recent studies with COVID-19 patients reported that there was a strong IgG antibody response against the nucleocapsid protein and a delay in eliminating the virus, leading to an increase in the severity of the infection and contributing to the hypothesis of ADE of SARS-CoV-2 (78).
In view of this, the geographic discrepancy in pathogenesis can be explained, since individuals who have experienced previous exposure to coronaviruses are experiencing the effects of ADE due to the heterogeneity of the antigenic epitope (79). In addition, the potential of human antibodies for vaccination will depend on whether antibodies play a role in disease progression or in protecting against viral infection (70).
As an evasion mechanism, CoVs use a glycan conformational shield to prevent the recognition of the virus by the immune system, and, for this reason, S glycoproteins are found in trimers form and require structural alterations to engage with cellular receptors. In most of the hCoVs described, these S trimers are found in a naturally closed conformation, however, this mechanism also causes a delay in the process of cell infection due to the need for major changes in the glycoprotein conformation. It was described that, in SARS-CoV-2, the S trimers seem to exist in a partially open state, which prevents recognition by the immune system, but accelerates the initiation of conformational changes in the receptor and the processes of binding and fusion (49).

Pathogenic Mechanisms
Considering the similarity between SARS-CoV-1 and SARS-CoV-2, it is likely that their biochemical interactions and pathogenesis are also similar (80, 81). Once SARS-CoV-2 was reported to use ACE2 to enter host cells, it is suggested that the virus may target a cell spectrum similar to SARS-CoV-1 (38, 82, 83). SARS-CoV-1 is known to mainly infect macrophages and pneumocytes in the lungs, as well as other extrapulmonary tissues that express ACE2, which can also be expected for SARS-CoV-2 (82–84). However, the affinity of SARS-CoV-2 to ACE2 is 10–20-fold higher than that of SARS-CoV-1, which could explain its higher transmissibility and demonstrate that it can bind more efficiently to host cells, having a robust infection in ACE2+ cells in the upper respiratory tract (7).
ACE2 is an enzyme belonging to the renin-angiotensin system, located on the cell surface of type II alveolar epithelial cells in the lungs and cells of other tissues, and plays a crucial role in controlling vasoactive effects in the body. Despite their similarities, ACE and ACE2 have different substrate specificities with distinct functionalities that perform opposite actions in the body. In brief, ACE cleaves angiotensin I to generate angiotensin II, the peptide that binds and activates angiotensin type 1 receptor (AT1R) to constrict blood vessels, thereby raising blood pressure. In contrast, ACE2 inactivates angiotensin II (Ang-II) while generating angiotensin 1-7 (Ang-1-7), a potent heptapeptide that acts in vasodilation and attenuation of inflammation (85).
Therefore, considering that SARS-CoV-2 uses ACE2 to enter cells, the main hypothesis of pulmonary pathology is that the increased activity of ACE (Ang-II) over ACE2 (Ang-1-7) may cause acute lung injury since the binding of the S protein to ACE2 leads to its blockage. Thus, the suppression of ACE2 occurs due to the increased internalization and release of ACE2 from the cell surface, which leads to a decrease in tissue ACE2 and the generation of Ang-1-7, and consequently higher Ang-II levels. Because of this, as shown in an experimental SARS-CoV-1 model, this process can drive an Ang II-AT1R-mediated inflammatory response in the lungs and potentially induce direct parenchymal injury (67, 80, 86, 87).
Another hypothesis states that SARS-CoV-2 infection blocks ACE2 function when binding to host cells, inhibiting its role of cleaving bradykinin and, as a consequence, bradykinin accumulates in the lung, promoting pulmonary edemas due to vasodilator activity and consequent respiratory failure. The increased bradykinin activation in the pulmonary endothelium can also induce neutrophil migration, enhancing tissue damage caused by the respiratory burst of these cells (88).
ACE2 is also highly expressed and co-expressed with TMPRSS2 in nasal epithelial cells, chalices, and hair cells (89). This finding is in accordance with the high detection of viral RNA in the upper airways present in nasal swabs and throats of both symptomatic and asymptomatic patients, demonstrating that the nasal epithelium is an important site for the infection to initiate and can represent an essential reservoir for viral dissemination and transmission (38).
Although the virus mainly affects the lungs, there are reports that SARS-CoV-2 also has organotropism, accompanied by dysfunction, in multiple organs, including the kidneys, liver, heart, and brain, which can influence the course of the disease and possibly worsen pre-existing conditions. It has been reported that ACE2, TMPRSS2, and cathepsin L can be expressed on glial cells and neurons, cardiomyocytes, liver cells, bile duct cells, and renal tubular cells (90, 91).
Evidence indicates that SARS-CoV-2 “neuroinvasion” can establish a direct entry along the olfactory nerve, mainly through the nasal olfactory epithelium, which expresses ACE2 and TMPRSS2, allowing access to the central nervous system (CNS). The spread of the virus through the hematogenous or transsynaptic pathway has also been widely discussed, however, it is known that the different levels of neurotropism and neurovirulence in patients with COVID-19 can be explained by a combination of viral factors and their interaction with the host (41, 92, 93).
Regarding the evolution of infected individuals, aging, comorbidities, and weakening of the immune system are factors that generally cause the infection to intensify at the acute phase, leading to the manifestation of more severe conditions (6). Thus, according to epidemiological studies, it is known that patients with chronic conditions, such as hypertension, diabetes, and chronic obstructive pulmonary disease (COPD), are more likely to develop a critical form of the disease (94–96).
The risk of applying medication commonly used in hypertension treatments to COVID-19 patients (97, 98) has raised different hypotheses over the issue of invoking a higher expression of ACE2 (99–101). A systematic review assessing the clinical outcomes for SARS-CoV-2-infected individuals regarding treatment using angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) concluded that these types of drugs have no deleterious effects and should continue to be used in COVID-19 patients (102), reinforcing the recommendations of several medical societies, including the American Heart Association (103) and European Society of Cardiology (104).
Respiratory diseases, such as COPD and asthma, cause a reduced lung function and greater susceptibility to lung inflammation, and are expected to show a potentially critical course of COVID-19. COPD patients are already considered more susceptible to the development of pneumonia based on the clinical characteristics exhibited, such as lung structural damage, alterations in local/systemic inflammatory response, impaired host immunity, microbiome imbalance, persistent mucus production, and the presence of potentially pathogenic bacteria in the airways (105). Additionally, in the scenario of COVID-19, smokers and individuals with COPD have shown to have increased airway expressions of ACE-2 (106). It is still worth mentioning that patients who have this type of disorder often use corticosteroid immune-suppressing drugs, whose effect of reducing the immunity to respiratory infections may represent another contributing factor to a higher risk of infection (107).

Clinical and Radiological Changes
Most COVID-19 patients exhibit mild to moderate symptoms, but approximately 15% progress to critical pneumonia and 5% eventually develop acute respiratory distress syndrome (ARDS), septic shock, multiple organ failure, and death (26, 108). Once the infection is installed, the spectrum of clinical presentations has been reported to range from asymptomatic infection to critical respiratory failure.
According to the severity of symptoms, patients can be classified as mild, severe, and critical. In general, the most commonly reported symptoms are fever, cough, myalgia, fatigue, pneumonia, dyspnea, as well as the loss of smell and taste, whereas less common reported symptoms include headache, diarrhea, hemoptysis, and a runny nose (108, 109). Most critically ill patients present progressive respiratory failure due to alveolar damage caused by hyper inflammation, which can result in lethal pneumonia (26).
A retrospective study conducted by Liu et al. (110) demonstrated that older patients with SARS-CoV-2 showed higher pneumonia severity index scores and had a higher chance of multiple lobe involvement compared with young patients. Elderly adults are more susceptible to SARS-CoV-2 and have a high risk of morbidity and mortality (111). This can be explained by factors such as physiological changes and multiple age-related comorbidities, in addition to associated polymedication (112).
Regarding the potential involvement of COVID-19 in the CNS, studies have investigated the neurological changes developed throughout the course of the disease. Nonspecific symptoms (dizziness, headache, and seizure) and specific symptoms (loss of smell or taste and stroke) were described (91, 113–115). Epidemiological studies have reported that some patients infected with SARS‐CoV‐2 did report headaches (8%), nausea, or vomiting (1%). A more recent study investigating 214 COVID‐19 patients found that about 88% of critically ill patients displayed neurologic manifestations, including acute cerebrovascular diseases and impaired consciousness (26, 116).
Among patients diagnosed with SARS-CoV-2, it has been reported that renal dysfunction is characterized by high levels of blood urea nitrogen, creatinine, uric acid, and D-dimer, associated with proteinuria and hematuria (90, 117–119). Recent studies have reported an incidence between 3-9% of acute kidney injury in COVID-19 patients, demonstrating renal abnormalities (94, 96, 111, 120). Cardiovascular complications are also associated with COVID-19 infection, including myocardial injury, myocarditis, acute myocardial infarction, heart failure, dysrhythmias, and venous thromboembolic events, being significant contributors to the mortality associated with this disease (121, 122).
Several studies found that CoVs can also affect other body regions, such as the gastrointestinal tract and ocular tissues (123, 124); some of them specifically investigated changes in the gastrointestinal tract and identified the presence of SARS-CoV-2 RNA in samples of anal/rectal swabs and feces of infected patients, establishing that the virus could be transmitted orally or fecally as well. Additionally, symptoms such as diarrhea, vomiting, and intestinal pain (125) have also been reported for SARS-CoV-2-positive patients, which can be associated with the expression of ACE2 in gastrointestinal epithelial cells, present especially in the small and large intestines, contributing to viral infection and replication in these cells (126).
Regarding ocular tissues, some studies have also identified the manifestation of conjunctivitis in patients with COVID-19 (<1%) (96), however, it is an underestimated number (127). Currently, it is still unclear how SARS-CoV-2 can cause conjunctivitis, but theories include: (i) conjunctiva can be a direct inoculation site for the virus, (ii) the virus can reach the upper respiratory tract through the nasolacrimal duct, or (iii) infection can occur via hematogenous through the lacrimal gland (123).
Histologically, biopsy samples of lungs reveal evident desquamation and hyaline membrane formation of pneumocytes, in addition to bilateral diffused alveolar damages along with cellular fibromyxoid exudate, indicating ARDS. In addition, the cytopathic effects found include multinucleated syncytial cells, increased atypical pneumocytes, and the presence of inflammatory infiltrates of mononuclear cells (26, 108).
More recently, reports on COVID-19 have included the occurrence of coagulation abnormalities in most critically ill patients (128–131). Tang et al. (132) reported the occurrence of disseminated intravascular coagulation in 71.4% of non-surviving COVID-19 patients and in only 0.6% of surviving patients, suggesting a high frequency in severe COVID-19 patients. Autopsies performed on patients with COVID-19 also demonstrated small fibrinous thrombi in pulmonary arterioles with endothelial tumefaction, the presence of megakaryocytes, and indications of coagulation cascade activation (133).
Although it is important to consider the direct procoagulant properties of SARS-CoV-2, the combination of immobility, systemic inflammation, platelet activation, endothelial dysfunction, and stasis of blood flow can lead to thrombotic complications that mimic systemic coagulopathies associated with severe infections, such as sepsis-induced coagulopathy (SIC), disseminated intravascular coagulation (DIC), and thrombotic microangiopathy (130). However, COVID-19 has some distinct features that may establish a new category of coagulopathy, denominated COVID-19 associated coagulopathy (CAC), whose main markers are higher D-dimer concentration and fibrinogen levels, a relatively lower platelet count, and longer prothrombin time (129). In COVID-19 patients, CAC has been associated with higher mortality (131).
Chest computed tomography (CT) in patients with COVID-19 has commonly demonstrated multifocal “ground-glass” opacity (GGO) in the lungs, which can occur concurrently with consolidation in posterior and peripheral areas, suggesting a pneumonia pattern in the organization of lung injury and indicating disease progression (134–136). Another important manifestation found through chest CTs is reticular pattern formation with interlobular septal thickening, which might be associated with interstitial lymphocyte infiltration and determine the disease course (108, 137, 138).
CT has highlighted many other alterations, including the “crazy-paving” pattern, which may result from the alveolar edema and interstitial inflammation in acute lung injury, and air bronchogram with a pattern of air-filled (low-attenuation) bronchi, but with gelatinous mucus and several airway changes, such as bronchiectasis and bronchial wall thickening resulting from the destruction of bronchial wall structure, proliferation of fibrous tissue, and fibrosis (137–140).