Transmission, pathogenesis, replication of SARS-CoV-2 (COVID-19)
SARS-CoV-2 is the virus that causes COVID-19, a respiratory disease that has become a global pandemic. Understanding how the virus spreads among people is crucial for preventing and controlling its transmission. According to the current scientific evidence, there are three main modes of transmission of SARS-CoV-2:
- Droplet transmission: This occurs when respiratory droplets (as produced when an infected person coughs or sneezes) are ingested or inhaled by individuals in close proximity (within 6 feet). Respiratory droplets are larger than 5 micrometers in diameter and can carry virus particles. They can also land on exposed mucous membranes (such as the mouth, nose, or eyes) or on surfaces and objects contaminated with the virus.
- Contact transmission: This occurs when a person touches a surface or object contaminated with the virus and then touches their own mucous membranes. The virus can survive on different materials for varying periods of time, depending on the environmental conditions. Contact transmission can be prevented by frequent hand washing and disinfection of high-touch surfaces.
- Aerosol transmission: This occurs when respiratory droplets mix into the air, forming aerosols, and cause infection while inhaling a high dose of aerosols into the lungs in a relatively closed environment. Aerosols are smaller than 5 micrometers in diameter and can remain suspended in the air for minutes to hours. They can travel farther than 6 feet and accumulate in poorly ventilated spaces. Aerosol transmission can be reduced by wearing masks, improving ventilation, and avoiding crowded indoor settings.
These modes of transmission are not mutually exclusive and can occur simultaneously. The risk of infection depends on several factors, such as the amount and duration of exposure, the viral load of the source, the susceptibility of the host, and the environmental conditions.
In addition to these modes of transmission, there is some evidence that SARS-CoV-2 can also be transmitted through other routes, such as fecal-oral transmission, blood transfusion, vertical transmission from mother to child, and animal-to-human transmission. However, these routes are considered less common and require further investigation.
Droplets transmission is one of the modes of transmission of SARS-CoV-2, the virus that causes COVID-19. It occurs when respiratory droplets (as produced when an infected person coughs or sneezes) are ingested or inhaled by individuals in close proximity . Respiratory droplets are larger than 5-10 micrometers (μm) in diameter and can travel short ranges between 3 to 6 feet before falling to the ground. These droplets carry virus and transmit infection by depositing on exposed mucous membranes in the mouth, nose, or eye of susceptible persons . Droplets transmission can also occur indirectly by touching mucous membranes with hands that have been soiled either directly by virus-containing respiratory fluids or indirectly by touching surfaces with virus on them.
The risk of droplets transmission is greatest within three to six feet of an infectious source where the concentration of these droplets is highest. Therefore, physical distancing, wearing masks, and avoiding crowded and poorly ventilated spaces are important preventive measures to reduce the spread of COVID-19 through droplets transmission . Additionally, frequent hand hygiene and environmental cleaning and disinfection can help prevent indirect contact transmission via contaminated surfaces .
Aerosol transmission occurs when respiratory droplets mix into the air, forming aerosols, and cause infection while inhaling a high dose of aerosols into the lungs in a relatively closed environment. Aerosols are very small droplets that can remain suspended in the air for minutes to hours and can travel farther than six feet from the source . Aerosol transmission is different from droplet transmission, which involves larger droplets that fall to the ground within seconds to minutes and usually do not travel more than six feet.
Aerosol transmission of SARS-CoV-2 is plausible under favorable conditions, particularly in relatively confined settings with poor ventilation and long duration exposure to high concentrations of aerosols. Examples of such settings include hospitals, nursing homes, restaurants, bars, gyms, schools, offices, and public transportation . Aerosol transmission may also be influenced by environmental factors such as temperature, humidity, sunlight, and air pollution .
To prevent aerosol transmission of SARS-CoV-2, it is important to reduce the generation and accumulation of aerosols in indoor spaces. This can be achieved by wearing masks that fit well and filter out aerosols, avoiding crowded and poorly ventilated places, increasing outdoor air supply and exhaust ventilation, using air purifiers and filters, and disinfecting the air with ultraviolet light . In addition, maintaining physical distance, washing hands frequently, and getting vaccinated are also effective measures to reduce the risk of infection by any mode of transmission.
Aerosol transmission occurs when respiratory droplets mix into the air, forming aerosols, and cause infection while inhaling a high dose of aerosols into the lungs in a relatively closed environment. Aerosols are very small droplets that can remain suspended in the air for minutes to hours and can travel farther than six feet from an infectious source. Aerosols are produced during normal breathing, speaking, singing, coughing, and sneezing by both asymptomatic and symptomatic people. The proportion of the droplet size distribution within the aerosol range depends on the sites of origin within the respiratory tract and on whether the distribution is presented on a number or volume basis. Evaporation and fragmentation reduce the size of the droplets, whereas coalescence increases the mean droplet size. Aerosol particles containing SARS-CoV-2 can also coalesce with pollution particulates, and infection rates correlate with pollution.
The risk of aerosol transmission depends on several factors, such as the amount of virus in the exhaled breath, the duration and proximity of exposure, the ventilation and air flow in the environment, and the use of personal protective measures (such as masks and face shields). The risk is higher in indoor settings with poor ventilation, where aerosols can accumulate over time . The risk is also higher in situations where people generate more aerosols, such as during singing, shouting, or exercising . The risk is lower in outdoor settings where aerosols are diluted by natural air currents . The risk is also lower when people wear masks that fit well and block or filter out most of the exhaled droplets and aerosols .
To prevent and control aerosol transmission of SARS-CoV-2, several measures can be taken at individual and community levels. These include:
- Avoiding crowded and poorly ventilated indoor spaces. If possible, meeting outdoors or opening windows and doors to increase natural ventilation .
- Wearing a mask that covers the nose and mouth snugly when in public or around people who are not from the same household. Masks should be made of multiple layers of breathable fabric and should not have valves or vents that allow exhaled air to escape .
- Maintaining physical distance of at least six feet from others who are not from the same household. Physical distancing reduces the chances of inhaling infectious aerosols from nearby people .
- Practicing good hand hygiene by washing hands frequently with soap and water or using alcohol-based hand sanitizer. Hand hygiene prevents the transfer of virus from contaminated surfaces or objects to mucous membranes .
- Getting vaccinated against COVID-19 when eligible. Vaccination protects against severe illness and reduces viral shedding, which may lower the risk of transmitting SARS-CoV-2 to others .
- Improving ventilation and air quality in indoor spaces by using mechanical systems (such as fans and filters) or natural methods (such as opening windows and doors). Ventilation and air quality can also be enhanced by using ultraviolet germicidal irradiation (UVGI) devices that can inactivate SARS-CoV-2 in aerosol form .
By following these measures, we can reduce the risk of aerosol transmission of SARS-CoV-2 and prevent further spread of COVID-19.
SARS-CoV-2 is a novel coronavirus that causes COVID-19, a respiratory disease that can range from mild to severe and even fatal. The pathogenesis of SARS-CoV-2 is the process by which the virus infects, replicates, and damages the host cells and tissues, leading to clinical manifestations and complications. The pathogenesis of SARS-CoV-2 is influenced by several factors, such as the viral characteristics, the host immune response, and the environmental conditions.
SARS-CoV-2 belongs to the genus Betacoronavirus, which also includes SARS-CoV-1 and MERS-CoV, two other zoonotic coronaviruses that cause severe respiratory infections in humans. SARS-CoV-2 shares about 80% sequence similarity with SARS-CoV-1 and 96% with a bat coronavirus, suggesting a bat origin for the virus. The most distinctive feature of SARS-CoV-2 is its spike (S) glycoprotein, which forms a crown-like structure on the viral envelope and mediates the attachment and entry of the virus into host cells. The S protein consists of two subunits: S1, which contains the receptor-binding domain (RBD) that recognizes the angiotensin-converting enzyme 2 (ACE2) receptor on human cells; and S2, which facilitates the fusion of the viral and cellular membranes. The S protein of SARS-CoV-2 has a higher affinity for ACE2 than SARS-CoV-1, which may explain its enhanced transmissibility. The S protein also has a furin cleavage site between the S1 and S2 subunits, which allows it to be activated by various host proteases and enhances its infectivity and cell tropism. In addition to the S protein, SARS-CoV-2 encodes other structural proteins, such as the membrane (M), envelope (E), and nucleocapsid (N) proteins, as well as non-structural proteins (NSPs) and accessory proteins that are involved in viral replication, transcription, assembly, and evasion of host immunity.
Host immune response
The host immune response to SARS-CoV-2 is crucial for controlling viral replication and clearing infection, but it can also contribute to tissue damage and disease severity. The innate immune system is the first line of defense against viral invasion and triggers the production of interferons (IFNs) and pro-inflammatory cytokines that activate the adaptive immune system. The adaptive immune system generates virus-specific antibodies and T cells that neutralize the virus and eliminate infected cells. However, in some cases, the immune response to SARS-CoV-2 can become dysregulated and excessive, leading to a cytokine storm syndrome that causes systemic inflammation, tissue injury, organ failure, and death. The factors that determine the balance between protective and pathological immune responses are not fully understood, but they may include viral load, genetic susceptibility, co-morbidities, age, sex, and environmental exposures.
The environmental conditions can also affect the pathogenesis of SARS-CoV-2 by influencing its stability, transmission, and exposure. For example, temperature, humidity, sunlight, ventilation, and surface materials can affect the survival of SARS-CoV-2 in aerosols and droplets that are generated by respiratory activities such as coughing, sneezing, talking, or breathing. These factors can also affect the dispersion and inhalation of viral particles in indoor or outdoor settings. Moreover, social distancing measures, personal protective equipment (PPE), hygiene practices, vaccination status, and testing strategies can affect the risk of exposure to SARS-CoV-2 among individuals or populations.
The incubation period is the time between exposure to the virus and the onset of symptoms. It varies depending on the strain of the virus, the age and health status of the infected person, and other factors. According to the World Health Organization (WHO), the incubation period for COVID-19 caused by the original strain of SARS-CoV-2 ranged from 2 to 14 days, with an average of 5 to 6 days. However, newer variants of the virus, such as Alpha, Beta, Delta, and Omicron, have been found to have shorter incubation periods. A recent study that analyzed data from 1.7 million confirmed cases in South Korea reported that the mean incubation period was 5.00 days for Alpha, 4.50 days for Beta, 4.41 days for Delta, and 3.42 days for Omicron. This suggests that these variants are more transmissible and can infect more people in a shorter time.
The symptoms of COVID-19 also vary depending on the variant and the individual. The most common symptoms reported by people with COVID-19 are fever, cough, shortness of breath, fatigue, headache, loss of taste or smell, sore throat, runny nose, and muscle or joint pain. However, some people may experience more severe symptoms, such as pneumonia, acute respiratory distress syndrome (ARDS), septic shock, blood clots, organ failure, or death. The risk of severe illness or complications increases with age and underlying medical conditions, such as diabetes, heart disease, lung disease, obesity, or immunosuppression. Some variants of the virus may also cause different or milder symptoms than others. For example, Omicron has been associated with more mild symptoms than Delta, such as sore throat, dry cough, fatigue, headache, and congestion. However, it is still unclear whether Omicron causes less severe disease or hospitalization than other variants.
The variation in incubation period and symptoms of COVID-19 makes it challenging to diagnose and control the spread of the virus. Therefore, it is important to follow public health measures such as wearing a mask, maintaining physical distance, avoiding crowded or poorly ventilated spaces, washing hands frequently, and getting vaccinated and boosted against COVID-19. If you have been exposed to someone with COVID-19 or have any symptoms suggestive of COVID-19, you should get tested as soon as possible and isolate yourself until you receive a negative result or complete your quarantine period. By doing so, you can protect yourself and others from this potentially deadly disease.
The alveoli are tiny air sacs in the lungs where gas exchange occurs. They are lined by a thin layer of epithelial cells that have two main types: type I and type II. Type I cells are flat and cover most of the alveolar surface, while type II cells are cuboidal and secrete surfactant, a substance that reduces surface tension and prevents alveolar collapse.
SARS-CoV-2, the virus that causes COVID-19, infects the alveolar epithelial cells by binding to a receptor called angiotensin-converting enzyme 2 (ACE2) that is expressed on their surface . The virus mainly targets type II cells, which have higher levels of ACE2 than type I cells. After binding to ACE2, the virus enters the cell and hijacks its machinery to replicate its genetic material and produce new viral particles. The infected cell then releases the new viruses, which can infect other cells or spread to the bloodstream.
The infection of alveolar epithelial cells has several consequences that impair lung function and cause respiratory distress. First, the infection triggers cell death by apoptosis (programmed cell death) or necrosis (uncontrolled cell death), which leads to alveolar damage and inflammation. Second, the infection reduces the production of surfactant by type II cells, which increases surface tension and causes alveolar collapse or atelectasis. Third, the infection induces a strong immune response that releases pro-inflammatory cytokines and chemokines, which attract immune cells such as macrophages and neutrophils to the site of infection. These immune cells can further damage the alveolar epithelium by releasing reactive oxygen species and proteases. Fourth, the infection disrupts the epithelial barrier that normally prevents fluid leakage from the capillaries into the alveoli. This results in pulmonary edema or fluid accumulation in the lungs, which impairs gas exchange and oxygen delivery.
In summary, SARS-CoV-2 affects the epithelial cells in the alveoli by infecting them via ACE2 receptor, causing cell death, reducing surfactant production, inducing inflammation, and disrupting epithelial barrier integrity. These effects contribute to the development of acute respiratory distress syndrome (ARDS), a life-threatening condition that requires mechanical ventilation and intensive care.
The innate immune system is the first line of host defense against most virus infections, with pathogen recognition receptors detecting SARS-CoV-2 RNA and protein components and initiating pro-inflammatory and antiviral responses. However, like many viruses, SARS-CoV-2 has evolved strategies to circumvent innate immune detection and antagonize the interferon (IFN) signaling pathway, which is crucial for limiting viral replication and dissemination.
One of the key mechanisms by which SARS-CoV-2 evades innate immune recognition is by reducing the exposure of its viral RNA to cytosolic sensors, such as RIG-I and MDA5. SARS-CoV-2 RNA has low cytosine-phosphate-guanosine (CpG) levels in the genome, which may reduce its immunogenicity. Moreover, SARS-CoV-2 RNA is shielded by nucleocapsid protein (N) and other viral proteins, such as nonstructural protein 14 (nsp14), which can bind to dsRNA and prevent its sensing by MDA5. Additionally, SARS-CoV-2 RNA synthesis occurs within double-membrane vesicles (DMVs) that are derived from the endoplasmic reticulum (ER) and are inaccessible to cytosolic sensors.
Another way by which SARS-CoV-2 escapes innate immune detection is by glycosylation of its spike protein (S), which is essential for viral entry into host cells via the angiotensin-converting enzyme 2 (ACE2) receptor. Glycosylation can shield essential elements of the S protein, such as the receptor-binding domain (RBD), from antibody recognition and neutralization. Furthermore, glycosylation can also mask potential PAMPs on the S protein, such as unmethylated CpG motifs, from endosomal sensors, such as Toll-like receptor 9 (TLR9).
Besides avoiding innate immune recognition, SARS-CoV-2 also actively impairs the IFN signaling pathway, which is responsible for inducing hundreds of IFN-stimulated genes (ISGs) that have antiviral and immunomodulatory functions. Several SARS-CoV-2 proteins have been shown to interfere with different steps of the IFN pathway, such as:
- nsp1: suppresses host gene expression, including IFN and ISGs, by inducing ribosome stalling and mRNA degradation.
- nsp3: deubiquitinates RIG-I and MDA5, impairing their activation and signaling.
- nsp6: blocks the fusion of DMVs with lysosomes, preventing the degradation of viral RNA and its exposure to endosomal sensors.
- nsp13: inhibits IRF3 phosphorylation and nuclear translocation, reducing IFN production.
- nsp15: cleaves dsRNA into shorter fragments that are less immunogenic for MDA5.
- nsp16: methylates viral RNA at the 5′ cap structure, mimicking host mRNA and avoiding recognition by RIG-I.
- ORF3b: binds to IRF3 and prevents its dimerization and nuclear translocation, reducing IFN production.
- ORF6: binds to karyopherin alpha 2 (KPNA2), a nuclear import factor for STAT1, impairing its nuclear translocation and ISG expression.
- ORF9b: targets mitochondrial antiviral-signaling protein (MAVS), a key adaptor for RIG-I and MDA5 signaling, for proteasomal degradation.
By evading innate immune detection and antagonizing IFN signaling, SARS-CoV-2 can delay or dampen the antiviral response of the host, facilitating viral replication and dissemination. However, this may also result in an excessive inflammatory response later in the infection, as uncontrolled viral replication triggers massive cytokine production by innate immune cells. This cytokine storm can cause tissue damage, organ failure and death in severe COVID-19 cases. Therefore, understanding the interaction between SARS-CoV-2 proteins and the innate immune system is crucial for developing effective diagnostic markers and therapeutic strategies for COVID-19.
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COVID-19 is a complex disease that affects multiple organ systems and causes a wide range of clinical manifestations. The main pathophysiology of COVID-19 is severe pneumonia, RNAemia, combined with the incidence of ground-glass opacities, and acute cardiac injury. The respiratory system is the primary target of SARS-CoV-2 infection, but other organs such as the heart, kidneys, liver, brain, and blood vessels can also be involved.
The respiratory symptoms of COVID-19 are mainly caused by the damage of the alveolar epithelial cells and the inflammatory response triggered by the viral infection. The virus enters the alveolar cells through the angiotensin-converting enzyme 2 (ACE2) receptor and replicates in the cytoplasm. The viral replication induces cellular stress and apoptosis, leading to the release of pro-inflammatory cytokines and chemokines that attract immune cells to the site of infection. The immune cells, such as macrophages, monocytes, and lymphocytes, produce more cytokines and reactive oxygen species that further damage the alveolar cells and the extracellular matrix. This results in increased vascular permeability, edema, hyaline membrane formation, and fibrin deposition in the alveoli. These changes impair gas exchange and cause hypoxia and respiratory failure.
The systemic manifestations of COVID-19 are largely mediated by the cytokine storm and the coagulation dysfunction induced by the viral infection. The cytokine storm is a hyper-inflammatory response characterized by elevated levels of cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in the blood. These cytokines can cause fever, fatigue, anorexia, and muscle pain. They can also activate the endothelial cells and disrupt the vascular integrity, leading to increased permeability, vasodilation, and hypotension. Moreover, they can stimulate the production of acute phase proteins such as C-reactive protein (CRP) and ferritin that reflect the severity of inflammation.
The coagulation dysfunction is a pro-thrombotic state characterized by increased levels of fibrinogen, D-dimer, and thrombin-antithrombin complex in the blood. These markers indicate the activation of both the intrinsic and extrinsic pathways of coagulation and the consumption of platelets and clotting factors. The coagulation dysfunction can cause microvascular thrombosis and organ ischemia in various tissues such as the lungs, kidneys, liver, brain, and heart. It can also increase the risk of venous thromboembolism (VTE) such as deep vein thrombosis (DVT) and pulmonary embolism (PE) in severe cases.
The cardiac complications of COVID-19 are multifactorial and include myocardial injury, myocarditis, arrhythmias, heart failure, cardiogenic shock, and cardiac arrest. The mechanisms of cardiac injury include direct viral invasion of cardiomyocytes through ACE2 receptor, hypoxia-induced myocardial damage due to respiratory failure, cytokine-mediated myocardial inflammation and dysfunction, microvascular thrombosis and ischemia due to coagulation dysfunction, and stress-induced cardiomyopathy due to catecholamine surge.
The renal complications of COVID-19 include acute kidney injury (AKI), proteinuria, hematuria, electrolyte imbalance, and renal replacement therapy requirement. The mechanisms of renal injury include direct viral invasion of renal tubular cells through ACE2 receptor, hypoxia-induced tubular necrosis due to respiratory failure, cytokine-mediated tubulointerstitial inflammation and fibrosis, microvascular thrombosis and ischemia due to coagulation dysfunction, rhabdomyolysis-induced acute tubular obstruction due to muscle injury.
The hepatic complications of COVID-19 include elevated liver enzymes (alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , gamma-glutamyl transferase ), hyperbilirubinemia, and liver failure. The mechanisms of liver injury include direct viral invasion of hepatocytes through ACE2 receptor or bile duct epithelial cells through CD147 receptor, hypoxia-induced hepatocellular damage due to respiratory failure, cytokine-mediated hepatic inflammation and dysfunction, drug-induced liver injury due to antiviral or immunomodulatory therapies, and ischemic hepatitis due to hypotension or cardiac failure.
The neurological complications of COVID-19 include headache, dizziness, anosmia, ageusia, encephalopathy, encephalitis, meningitis, stroke, Guillain-Barré syndrome, and Miller Fisher syndrome. The mechanisms of neurological injury include direct viral invasion of neurons or glial cells through ACE2 receptor or olfactory epithelium through TMPRSS2 receptor, hypoxia-induced cerebral damage due to respiratory failure, cytokine-mediated neuroinflammation and blood-brain barrier disruption, microvascular thrombosis and ischemia due to coagulation dysfunction, and immune-mediated demyelination or neuropathy due to molecular mimicry or autoimmunity.
The pathophysiology of COVID-19 is complex and dynamic, involving multiple organ systems and molecular pathways. A better understanding of the mechanisms of viral infection and host response is essential for developing effective diagnostic, preventive, and therapeutic strategies for this emerging disease.
Coronavirus (SARS-CoV-2) is a positive-sense single-stranded RNA virus with an envelope and a spike protein on its surface. The spike protein mediates the attachment and entry of the virus into human cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. The replication of coronavirus involves several steps, including:
- Translation of the genomic RNA into two large polyproteins, pp1a and pp1ab, which are cleaved by viral proteases into 16 nonstructural proteins (nsps).
- Formation of membrane-bound replication-transcription complexes (RTCs) that synthesize negative-sense RNA intermediates and subgenomic RNAs.
- Translation of the subgenomic RNAs into structural and accessory proteins, such as spike, envelope, membrane and nucleocapsid proteins.
- Assembly of the viral genome and structural proteins into nucleocapsids that bud into the endoplasmic reticulum-Golgi intermediate compartment (ERGIC).
- Maturation and release of the virions by exocytosis.
In this section, we will briefly describe each step of coronavirus replication and highlight some of the features that distinguish it from other RNA viruses.
One of the key steps in the infection process of SARS-CoV-2 is the binding of its spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of human cells. ACE2 is a transmembrane protein that has a catalytic domain that converts angiotensin II to angiotensin 1–7, a peptide that has vasodilatory and anti-inflammatory effects. ACE2 is expressed in various tissues, such as the lungs, heart, kidneys, intestines and liver , and plays a role in regulating blood pressure, inflammation and tissue repair.
The S protein of SARS-CoV-2 is a trimeric glycoprotein that consists of two subunits: S1 and S2. The S1 subunit contains the receptor-binding domain (RBD), which is responsible for recognizing and binding to ACE2. The S2 subunit contains the fusion peptide, which mediates the fusion of the viral and cellular membranes. The S protein undergoes a series of conformational changes during the entry process, which are triggered by proteolytic cleavage and receptor binding.
The first step in SARS-CoV-2 entry is the attachment of the virus to the cell surface, which can be facilitated by accessory molecules, such as heparan sulfate proteoglycans, sialic acids and neuropilins. The attachment enhances the exposure of the RBDs on the S protein and increases the probability of interaction with ACE2. The RBDs can adopt two conformations: up and down. The up conformation exposes the binding site for ACE2, while the down conformation hides it. Only one RBD per trimer needs to be in the up conformation for ACE2 binding to occur.
The second step in SARS-CoV-2 entry is the binding of the RBD to ACE2. The RBD interacts with ACE2 through a network of hydrogen bonds and hydrophobic contacts, mainly involving residues in the helix 31–42 and β-sheet 1–7 regions of ACE2. The binding affinity of SARS-CoV-2 RBD for human ACE2 is about 10- to 20-fold higher than that of SARS-CoV RBD, which may contribute to its higher transmissibility. The binding of RBD to ACE2 induces conformational changes in both proteins, which facilitate the subsequent steps of entry.
The third step in SARS-CoV-2 entry is the proteolytic activation of the S protein. This step involves two cleavage sites on the S protein: S1/S2 and S2`. The S1/S2 site is located at the boundary between the S1 and S2 subunits and is cleaved by furin-like proteases during virus assembly or upon virus release from infected cells. This cleavage generates two separate subunits that remain non-covalently associated on the virion surface. The S2` site is located within the S2 subunit and is cleaved by cell surface or endosomal proteases, such as transmembrane protease serine 2 (TMPRSS2) or cathepsin L, upon virus binding to ACE2. This cleavage exposes the fusion peptide and primes the S protein for membrane fusion.
The fourth step in SARS-CoV-2 entry is the endocytosis and membrane fusion of the virus. Depending on the cell type and availability of proteases, SARS-CoV-2 can enter cells either via clathrin-mediated endocytosis or macropinocytosis. Once inside endosomes, the virus encounters low pH conditions that trigger further conformational changes in the S protein. The fusion peptide inserts into the endosomal membrane, while another region of the S protein called heptad repeat 1 (HR1) forms a coiled-coil structure that interacts with heptad repeat 2 (HR2) to form a six-helix bundle. This brings the viral and endosomal membranes into close proximity and allows their fusion. Alternatively, if TMPRSS2 is present on the cell surface, it can cleave both S1/S2 and S2` sites and enable the S protein to fuse directly with the plasma membrane without endocytosis.
The final step in SARS-CoV-2 entry is the release of the viral genome into the cytoplasm, where it can initiate the replication and transcription of viral RNA and the production of new viral particles.
One of the key steps in the infection cycle of SARS-CoV-2 is the attachment and entry of the virus into human cells. This process involves the interaction of the viral spike (S) glycoprotein with its cellular receptor, angiotensin-converting enzyme 2 (ACE2), and the subsequent fusion of the viral and cellular membranes. The S glycoprotein is a trimeric protein that consists of two subunits, S1 and S2. The S1 subunit contains the receptor-binding domain (RBD) that recognizes and binds to ACE2, while the S2 subunit mediates membrane fusion through its fusion peptide, heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains.
The attachment and entry of SARS-CoV-2 into human cells can be divided into four main steps: (1) binding of the RBD to ACE2, (2) proteolytic activation of the S protein by host proteases, (3) conformational changes of the S protein to expose the fusion peptide, and (4) membrane fusion by the formation of a six-helix bundle between HR1 and HR2.
- Binding of the RBD to ACE2: The RBD is located at the distal end of the S1 subunit and can adopt two conformations: an up conformation that is accessible for ACE2 binding, and a down conformation that is hidden from ACE2. The RBD undergoes dynamic transitions between these two states, which affects its binding affinity and avidity for ACE2. The binding of the RBD to ACE2 is influenced by several factors, such as the amino acid residues at the interface, the glycosylation patterns on both proteins, and the presence of other attachment factors on the cell surface. The binding of one RBD to ACE2 induces conformational changes in the neighboring RBDs, resulting in a more stable binding of the S trimer to ACE2 .
- Proteolytic activation of the S protein by host proteases: The binding of the RBD to ACE2 facilitates the exposure of two cleavage sites on the S protein: S1/S2 and S2`. The S1/S2 site is located at the junction between the S1 and S2 subunits, while the S2` site is located upstream of the fusion peptide in the S2 subunit. The cleavage at these sites by host proteases is essential for activating the membrane fusion potential of the S protein. The cleavage at the S1/S2 site separates the S1 subunit from the S2 subunit, allowing for conformational changes in the latter. The cleavage at the S2` site exposes the fusion peptide, which inserts into the target cell membrane. Different types of proteases can cleave these sites depending on the cell type and tissue location. For example, furin-like proteases can cleave both sites in a pre-activation manner during virus assembly or egress, while transmembrane protease serine 2 (TMPRSS2) and cathepsin L can cleave both sites in a post-attachment manner during virus entry .
- Conformational changes of the S protein to expose the fusion peptide: After proteolytic activation, the S protein undergoes a series of conformational changes that enable membrane fusion. First, the S1 subunit dissociates from the S2 subunit, exposing a hydrophobic region called FP1 in HR1. Second, FP1 interacts with FP2, another hydrophobic region located downstream of HR1, forming a loop structure called FP loop. Third, FP loop inserts into
The replication process of SARS-CoV-2 genomic RNA involves several steps that are mediated by the viral replicase-transcriptase proteins and other viral and host factors. The replicase-transcriptase proteins are encoded by the open reading frames 1a and 1b (ORF1a and ORF1b) of the viral genome and are synthesized as two large polyproteins, pp1a and pp1ab. These polyproteins are cleaved by viral proteases into 16 nonstructural proteins (nsp1 to nsp16), which form the core of the replication-transcription complexes (RTCs) that are responsible for viral RNA synthesis .
The RTCs are membrane-bound structures that accumulate at perinuclear regions and are associated with double-membrane vesicles . The RTCs use the genomic RNA as a template to produce both negative-sense and positive-sense RNA intermediates. The negative-sense RNA intermediates serve as templates for the synthesis of new genomic RNA and subgenomic RNAs (sgRNAs), which encode the structural and accessory proteins of the virus . The positive-sense RNA intermediates serve as templates for the synthesis of nested sgRNAs, which are capped and polyadenylated at their 5′ and 3′ ends, respectively .
The synthesis of sgRNAs involves a discontinuous transcription mechanism that is unique to coronaviruses. This mechanism involves the recognition of conserved transcription regulatory sequences (TRSs) that are located at the 5′ end of each gene and at the 3′ end of the leader sequence, which is a short sequence at the 5′ end of the genomic RNA . During negative-strand synthesis, the RTCs switch templates from one TRS to another, resulting in the generation of fused negative-strand sgRNAs that contain the leader sequence at their 5′ end. These fused negative-strand sgRNAs then serve as templates for positive-strand sgRNA synthesis .
The newly synthesized genomic RNA and sgRNAs are then packaged into viral particles by interacting with the nucleocapsid protein (N), which binds to specific packaging signals on the RNA molecules . The viral particles also contain the spike protein (S), which mediates attachment and entry into host cells; the envelope protein (E), which is involved in virus assembly and release; and the membrane protein (M), which provides structural stability and interacts with other viral proteins . The assembly and release of viral particles occur at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where the viral proteins and RNA are transported by vesicles .
The replication process of SARS-CoV-2 genomic RNA is a complex and dynamic process that is regulated by multiple factors and interactions. Understanding this process at a molecular level can provide insights into the biology and pathogenesis of SARS-CoV-2, as well as potential targets for antiviral intervention.
After the replication of SARS-CoV-2 genomic RNA and the synthesis of viral proteins, the next step in the viral lifecycle is the assembly and release of new virions from the infected cells. This process involves the following steps:
- The structural proteins of SARS-CoV-2, namely the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, are translated in the cytoplasm and transported to the endoplasmic reticulum (ER) or the ER-Golgi intermediate compartment (ERGIC) .
- The S protein is cleaved by a host protease, furin, into two subunits, S1 and S2, which remain associated by non-covalent interactions . The S1 subunit contains the receptor-binding domain (RBD) that binds to ACE2 on the host cell surface, while the S2 subunit mediates the fusion of the viral and cellular membranes .
- The M protein is the most abundant structural protein and plays a key role in virus assembly by interacting with other viral proteins and the viral RNA . The M protein has three transmembrane domains and a short cytoplasmic tail that contains a tyrosine-based sorting signal that directs it to the ERGIC .
- The E protein is a small integral membrane protein that forms ion channels and has multiple functions in virus assembly, release, and pathogenesis . The E protein also has a cytoplasmic tail that contains a PDZ-binding motif that may interact with host proteins involved in membrane trafficking .
- The N protein binds to the viral RNA and forms helical nucleocapsids that are packaged into the virions . The N protein also interacts with the M protein and may facilitate the encapsidation of the viral RNA .
- The assembly of SARS-CoV-2 occurs in the ERGIC, where the viral proteins and RNA are concentrated and form budding complexes . The M protein acts as a scaffold that recruits the E and S proteins to the budding site, while the N protein associates with the M protein through its RNA-binding domain .
- The budding of SARS-CoV-2 into the ERGIC lumen results in the formation of virus-containing vesicles that are transported to the cell surface along the secretory pathway . The release of SARS-CoV-2 from the infected cells occurs by exocytosis of these vesicles through fusion with the plasma membrane .
- Alternatively, some SARS-CoV-2 particles may be released by cell-to-cell spread through direct fusion of infected cells with neighboring cells or through tunneling nanotubes that connect distant cells . This mode of transmission may facilitate viral escape from neutralizing antibodies and innate immune responses .
The COVID-19 pandemic has posed unprecedented challenges to the global health and socio-economic systems. Understanding the transmission, pathogenesis, and replication of SARS-CoV-2 is crucial for developing effective preventive and therapeutic strategies against this novel coronavirus. In this article, we have outlined the current knowledge on these aspects of SARS-CoV-2 infection, based on the available scientific literature and official sources. However, there are still many gaps and uncertainties that need to be addressed by further research. Some of the key questions that remain unanswered are:
- What are the exact mechanisms and factors that determine the transmissibility and virulence of SARS-CoV-2 variants?
- What are the long-term consequences and complications of COVID-19 infection on different organs and systems?
- How long does the immunity conferred by natural infection or vaccination last and how effective is it against emerging variants?
- What are the best combinations and regimens of vaccines and antiviral drugs to prevent and treat COVID-19 infection?
- How can we improve the global surveillance, response, and preparedness for future pandemics of similar or greater magnitude?
The COVID-19 pandemic has also highlighted the importance of international collaboration, coordination, and communication among scientists, health professionals, policymakers, and the public. Only by working together can we overcome this crisis and prevent or mitigate future ones. We hope that this article has provided a comprehensive and updated overview of SARS-CoV-2 infection and stimulated further interest and inquiry into this topic.
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