Recognition
In December 2019, a novel virus of the genera beta-coronaviridae was identified as the causative agent of an acute atypical respiratory disease that disproportionately affected elderly patients who presented with complaints of dry cough, fever, and dyspnea, along with non-specific symptoms like headache, fatigue, dizziness, diarrhea, and vomiting. It was evident that the principal symptoms were respiratory symptoms which pointed to a respiratory infection. Also, these symptoms worsened quickly to cause hypoxia that was associated with fluid accumulation in the lungs. These symptoms were also associated with acute alveolar injury (AAI). The triad of hypoxia, AAI, and fluid accumulation in alveolar sacs (a form of diffuse pneumonia) is associated with acute respiratory distress syndrome (ARDS). However, this ARDS occurred on a background of viral pneumonia. Moreover, the duration from onset of symptoms to ARDS was noted – by Health Authorities in Wuhan – to be as short as 9 days. In fact, a 61-year old male patient who presented with complaints of cough and fever on December 20, 2019, saw his symptoms worsening and by December 27, he was diagnosed with respiratory distress which worsened within a period of 2 days to mandate assisted positive-pressure ventilation via mechanical ventilation, but his prognosis did not improve and he passed away on January 9, 2020.
The Chinese health authorities recognized that they were dealing with an aggressive form of atypical respiratory disease that was of viral origin. Laboratory tests were done on cough samples, and a viral sample was isolated and then subjected to a polymerase chain reaction (PCR) to get copies of the viral genome for further study. This allowed Chinese scientists to read the genetic sequence of the virus and later publish it.
The viral genome was found to share 79% of its genetic sequence with a beta-coronavirus that had caused an outbreak of severe acute respiratory syndrome (SARS) in 2003. This beta-coronavirus was named SARS-coronavirus (SARS-CoV). Additionally, the mean incubation period of this new disease was estimated to be 5.2 days, which is quite close to the mean incubation period of 4.4 days for SARS caused by SARS-CoV. Interestingly, the mean incubation period for Middle East Respiratory Syndrome (MERS), a respiratory illness caused by coronavirus, is 5.5. days. Likewise, because SARS-CoV originated from a bat, it can be expected that this new virus also originated from a bat. Genome analysis was done comparing the genome of the new CoV strain and that of Bat coronavirus, RaTG13, and it was found that there was 96.2% shared genetic homology. It was evident that this new virus was a new strain of coronavirus (CoV) that shared 79% genetic homology with SARS-CoV virus, and it was named the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2).
It was suspected that SARS-CoV-2 originated from a bat, and underwent homologous recombination in an intermediate mammalian host so as to acquire the capacity to infect humans.
SARS-CoV-2
Like SARS-CoV-1, SARS-CoV-2 belongs to the genera beta-coronavirus, which is part of the Orthocoronavirinae subfamily of the family Coronaviridae. The coronavirus has an envelope around the nucleocapsid that encases a single-strand of positive-sense ribonucleic acid (RNA) with about 30,000 nucleic acid bases (~30kb).
Coronaviruses that belong to the alphacoronavirus and beta-coronavirus genera infect mammals such as humans, bats, and pangolins. In fact, it is the NL63 and 229E strains of alphacoronavirus that cause croup and common cold in humans. However, unlike alphacoronavirus which causes non-lethal infection, beta-coronavirus causes lethal respiratory infections, and the coronavirus strains responsible for SARS and Middle-East Respiratory Syndrome (MERS) belong to the beta-coronavirus genus.
There are 2 sub-strains of the strain SARS-CoV-2 that have been identified as responsible for causing the Coronavirus Disease of 2019 (COVID-19). These sub-strains have been designated as the S-type and the L-type, and they are explained below:
- S-type SARS-CoV-2 accounts for about 30% of SARS-CoV-2 infections and causes mild COVID-19 symptoms. It is suspected to be the original sub-strain that caused the initial infection following a suspected zoonotic transmission of this virus from a mammalian host to a human being in Wuhan city in Hebei province in China.
- L-type SARS-CoV-2 accounts for 70% of infections and causes more severe symptoms and is more aggressive than the S-type. The S-type sub-strain evolved into the L-type sub-strain, and this allowed for human-to-human transmission. Moreover, the S-type SARS-CoV-2 usually lacks the furin cleavage sequence, which is normally found in the L-type SARS-CoV-2. The furin cleavage sequence is discussed later.
Viral Structure
As mentioned above, SARS-CoV-2 is a positive-sense, single-stranded RNA (ssRNA) virus encased inside a nucleocapsid that is then enveloped. The nucleocapsid and envelop are made of proteins known as structural proteins because they house the viral genome and give the virus its volumetric shape. This enveloped virus comes with a lipid bilayer envelop that holds unique proteins called envelope (E) proteins. The virus needs this envelope to remain viable.
This lipid bilayer can be dissolved by a lipophilic substance that breaks up the lipophilic chains in the envelope. Soap is an amphiphilic compound that contains both lipophilic and hydrophilic qualities, and it can therefore disintegrate the SARS-CoV-2 envelop, as well as allow the disintegrated virus to be washed away by soapy water. It is for this reason that people are advised to wash their hands with soap and water so as to remove the virus from their hands. If soap is not available, another lipophilic compound that can be used is alcohol. Usually, this alcohol is dissolved in water and combined with gel, principally glycerin, to form alcohol-gel that is popularly known as hand sanitizer. The gel increases the viscosity of the alcohol and reduces the rate of alcohol evaporation, therefore, increasing the duration that the alcohol acts on SARS-CoV-2 viruses on the hand and killing them.
SARS-CoV-2 has 5 structural proteins, and they are the dimer of Hemagglutinin Esterase (HE), Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) proteins. The structural proteins principally involved in infection are the S and HE proteins. The presence of HE proteins which are normally found in the influenza C virus provides a novel target for managing SARS-CoV-2 infection by using anti-influenza antibodies. There is a need for studies to find out if antibodies against HE in influenza C can be used to reduce SARS-CoV-2 infection in exposed persons. Also, there is a need to recall that the Influenza C virus causes a severe form of flu – as compared to Influenza A and Influenza B viruses – which can cause epidemics; and the role of HE protein in increasing the infectivity of a virus in humans should be considered.
The envelope encloses the virion, which is the virus particle. The N protein encloses the RNA to form a virion. This virion can only survive if it is encased in the envelope. The structural protein considered to play the most significant role in SARS-CoV-2 infectivity is not the HE protein, but it is the S protein.
The Spike (S) protein is a transmembrane glycoprotein that is the primary target of antibodies produced against SARS-CoV-2, and this reveals that it is involved in the infection process. This S protein is a trimeric glycoprotein that protrudes through the envelope, and this allows it to be attached to the host cell. Expectedly, the types of cells that this S protein can be attached to determine which cells can be infected by the virus, and this choice of cells is called host tropism.
The S protein has 2 functional subunits, the S1 and S2 subunits. S1 subunit binds the S protein to a specific protein on the host cell called the receptor. After the S1 subunit binds to its receptor, the S2 subunit causes the viral membrane and host cell membrane to fuse, and this leads to the viral RNA being injected into the host cell as part of an endosome. The receptor protein for SARS-CoV is known to be the enzyme, angiotensin-converting enzyme 2 (ACE2), which is expressed in cells found in the arteries, lungs, heart, kidney, ileum, and bladder.
Normally, when ACE2 binds to its physiological ligand, the vasoconstrictor angiotensin II, it converts it into the vasodilatory heptapeptide, angiotensin I (1-7), which causes the smooth muscles in arteries to relax, which results in dilation of the arteries. As expected, ACE2 is richly expressed in arteries. Also, ACE2 is richly expressed in lung epithelial cells, principally the Type II pneumocyte.
What is COVID-19 – an Infection or a Disease?
The structure of SARS-CoV-2 virus has been described above, and it is known that the S and HE proteins allow the virus to attach to healthy human cells, and then the viral and host cell membranes are fused and the virus is absorbed into the cell. In the cell, this virus reproduces and the new viruses exit the cell to go infect other healthy cells. The initial phase of viral entry into cells and reproduction inside cells is called an infection, and if the cell is damaged in the process, then a disease is said to have occurred. This shows that disease describes the pathophysiological processes that occur subsequent to cell damage. So, if SARS-CoV-2 has entered a cell but has not damaged that cell, then no disease has occurred and the patient is normally asymptomatic.
SARS-CoV-2 causes an infection, and the resulting cell damage initiates a number of pathophysiologic processes that manifest as diseases. The group of diseases caused by SARS-CoV-2 was collectively designated by the World Health Organization (WHO) as Coronavirus Disease of 2019 (COVID-19).
As mentioned earlier, SARS-CoV was identified as the cause of acute respiratory distress syndrome (ARDS) that ravaged the world in 2002-2003. Its 79% genome homology with SARS-CoV-2 means that SARS-CoV-2 can cause ARDS and other respiratory diseases that can lead to respiratory failure. This has been proved as severe COVID-19 is associated with respiratory failure and ARDS. In the critical phase of COVID-19, there is multi-organ failure associated with septic shock.
The morbidity and mortality figures for COVID-19 are being tracked, collected, and collated by the research collective, Center for Systems Science and Engineering (CSSE), which is based at John Hopkins University. Even so, why does this pandemic exist in the first place? This pandemic exists because there is no definitive targeted therapy. Currently, supportive management forms the mainstay of COVID-19 management.
As described earlier, infection and disease are two different concepts. Even so, there are risk factors that cause an infection to develop into an aggressive disease. Univariable analysis done by Zhou et al (2020) showed that hypertension, coronary artery disease (CAD), and diabetes mellitus (DM) are risk factors for patients infected with SARS-CoV-2 developing severe COVID-19 symptoms.
Forms of COVID-19
It was earlier mentioned that the patients who were diagnosed with COVID-19 presented with a wide range of symptoms. Heterogenous symptomatology allows for differentiation of COVID-19 into five (5) forms: asymptomatic, mild, moderate, severe, and critical COVID-19. These forms are described below:
- Asymptomatic Form: The patient has a positive COVID-19 nucleic acid test but does not present with any symptoms and chest radio-imaging shows a normal lung. This indicates that infection has occurred but very few cells have been damaged.
- Mild Form: It manifests with fever, cough, sore throat, loss of smell (dysgeusia), sneezing, and runny nose, all of which are symptoms of acute upper respiratory tract infection (URTI). Other symptoms are fatigue, nausea, myalgia, diarrhea, vomiting, and abdominal pain.
- Moderate Form: There is pneumonia without hypoxemia, and chest radio-imaging – in particular, computed tomography (CT) scanning – reveals pulmonary lesions.
- Severe Form: The pneumonia is associated with hypoxemia (which is low arterial oxygen saturation (SpO2), that is [i.e] SpO2 levels of less than 92%). Another interesting observation is that patients with severe COVID-19 have prolonged thrombin time, and high levels of fibrinogen and d-dimer.
- Critical Form: There is ARDS associated with encephalopathy, acute kidney injury (AKI), coagulation dysfunction, myocardial infarction, shock, and heart failure.
An Idea about Infection Control
The initial zoonotic transmission created the index case (the original patient) who then infected other people, thus confirming that human-to-human transmission had been established. Our thinking is that a genetic mutation that affected the expression of surface proteins played a key role in ensuring that the virus that had entered the human body as a result of a zoonotic infection was now capable of expressing surface proteins and envelope proteins that allowed it to be transmitted between humans in an aerosolized form. If this mutation occurred, then it is possible that another mutation can increase viral infectivity following human-to-human transmission, and this has happened as attested by the emergence of the Omicron and Delta variants of SARS-CoV-2. Moreover, mutations do play a role in establishing drug resistance.
Equally, these mutations show that the SARS-CoV-2 virus is undergoing homologous recombination inside the human body. Therefore if a genetically-engineered SARS-CoV-2 RNA – whose genome has been tweaked to encode defective HE and S proteins (or even encoding antiviral proteins or simply missing genes for the S protein) – is introduced into the body of an infected person, then the pathogenic SARS-CoV-2 virus can exchange genetic material with this bioengineered SARS-CoV-2 RNA. This exchange can happen via homologous recombination, which would allow for the new viruses formed to be non-infective or non-pathogenic. This homologous recombination can cause a loss of function mutation in the next generation of SARS-CoV-2 virus, and if the sub-strain affected is the L-type SARS-CoV-2, then the severity of COVID-19 can be reduced and disease progression suppressed. The bioengineered SARS-CoV-2 RNA can be introduced into the body of an infected patient through viral nanoparticles.
As mentioned earlier, SARS-CoV-2 has proteins that allow it to attach to cells in the respiratory tract and alveoli of humans. Relatedly, this implies that targeting these receptors in human cells can prevent infection, and because the main implicated receptor for the S protein is the angiotensin-converting enzyme-2 (ACE-2) protein expressed on cells, then ACE-2 blockers can inhibit infection.
How SARS-CoV-2 infection occurs is described below.
SARS-CoV-2 Lifecycle – From Infection to Cell Damage
SARS-CoV-2 gains entry into the body when a person inhales a droplet containing the virus. This is the direct route of infection because inhalation allows the virus to reach the alveolus. This virus can also adhere to fomites such as countertop surfaces or staircase rails hence the need for surface disinfection to prevent uninfected people from picking up the virus with their hands when they touch the contaminated fomites. Relatedly, hand washing is recommended as one of the ways to disrupt fomite transmission.
The principal cell infected by SARS-CoV-2 in the alveolus is the Type II pneumocyte (also called Type 2 alveolar cell), and this lifecycle focuses on infection of this alveolar cell.
The lifecycle of SARS-CoV-2 has 6 phases which are described below:
- Attachment: The HE protein and the S1 subunit of the S protein of SARS-CoV-2 binds to the ACE2 trans-membrane protein on the cell. This binding causes the S protein to undergo protease cleavage at the S1/S2 cleavage site which breaks the covalent bonds between the 2 subunits of the S proteins, but still ensures that the two subunits remain bounded together non-covalently. This allows the cleavage to cause a conformational change in the S protein.
- Penetration: S1/S2 cleavage primes S2 subunit for further cleavage. This subsequent cleavage activates the S2 subunit, which results in a conformational change that enables this subunit to fuse the viral membrane to the host cell membrane. Evidently, the S1 subunit can be described as the receptor-binding domain, while the S2 subunit can be described as the membrane-fusion domain.
An interesting fact is that furin – a protease enzyme that is endogenously produced in the human body – can cause S1/S2 cleavage that exposes the fusion peptides in the S2 subunit, thus allowing them to fuse the viral membrane to the cell membrane, hence permitting virus entry into the host cell. This means that furin facilitates the entry of the virus into the host cell by promoting the activation of the S2 subunit. Interestingly, furin cannot cleave the S-protein subunits in the MERS-like CoV that was isolated from bats in Uganda, and as expected, this CoV could bind to the human cell but could not enter it, hence cell attachment could occur but penetration could not happen, and thus the virus did not cause respiratory disease. This reveals that cleavage of S1/S2 is needed for viral penetration and initiation of the disease process, and the absence of amino acid sequences that can be cleaved by furin creates a barrier to zoonotic transmission of coronavirus disease. Expectedly, the presence of furin cleavage sequence in the S1/S2 cleavage site allows for zoonotic transmission of CoV, and because SARS-CoV-2 has this sequence, then it can be transmitted from non-primate mammals to humans.
Relatedly, the absence of the furin cleavage sequence in some CoV strains shows that this sequence was acquired by infectious strains of CoV, such as SARS-CoV-2. This acquisition of a new sequence of amino acids can occur through genetic mutation, particularly an insertion mutation where new genes are introduced to the viral RNA genome, or through genetic exchange via homologous recombination where two (2) viruses existing in the same host exchange gene materials which are then integrated into the genomes of the viruses. According to Synowl.com, an insertion mutation is the most likely cause for the introduction of the furin cleavage site that allowed the bat CoV to acquire the capability to cause human diseases. This mutation that causes a bat CoV to jump across species and infect humans can be described as a gain of function mutation.
Another host cell protein that primes the S protein for S1/S2 cleavage is the transmembrane serine protease, Type II, which is also referred to as Transmembrane protease, serine 2 (TMPRSS2) because it is encoded by TMPRSS2 gene.
- Uncoating: The fusing of the host cell membrane with the viral envelope allows the cell membrane to enclose the virus in a vesicle that buds off the plasma membrane and enter the cell. This process is called endocytosis and the vesicle is called an endosome. Inside the endosome, the pH is low and there are proteases that break down the structural proteins to release the nucleocapsid. These proteases break down the envelope, while the low pH allows the envelope and endosomal membrane to fuse, and then inject the nucleocapsid into the cytoplasm. Inside the cytoplasm, the nucleocapsid is broken down by cytoplasmic proteases to release the ssRNA of SARS-CoV-2. This process is called uncoating.
Relatedly, if the pH inside the endosome is raised, then the envelope and the endosomal membrane will not fuse, and proteases inside the endosome will have enough time to degrade the envelope and nucleocapsid proteins, and then degrade the ssRNA hence killing SARS-CoV-2 inside the endosome. The antimalarial drug, hydroxychloroquine, can enter the infected cell and get into the endosome and raise the endosomal pH hence killing some of the SARS-CoV-2 that have infected the cell. Nonetheless, a study quoted in 2020 by Dr.Justin Meyer of the University of California, San Diego Campus, showed that Hydroxychloroquine did not reduce the mortality rates of infected patients if it was used alone to manage SARS-CoV-2 infection.
- Biosynthesis: In the cytoplasm, the ssRNA binds to ribosomes. Because this single-strand RNA is positive-sense, it serves as a messenger RNA (mRNA) that the ribosomes can decode and translate into amino-acid sequences that form polypeptides which are then folded into viral proteins. Ribosomes can exist as free ribosomes in the cytoplasm, or they can be attached to the endoplasmic reticulum to form the rough endoplasmic reticulum (RER).
One of the proteins produced is an important enzyme called RNA-dependent RNA polymerase (RdRNA or simply RNA Replicase), which is used in the production of viral RNA. This enzyme is not found in the host cell and it must be present for viral RNA to be formed. Moreover, this enzyme lacks a proofreading function and this allows for high error rates during RNA replication, which results in high mutation rates. This replicase allows for the synthesis of a new negative-sense RNA (nsRNA) from the ssRNA, and then uses this nsRNA to create a new positive-sense RNA. Because the ssRNA and replicase are not consumed, then nucleotides in the host cell can be used to produce many new positive-sense ssRNAs. There are also interesting findings associated with the Gorbelyna group which indicated that viral synthesis is related to the encoding of 3’ to 5’ exoribonucleases that help proofread some of the errors of the RdRNA.
Therefore, during biosynthesis, viral proteins and new positive-sense ssRNAs are produced. Even so, if the activity of replicase is inhibited, then no new viral RNAs are produced and new virions cannot form in the cell. There are 2 drugs that can inhibit the activities of RNA-dependent RNA polymerase, and they are alovudine and remdesivir. Even so, alovudine has a poor safety profile due to its high toxicity, and its isotopically-enriched compound, Fluorothymidine F-18 cannot be used in managing COVID-19, which leaves remdesivir as the most promising replicase inhibitor. Another drug that inhibits replicase activity is favipiravir.
Remdesivir (GS-5734) is a prodrug that is metabolized into an alanine metabolite that is then hydrolyzed into nucleoside (GS-441524) monophosphate, which is then phosphorylated into an active nucleoside (GS-441524) triphosphate. RNA-dependent RNA polymerase takes this nucleoside (GS-441524) triphosphate and adds it into a growing RNA chain, and because it cannot be bound to another nucleotide, then the RNA chain is terminated, and an unviable viral RNA is synthesized. Therefore, remdesivir acts by causing chain termination of new RNA being produced by replicase.
- Maturation: This involves the assembly of the viral proteins around the viral RNA to form a nucleocapsid that is then covered by an envelope to form a new viable SARS-CoV-2 virus. During biosynthesis, polyproteins are produced, and these polyproteins need to be broken down by endopeptidases (proteases) to release the 5 structural proteins that were described above as the S, M, HE, N, and E proteins. If these proteases are inhibited, then the structural proteins are not generated from the cleavage of their parent polyproteins, and thus the nucleocapsid cannot form, and the (already) formed viral RNAs are left to wither and disintegrate inside the host cell. Therefore, protease inhibition prevents replication of SARS-CoV-2 inside the infected cell. The drugs that cause protease inhibition are called protease inhibitors, and they include danoprevir and lopinavir. After normal protease-mediated cleavage of polyproteins, the structural proteins and ssRNA are transported into the Golgi apparatus where they are packaged and assembled into vesicles that bud off as new viruses enveloped by the lipid bilayer of the Golgi apparatus. Then, the lipid bilayer of a vesicle containing a virus fuses with the plasma membrane and extrudes the virus out of the cell. This process is called exocytosis.
- Release: The new SARS-CoV-2 viruses are released from the host cell, usually resulting in cell lysis or cell death. This results in tissue damage. An interesting observation is that these new SARS-CoV-2 viruses are already cleaved at their S1/S2 cleavage site so that these new viruses are released with their S2 subunit (of the S protein) already activated to cause fusion of viral and cell membranes, and thus these new viruses easily infect the surrounding susceptible cells. This accounts for rapid spread of SARS-CoV-2 infection after the incubation period (when the initial biosynthesis occurred), and this explains why COVID-19 symptoms rapidly worsen after the disease shifts from Asymptomatic Form to the Mild Form. It also explains the rapid spread of human-to-human transmission of SARS-CoV-2 because the SARS-CoV-2 created inside the human body is more infective than the virus acquired from the bat, and the reason for this is that the membrane-fusion (S2) domain of the S-protein is already activated to easily cause fusion of viral and host cell membranes after the virus is attached to the host cell by the receptor-binding (S1) domain.
