Coronaviruses (CoVs) are very diverse pathogens that belong to the order Nidovirales, family Coronaviridae, and subfamily Coronavirinae. The subfamily Coronavirinae is composed of four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Of the four genera, alphacoronaviruses and betacoronaviruses more commonly infect the human body. Human coronaviruses (HCoV) affect the respiratory system. Mild HCoVs include: HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63. In 2002, a betacoronavirus was discovered that jumped species and caused a new disease, called Severe Acute Respiratory Syndrome (SARS). The disease caused pneumonia-like symptoms including fever, cough, dyspnea, and diarrhea. In 2012, another betacoronavirus was discovered that caused middle east respiratory syndrome (MERS). SARS and MERS affect the respiratory and gastrointestinal (GI) systems and are predicted to have originated from animals, specifically bats. SARS-CoV and MERS-CoV are closely related to other bat CoVs on the phylogenetic tree. Sequence analysis has revealed that these RNA viruses have high mutation rates, which may likely lead to host range expansion among phylogenetically related species. Coronaviruses are found in many animals. Wuhan Municipal Health commission in China first reported SARS-CoV-2, with the highest exposure rate in Huanan Seafood Wholesale Market where poultry, bats, snakes, and other animals are being sold. Coronavirus can affect a wide array of animals such as swine, cattle, horses, camels, cats, dogs, rodents, birds, bats, rabbits, ferrets, mink, snakes, etc.
The new SARS-CoV-2 has become the new addition to the coronavirus family. Due to mutation adaptation, the virus causes mild diseases in the reservoir host. However, upon entry into humans, SARS-CoV-2 starts a new adaptation process and causes a severe disease. Bats are known to be reservoir hosts of SARS-CoV-2 as well as other viruses such as Ebola virus. Bats and other animals serve as SARS-CoV-2 transmission vectors which are potentially transmitted to humans. The potential of animal to human and human to human transmission poses a major threat to public health.
In December 2019, a betacoronavirus infection was observed in Wuhan, China, that is predicted to have originated from bats and crossed to humans. The World Health Organization (WHO) named the novel coronavirus 2019-nCoV, also referred to as coronavirus disease 2019 (COVID-19). The virus causing COVID-19 was identified as SARSCoV-2. SARS-CoV-2 has a positive single-stranded RNA (+RNA) genome with 5’-cap and 3’-poly-A tail. CoVs have crownlike spike (S) glycoproteins on their envelope that target receptors in the human body. SARS-CoV and SARS-CoV-2 target the human angiotensin-converting enzyme 2 (ACE2) which is found in many tissue and cell types. Apart from the oral and nasal mucosa, the ACE2 receptor is also found in numerous human organs, such as bone marrow, brain, colon, kidney, liver, lung, lymph nodes, nasopharynx, skin, small intestine, spleen, stomach, and thymus. Whereas, MERS-CoV binds to dipeptidyl peptidase 4 (DPP4) found in the kidneys, lower respiratory, and gastrointestinal tract.
Similar to SARS-CoV and MERS-CoV, patients infected with SARSCoV-2 present with symptoms involving both the respiratory and gastrointestinal systems. In a study including 164 participants, 96% of patients had 3 main symptoms including fever, cough or shortness of breath. Other SARS-CoV-2 symptoms include dyspnea, headache, sore throat, diarrhea, nausea, vomiting, and abdominal discomfort. Although, not all individuals infected with coronavirus will present signs and symptoms, making them asymptomatic carriers. The first case of asymptomatic carriers of SARS-CoV-2 was observed when a family of six traveled to Wuhan; only five were infected with SARS-CoV-2. One of the family members (a 10-year-old child) did not travel to Wuhan yet had contact with infected family members. The child tested positive for SARS-CoV-2, however, was an asymptomatic carrier.
An individual’s status of the immune system is a determining factor in combating the infection; immuno-compromised patients may develop severe illness or die. SARS-CoV-2 affects immunocompromised older males.
As of December 9, 2020, there have been over 69 million confirmed positive COVID-19 cases and over 1 million deaths. Laboratory testing is an important factor in diagnosing CoVs. Diagnostic testing includes detection of the virus from nasal or oral samples using reverse transcription polymerase chain reactions (RT-PCR) or using blood samples in serology tests. RT-PCR detects nucleic acid targets for the novel coronavirus. Serology tests are not as reliable for diagnostic purposes because the development of protective antibodies in response to infection requires some time. Thus, RT-PCR using respiratory secretions is the preferred method for virus detection.
Currently no specific antiviral therapies for CoVs exist. The coronavirus evolving creates the need for vaccines and effective treatments. Current clinical trials aim to create vaccines by adapting SARS-CoV and MERS-CoV approaches, amongst other diseases. It is suggested that mortality and complications due to infection or co-infection may be reduced by providing patients with antibiotic treatments and steroids early during the infection. Moreover, early detection and treatment are crucial to prevent further spread of coronavirus in severe cases. Management of the virus includes providing supplemental oxygen and other symptom relief methods.
The outbreak of the novel coronavirus addresses the importance of preparedness for viral diseases. With no potential cures yet available for CoVs, prevention of the disease is very important to minimize the spread of the infection. Public health officials have initiated prevention practices based on previous zoonotic coronavirus outbreaks. Best practices for prevention include controlling the source of infection, good hygiene, wearing a fitted face mask, and avoiding crowds.
Isolation is recommended to further minimize contamination for those exposed to the virus. Since HCoVs mainly spread from human to human, social distancing and quarantine practices have been strongly recommended to the public. Also, remaining at least 6 feet away from another person minimizes the chance of spreading the virus through respiratory droplets when not wearing protective equipment. Good hygiene includes proper and frequent handwashing with soap and water for a minimum of twenty seconds and frequent cleaning of touched objects. Furthermore, prevention of future diseases can be put into practice by studying the evolution of the host range for CoVs. Meanwhile, it has become clear that detailed understanding of the pathogenesis of COVID-19 may provide more insight on identification of the potential targets that may hinder or prevent the infection and transmission of the virus.
Coronavirus is the cause of SARS, MERS, and the novel coronavirus 2019 (COVID-19). Coronavirus is part of the coronaviridae family which is commonly found in mammals and birds. The virus has single stranded positive RNA and is the second largest RNA genome compared to all other RNA viruses. The genome includes 5’ cap and 3’ poly A tails. Virus RNA replicase gene is adjacent to the polyprotein la and lab genes which code for proteins important for genome replication. Viral RNA encodes both structural and nonstructural proteins with different functions on the 3’ end of the viral genome. The genome and subgenome contain at least six open reading frames (ORF); the first ORF codes for nonstructural proteins. The ORF at the 3’ end encodes four main structural proteins such as spikes (S), membrane (M), envelope (E) and nucleocapsid (N) proteins. The other ORFs contain small noncoding regions which cause some of the ORFs to overlap. The end of the genome contains 5’ untranslated region (UTR), also referred to as the leader sequence, with about 210-530 nucleotides and 3’ contains about 270-500 nucleotides. Due to the genome being positive sense RNA, it is more infectious compared to viruses with negative sense RNA genome. Positive sense RNA is more infectious because the RNA genome could serve as messenger RNA (mRNA) to produce virions and could be directly translated by the host.
VIRAL ENTRY AND REPLICATION
Like other CoVs, SARS-CoV-2 utilizes its S protein to bind to host (target) cell receptors and fuse into host cell plasma membranes following priming by host cell proteases. Specifically, SARS-CoV-2 utilizes the C-terminal domain on the S1 subunit (SARS-CoV-2-CTD or otherwise referred to as SARS-CoV-2-RBD) to bind to human ACE2 receptor and applies type II transmembrane serine protease, TMPRSS2, for S protein priming and lysosomal cathepsins for cell entry activation.[20-22] A recent study discovered SARS-CoV-2 entry depends on the receptor binding domain (RBD) of the S protein to interact with ACE2 and negatively charged cellular heparan sulfate (HS), a copolymer. The HS and ACE2 binding sites are adjacent to one another and to the components of the host cell surface/extracellular matrix. Consequently, HS acts as co-receptor/co-factor for SARSCoV-2, by initially binding to the S protein’s RBD subunit S1, thus, priming the S protein by altering the structure of the RBD into an open conformation. This change allows the RBD open conformation to bind to the host cells’ ACE2 receptor.
Comparison of the SARS-CoV-2-CTD and human ACE2 complex to their homolog, SARS-CoV receptor binding domain (SARS-RBD), shows increased intermolecular interactions and a 4-fold increase in affinity towards human ACE2 by SARS-CoV-2-CTD. However, another study suggests that the overall binding strength of the SARSCoV-2 S protein is comparable to or even lower than that of the SARSCoV’s S protein. These conflicting binding affinities were explained by cryo-EM results revealing that the RBD of SARS-CoV-2 is primarily displayed in a lying-down, rather than standing-up, state, whereas the opposite is seen in SARS-CoV. This leads to an overall lower RBD accessibility, thus, explaining the comparable/lower binding strength of the SARS-CoV-2 S protein. Following the binding to human ACE2, pre-activation of the S protein by proprotein convertase (PPC), furin occurs. Subsequently, TMPRSS2 and cathepsins activate the S protein and facilitate viral entry through fusion into the host cell membrane. Viral genome is then uncoated and released into the cytoplasm where viral genome transcription protein translation will occur. First, ORF1a and ORF1b are transcribed from the genomic RNA and subsequently translated to polypeptides 1a and 1ab (pp1a and pp1ab). These proteins are cleaved by viral proteases into nonstructural proteins which form RNA-dependent RNA polymerase (RdRp). RdRp then uses the positive sense RNA genomic RNA (gRNA) to synthesize negative-sense RNA intermediates. These intermediates are then used as a template to form gRNA as well as subgenomic RNA (sgRNA). Transcriptome analysis obtained via sequencing-by-synthesis (SBS) and nanopore-based direct RNA sequencing (DRS) reveals transcription of 9 canonical sgRNAs: S, 3a, E, M, 6, 7a, 7b, 8, and N. Newly synthesized viral envelope glycoproteins are inserted into the rough endoplasmic reticulum (ER) or Golgi apparatus. Assembled viral particles then enter the ER-Golgi intermediate compartment (ERGIC) and are released out of the host cell via exocytosis.[26,27]
MEANS OF COVID-19 TRANSMISSION
There are more than 69 million cases of COVID-19 as of December 9, 2020 globally and over 1 million deaths. SARS (in 2003) and MERS (in 2012) both shared the same modes of transmission through droplets and direct contact. Respiratory infection can be transmitted through droplets. Inhaled droplets reside in the upper respiratory tract which could be removed by mucus through the nose or move up the mucociliary escalator. SARS-CoV-2 is transmitted through droplets and by direct and indirect physical contact. Direct physical contact occurs when an individual has contact with another individual who either has the infection or is a carrier, such as shaking an infected individual’s hand. Indirect physical contact occurs when touching contaminated surfaces such as doorknobs. Droplet transmission occurs when one person is close to another person that is coughing or sneezing. Then an individual may become infected if their eyes, nose, and mouth are exposed. Many believe that COVID-19 is airborne, however, the primary route of transmission is via droplets and contact. The difference between the two types of transmission is that airborne transmission contains aerosols that are much smaller than droplets, which last longer in the air than droplets. However, it is also possible for a person to be exposed to COVID-19 through airborne transmission by treatment ventilators.
Additionally, there were some cases where patients showed signs of diarrhea, vomiting and stomach pain, raising the question whether COVID-19 could be transmitted through fecal-oral transmission? Two laboratories in China successfully isolated live COVID-19 from infected patients’ stool samples, suggesting that COVID-19 may potentially be transmitted through fecal-oral transmission. Therefore, the respiratory system and potentially the digestive system play a role in transmitting the virus.
There is currently a controversy whether SAR-CoV-2 droplets can land on exposed human eyes (ocular surface) and infect the host. A male individual became infected by the virus for only wearing a mask and not eye protection during an inspection at Wuhan. A more recent study also showed that human eyes may be susceptible to SARS-CoV-2 entry. SARS-CoV-2 uses the same receptor as SARS; both use ACE2. Immunohistochemical analysis showed that post-mortem eyes samples express ACE2 and TMPRSS2 (surface protease that aids in viral entry) on the conjunctiva, limbus, and cornea, with higher expression levels on the surface of conjunctiva and corneal epithelium, suggesting that human eyes may serve as a port of entry for SAR-CoV-2 through droplets transmission.
With seven HCoVs now identified, understanding the transmission of coronaviruses is crucial. SARS-CoV-2 targets ACE2, which can be found in the lungs, kidneys, GI tract, and liver. New studies found that fecal-oral transmission of the virus is possible. Moreover, replication of the virus can happen in the respiratory and digestive system. SARSCoV and MERS-CoV can withstand various environmental conditions and encourage fecal-oral transmission. Also, traces of SARS-CoV RNA can remain in the stool more than 10 weeks, leading to the premise that SARS-CoV-2 potentially has the same characteristic. Stool analysis is recommended for diagnosing patients who present with GI symptoms. The first confirmed patient in the United States presented with GI symptoms for two days, and the nucleic acid analysis of stool and respiratory samples revealed positive results for COVID-19. Detection of viral RNA was likely due to its presence in enterocytes of the ileum and colon. It is believed that a person infected through fecal transmission, can also spread the virus through respiratory droplets or feces. Moreover, those with GI symptoms can still spread the virus after respiratory symptoms have improved. Further studies are necessary to determine the connection between fecal viral load and the presence of GI symptoms as well as disease severity.
There have been less reported cases of children of the age group of 0-17 years old affected by SARS-CoV-2 in the United States, likely attributed to the closure of schools. However, a study conducted in Utah and Wisconsin on children infected with SARS-CoV-2 and their household contacts, showed the transmission patterns in the household and explained how children acquire SARS-CoV-2 by exposure to household contacts. Children have shown a high potential of being asymptomatic carriers of SARS-CoV-2, with a notable 16% being asymptomatic, which may play a role in transmitting the virus to others including their household.
PREVENTION OF COVID-19
Since the virus is spreading rapidly across the globe, specific measures are recommended by the US Center of Disease Control and Prevention (CDC). Virus spreads through droplets and through contact routes; it is suggested that people should stay six feet apart to avoid infecting others through droplet transmission. COVID-19 could spread via asymptomatic carriers. To prevent viral spread, individuals should have healthy hygiene by frequently washing their hands with soap and water for at least 20 seconds and use hand sanitizer. A person should frequently wash their hands in their homes and out in public places if they blow their nose, cough, or sneeze. People should also distance themselves and avoid close contact with others. Individuals should avoid crowded places and do not touch their face while conducting errands. It is advised to wear a cloth face covering (surgical face mask) in public. Cloth face coverings should not be placed on children under 2 years old and on individuals that have difficulty breathing. People should avoid using public transportation, limit traveling, and practice self-isolation to help minimize/stop the spread of the virus.
Surfaces such as doorknobs, toilets, light switches, countertops, desks, phones, keyboards, and sinks should be decontaminated by disinfectants. Globally, the practice of isolation and containment policy and whole countries' quarantines have been enforced in Italy and Iran, respectively to stop the spread of COVID-19.
VIRAL PATHOGENESIS AND PATHOLOGICAL FEATURES OF COVID-19
In order to enter the host cell, SARS-CoV-2 binds to HS and ACE2. While HS is ubiquitously present in all cells, ACE2 is abundant in lung and small intestine epithelia.[41,42] Combined with the virus's route of transmission, this would confer greater susceptibility of the pulmonary system, explaining why the lungs can be considered as the classical target organ in the setting of SARS-CoV-2 infection.
ACE2 is present in many human organs and tissues, including the brain, lungs, liver, kidneys, spleen, heart, blood vessels, GI tract, skin, as well as arterial and venous endothelium,[42,43] leading to the possibility of multiple organs and tissues being affected. SARS-CoV-2 infection and subsequent inflammatory changes affecting endothelial cells can induce endotheliitis in several organs, which can ultimately result in cell death. Endothelial cell apoptosis and dysfunction have also been reported in COVID-19 patients. The endothelium plays a role in preventing thrombosis by acting as a surface of which prevents cell and clotting protein attachment. As a result, endothelial involvement in SARS-CoV-2 infection may increase the likelihood of thromboembolism formation.
New studies suggest thromboembolism may play a key role in mortality of COVID-19 patients. In a study, autopsies were performed on patients infected with SARS-CoV-2 in order to understand the histopathological changes. Similar to SARS and MERS, it was discovered that patients infected with SARS-CoV-2 show small vessel thrombosis with alveolar hemorrhage postmortem. Thrombosis was present in many other organ systems due to immunothrombosis. When advanced, thrombosis can lead to multiple organ failure due to disseminated intravascular coagulation (DIC). However, a very low percentage of COVID-19 patients meet the criteria for DIC. Moreover, pulmonary intravascular coagulopathy was coined to differentiate it from DIC. Pulmonary intravascular coagulopathy suggests pulmonary inflammation, vasculopathy and thrombosis. Pathological changes were observed in various organs.
COVID-19 poses a threat to those with comorbidity or immunocompromised individuals. There is a correlation between mortality and a patient's MuLBSTA score. MuLBSTA considers multilobe infiltrate, absolute lymphocyte count, bacterial coinfection, smoking history, hypertension history, and age in a scoring system. This model is used to predict mortality in viral pneumonia patients but has been used for COVID-19 patients. SARS-CoV-2 is more likely to infect those with a weak immune response.
CLINICAL SYMPTOMS OF COVID-19
In March 2003, an outbreak of SARS occurred in Hong Kong. It presented as a respiratory illness that led to death. The symptoms for SARS included fever, headache, fatigue, dry cough, and muscle aches. It was also common for individuals to have symptoms such as diarrhea and nausea. Patients eventually developed pneumonia and had to be placed under ventilation support. SARS was caused by coronavirus which infects animals and spreads to humans. MERS first occurred in 2012 in Saudi Arabia and is associated with coronavirus as well. It is known as a respiratory tract disease in which an infected patient shows symptoms of fever, cough, difficulties in breathing, and development of pneumonia.
The first cases of COVID-19 were diagnosed in Wuhan, China and patients experienced flu-like symptoms. In a study, 47 out of 99 patients who had a prior history of a chronic disease were exposed to a Wuhan seafood market. The patients commonly exhibited symptoms of pneumonia, fever, cough, and shortness of breath. Some also had muscle ache, headache, confusion, chest pain, and diarrhea at the time of COVID-19 diagnosis. In general, COVID-19 symptoms are similar to common “cold” symptoms. However, it is a severe illness that leads to death. People who are at a higher risk include those who have underlying diseases, are obese, immunocompromised, and those who are 65 years and above. After exposure to the virus, it takes 2-14 days to develop symptoms such as fever, dry cough, and shortness of breath. Drastic signs of COVID-19 that require immediate medical attention are when the patient has difficulty breathing, pain and pressure in the chest, unable to lift their body, and if the patient’s face and lips turn bluish due to the loss of oxygen.
COVID-19 has a wide variation of symptoms and many symptoms are mistaken for the “common cold.”. In the United States, COVID-19 symptoms are limited and vary among patients. Between January 14-April 14, 2020, CDC conducted a questionnaire to better evaluate the variations of symptoms in the United States. 158 out of 164 patients had three symptoms of fever, cough, and shortness of cough. Total of 57 adult patients were hospitalized and 39 of them had these three symptoms, while 25 non-hospitalized patients commonly experienced symptoms such as headache, abdominal pain, and diarrhea. Age and sex were also are taken into consideration. The results showed that the presentation of the three common symptoms of fever, cough, and shortness of cough directly correlated with the age of patient.
DIAGNOSIS OF COVID-19
Diagnosis of SARS-CoV-2 infection requires the detection of the virus in a patient-derived sample, which can be done either through real-time RT-PCR or serological assays. RT-PCR is currently the main method for diagnosis, which uses a nasopharyngeal (NP) swab obtained from the patient. The NP swab is processed and the RNA (if present) is isolated and reverse-transcribed into DNA. The DNA will undergo PCR amplification using SARS-CoV-2 specific primer. The targeted sequences consist of genes that code for the viral RNA-dependent RNA polymerase (RdRp), the viral envelope (E genes), and the viral nucleocapsid (N genes). Presence of the targeted sequences constitutes a positive result. Another assay available is a rapid antigen test, which is an option for point of care testing. Antigen tests are used to detect antigens rather than antibodies or nucleic acid. Antigen tests are used for those with an active infection. Rapid antigen tests can be used for CoV patients and will produce results within minutes. Faster results are significant to help slow the spread of the virus. However, antigen tests have a higher chance of missing an active infection and may require a follow up test using RT-PCR.
Serological assays involve detection of IgG/IgM antibodies being generated by the host in response to a SARS-CoV-2 infection. Various types of serological assays include rapid diagnostic tests (RDT), enzyme-linked immunosorbent assay (ELISA), and neutralization assays. While RT-PCR and antigen test can only detect active infections with detectable viral load, serological tests can inform whether a person has been previously infected and therefore recovered. As a result, this method can also aid in epidemiological studies in confirming the number of cases. However, serological assays are limited in their diagnostic ability as they would not be able to accurately diagnose a positive patient in the setting of an early infection. This is because the patient likely would not have developed antibodies at the time of testing. Consequently, both methods of diagnostic testing have their own advantages in this current pandemic.
POTENTIAL TREATMENT OPTIONS FOR COVID-19
COVID-19 is considered as a newly emerged disease therefore, no definite line of treatment has been established yet. As with many viral infections, medical management involves symptomatic relief and supportive care. Given that COVID-19 primarily affects the respiratory tract, supportive care consists of administration of supplemental oxygen and providing mechanical ventilatory support. To attempt combating the widespread and persisting number of emerging cases, various pharmacological agents have been brought into question while simultaneous vaccine development efforts are under scrutiny. To this end, hydroxychloroquine or chloroquine quickly became a major drug to be considered. Hydroxychloroquine is a known pharmacological agent used to treat malaria and autoimmune conditions such as rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Hydroxychloroquine is a weak base which can accumulate within lysosomes and subsequently increase lysosomal pH to ultimately interfere with lysosomal activity and autophagy. Additionally, it allows influx of zinc to the host cell which inhibits in vitro RNA-dependent RNA polymerase activity.[53-56] The compound also impacts the immune system by reducing toll-like receptor (TLR) signaling and cytokine production.
Hydroxychloroquine is a weak base, thus, the increase in intracellular pH can influence viral release from endosomes following entry into host cells. The reduction in cytokine production may also have implications in combating cytokine storms which is speculated to be the cause of the sudden and rapid declination of COVID-19 patients. However, following the release of the Emergency Use Authorization (EUA) for hydroxychloroquine/ chloroquine, clinical trials are continuing to further investigate its effects on infected patients. Results from such clinical trials prompted the Food and Drug Administration (FDA) to release a statement on April 24, 2020 regarding the drug’s safety concerns involving cardiac complications like arrhythmias and prolonged QT intervals.
On May 1, 2020, the FDA released another EUA for antiviral remdesivir following promising in vitro and clinical trials findings.[57-59] Remdesivir is an existing antiviral indicated for use against other retroviruses such as the Ebola virus and MERS-CoV. Remdesivir inhibits viral RdRp by acting as a nucleoside analogue of adenosine, resulting in chain termination during RNA synthesis. The drug also appears to have specificity towards coronaviruses; one study found similar RdRp inhibition in SARS-CoV and MERS-CoV, and selectivity over adenosine during RNA synthesis.
Several other pharmacological agents have been studied including lopinavir/ritonavir (LPVr) and ivermectin. Lopinavir is a protease inhibitor typically used for the treatment of human immunodeficiency virus (HIV). Ritonavir is included in combination therapy protocols as it increases the half-life of lopinavir by inhibiting cytochrome P450 3A, which would otherwise quickly metabolize lopinavir. While there were some suggestions of antiviral activity of lopinavir/ritonavir (LPVr) treatment against SARS-CoV-2, there are also discussions of how coronavirus proteases do not possess the targeted C2-symmetric pocket that HIV proteases have, implying potential decreased LPVr potency against CoVs. Furthermore, a trial conducted on 199 hospitalized patients with severe COVID-19 in China showed no benefits of LPVr treatment. Ivermectin is an FDA-approved anti-parasitic agent with antiviral activity against various viruses, including RNA viruses such as HIV, simian virus SV40, dengue virus, and west Nile virus. One mechanism of action includes inhibition of nuclear import of host and viral proteins and targeting IMPα/β1 cargo proteins. In SARSCoV, IMPα/β1 is involved in a signal-dependent nucleocytoplasmic shutting of the virus nucleocapsid protein during infection, thus, negatively affecting host cell division. IMPα/β1 is also sequestered by SARS-CoV accessory protein ORF6, into the rough ER/golgi membrane to antagonize STAT1 function within interferon signaling. Based on genome similarity of SARS-CoV-2 and SARS-CoV, it is hypothesized that ivermectin displays anti-SARS-CoV-2 activity. Indeed, an approximate 5000-fold reduction in viral load 48 hours post-ivermectin treatment in vitro is reported. Despite these findings, ivermectin must undergo clinical trials before any further conclusions can be made.
Steroid Therapy represents yet another potential treatment option. Patients with severe COVID-19 can develop systemic inflammatory response leading to lung injury and multisystem organ dysfunction. The potent anti-inflammatory effects of corticosteroids might prevent or mitigate these deleterious effects. Randomized Evaluation of COVID-19 Therapy (RECOVERY) trial included 2,104 people hospitalized for the illness who were randomly assigned to receive the common corticosteroid drug dexamethasone showed that dexamethasone (commonly used to treat rheumatoid arthritis and other inflammatory conditions) reduced deaths by 30% among patients on ventilators and by 20% among those receiving oxygen therapy alone. It had no benefit for patients who did not need breathing support.
Cellular HS (along with ACE2) acts as SARS-CoV-2 co-receptor for successful infection. This prompted targeting S protein-HS complex and use of exogenous heparin (as an inhibitory agent) as potentially effective therapeutic approaches against the infection and prevention of SARS-CoV-2. Heparin along with heparin lyases, non-anticoagulant heparin, unfractionated heparin (UFH), and human lung/tissue HS are potential novel therapeutic targets by strongly blocking S protein RBD from binding to ACE2 receptor, ultimately preventing viral infection. The positively charged amino acid residues located in the RBD subdomain create a docking site allowing heparin/HS to bind to the RBD of SARS-CoV-2 S protein. Thus, heparin and non-anticoagulant derivatives strongly block S protein binding and viral infection induced by the pseudotyped virus as well as live SARS-CoV-2 virus. Figure 1 depicts currently available various treatment options for SARS-CoV-2 infected individuals.
IMMUNE RESPONSE AND VACCINE DEVELOPMENT
Similar to other infectious diseases, innate and adaptive immune responses should be effectively employed to combat SARS-CoV-2. In the setting of SARS-CoV-2 infection, innate immune system recognizes viral genome as pathogen-associated molecular patterns (PAMPs) by endosomal RNA receptors, TLR8 (toll-like receptor), TLR7, and RIG/ MDA5 (retinoid-inducible gene/melanoma differentiation-associated gene). This recognition further activates other signaling pathways and transcription factors, such as interferon regulatory transcription factor 3 and 7 (IRF3 and IRF7) to encode for proinflammatory cytokines and chemokines. IRF3 and IRF7 stimulate the production of type-1 interferons (IFN-1) to suppress viral replication, however, this is not observed with COVID-19.[62-65] Severe cases of COVID-19 patients with pneumonia or acute respiratory distress syndrome (ARDS) are reported due to dysregulated immune response driving inflammation and cytokine release syndrome (CRS).
The chemotactic molecule CCL5 (chemokine C-C motif ligand 5) is up-regulated in SARS-CoV infected epithelial cells and macrophages, mediating robust inflammatory responses.[67,68] Therefore, disruption of CCL5-CCR5 (chemokine C-C motif receptor 5) pathway could prevent and reverse the effects of CRS. CCR5-specific human IgG4 monoclonal antibody (mAb), leronlimab, competitively binds to CCR5, preventing CCL5-induced activation and ultimately lymphocyte chemotaxis involved in CRS. Treatment with leronlimab for critical COVID-19 patients, through an FDA-approved emergency investigational new drug request, reduces interleukin-6 (IL-6) levels, normalizes CD4/CD8 cell ratio, and lowers SARS-CoV-2 viral load, suggesting potential anti- SARS-CoV-2 activity.
The humoral immune response plays a role in combating infection by producing IgG and IgM antibodies, as well as neutralizing antibodies. Convalescent plasma extracted from COVID-19 patients both inhibits the infection of SARS-CoV-2 and relieves symptoms of infected patients, indicating the efficacy of neutralizing monoclonal antibodies (mAbs). SARS-CoV-2 relies on the S glycoprotein to penetrate host cells, however, the receptor binding domain (RBD) of the S glycoprotein initiates the binding of the S protein to the ACE2 receptor, causing the conformational change in subunit S2 and the development of a six helical-bundle. As a result, the viral and host cell membranes fuse together. Thus, developing a potent neutralizing human mAbs such as fragment antigen binding (Fab), single-chain variable fragment (scFv), and heavy chain variable domain (VH) can be considered as potential therapeutic modalities. Recently, the IgG1 VH domain was shown as a promising candidate due to its small size (15 kDa), high stability and affinity to antigens. The ab8 region of human IgG1 crystallizable fragment (Fc) exhibits strong neutralizing activity and specificity against the virus in vitro and in animal models. The VH ab8 was fused to the human IgG1 Fc resulting in the formation of a bivalent antibody (VH-Fc ab8) which extended VH ab8 in vivo half-life and increased its avidity. This allowed VH-Fc ab8 to effectively surpass ACE2-Fc for binding to RBD due to its enhanced binding properties. VH-Fc ab8 drastically reduced the presence of the virus in ACE2-adapted SARSCoV-2 mouse and hamster models by 10-folds at a dose of 2 mg/kg. Additionally, viral load in the lungs was alleviated which reduced the occurrence of pneumonia and notably decreased the upper airway shedding, resulting in reduced SARS-CoV-2 transmission. VH-Fc ab8 is also advantageous due to its small size (80 kDa), allowing the treatment to require smaller quantities of VH-Fc ab8 to produce a better outcome compared to IgG1 which is twice the size of VH-Fc ab8.
The size of the bivalent antibody is crucial for diffusion through different tissues, thus, allowing VH-Fc ab8 to diffuse through the peritoneal cavity to the blood and lungs quicker than IgG1 ab1. Although 2-3 mg/kg of VH-Fc ab8 was used to treat the small animal models, human subjects may require a higher dosage to produce a similar outcome. Considering VH-Fc ab8’s potent specificity and consequential neutralizing activity due to its bivalency, VH-Fc ab8 would bind to the S trimer ectodomain consequently overlapping the area of which ACE2 would typically bind to the S glycoproteins. Taken together, VH-Fc ab8 potently neutralizes SARS-CoV-2 in vitro and in vivo while outcompeting ACE2 binding to RBD.
The development of a vaccine against SARS-CoV-2 is imperative in preventing further person-to-person spread, especially considering the lack of an established, standardized treatment regimen. Vaccines utilize humoral immunity to produce neutralizing antibodies to limit the current infection and/or prevent re-infections. Various types of vaccines can be utilized, including inactivated vaccines, live-attenuated vaccines, S protein-based vaccines, vectored vaccines, nucleic acid vaccines.[68,70] Inactivated vaccine for SARS-CoV was deemed safe and well-tolerated in phase I clinical testing in 2007, thus, a similar approach can be taken for SARS-CoV-2. Inactivated vaccines can elicit antibody-dependent enhancement (ADE) in addition to the production of neutralizing antibodies, however, the mechanisms of ADE and its effect on immunity should be further scrutinized. Live-attenuated vaccines involve the incorporation of mutations to construct a less virulent strain of the virus. The major concern about using this approach is the possibility of transmission of the mutated strain from vaccinated individuals, which may encounter the wild-type strain and recombination events might occur. To combat this caveat, solutions such as the construction of a recombination-resistant genome may be explored.
S protein-based vaccines involve targeting the S glycoprotein, present on viral envelope which facilitates host cell entry, to induce an ADE response. Adenovirus type-5 (Ad5)-based vaccine targeting the S glycoprotein is well- tolerated and immunogenic 28 days-post vaccination, suggesting the need for further evaluation and testing[74-76]
Nucleic acid vaccines consist of DNA vaccines and mRNA vaccines. The U.S. National Institute of Allergy and Infectious Disease (NIAID) in collaboration with biotech company Moderna Therapeutics is conducting a phase I clinical trial for an mRNA vaccine. This mRNA vaccine, also known as the mRNA-1273 vaccine, encodes the S-2P antigen, which is comprised of SARS-CoV-2 glycoprotein with a transmembrane anchor and an S1-S2 cleavage site. The mRNA-1273 vaccine is constructed with modified nucleotides to prevent early intracellular activation of interferon-associated genes and is delivered with a lipid nanoparticle capsule for increased potency. BioNTech and Pfizer collaborated to construct their own mRNA vaccine like the Moderna vaccine. This mRNA vaccine (referred to as BNT162b1) consists of a nucleoside modified mRNA transcript encoding the receptor-binding domain of SARS-CoV-2 S-protein fused to a T4 fibritin-derived ‘foldon’ trimerization domain for increased immunogenicity. The mRNA transcript is encapsulated by lipid nanoparticles similar to Moderna’s approach.[77,78] These two vaccines greatly differ in their storage conditions, raising concerns regarding distribution for usage.
Moderna’s vaccine can be shipped and stored long-term at -20°C for 6 months and will remain stable for up to 30 days at standard refrigerator temperatures of 2-8°C. On the other hand, Pfizer requires much lower temperatures, recommending storage at -70°C (±10°C) for up to 6 months and can remain stable at 2-8°C for 5 days.
However, to aid transport in the extremely low temperature required, Pfizer constructed their own thermal shipper (with a GPS-enabled thermal sensor for constant location and temperature monitoring) that can maintain the required -70°C for up to 10 days if unopened. If an ultra-low-temperature freezer is unavailable to reach the maximum 6 months long-term storage time, this thermal shipper can be used for another 20 days by refilling it with dry ice every 5 days. The reason behind the storage temperature discrepancies between the two mRNA vaccine candidates is unclear. However, Moderna’s spokesperson attributed their higher storage temperature (in relation to Pfizer) to their own experience with creating mRNA vaccines.
Similarly, DNA vaccines face their unique circumstances. Administration of DNA vaccines can be accomplished via four routes: intramuscular (IM) injections, gene-gun delivery, electroporation delivery, and a needle-free delivery (as seen for the Ebola virus and Lassa Fever virus). These methods, however, have insufficient efficacy. IM injections result in accumulation of the vaccine in the intracellular space, with minute amounts being ultimately translated into protein. Genegun and electroporation delivery methods have higher accompanying costs and are reportedly painful processes. Finally, the needle-free delivery is still undergoing investigation. This method has comparable immunological effects to delivery via gene-gun and electroporation while also being a low-cost option, therefore, can be further explored for delivery of DNA vaccines. An example of this delivery method is seen by using a needle-free injection device, Bioinjector® 2000™, which has shown increased immune response compared with delivery via needle and syringe.
Throughout the recent history, pandemics due to members of the Coronaviridae family (MERS, SARS, and now SARS-CoV-2) have had major impacts on the global health/population. The genome of coronavirus is large, positive, single-stranded RNA. To enter the host cell, SARS-CoV-2, like closely related SARS-CoV, binds to hACE2 receptors via its S protein. Proprotein convertase, furin, then pre-activates the S protein, and then TMPRSS2 and cathepsins activate the S protein to further facilitate viral entry and membrane fusion. The virus then replicates in the cytoplasm and exits the host cell via budding. The novel coronavirus is transmitted through droplets, direct and indirect physical contact. Furthermore, COVID-19 may be aerosolized and possibly transmitted along with fecal-oral transmission (pending more testing to confirm). Infected patients with COVID-19 show symptoms such as fever, cough, and shortness of breath which are similar to the symptoms seen in the common cold, SARS, and MERS. Real-time RTPCR is the golden standard method for the diagnosis of COVID-19, while serological assays have also been released for use. In order to prevent a person from contracting the virus or further transmitting the virus, it is advised to keep a healthy hygiene, practice social distancing, and avoid non-essential traveling. These practices are implemented with the intention of reducing the spread of COVID-19 and “flattening the curve” to prevent overwhelming healthcare systems, especially considering the current limitations in treatment options. Concomitant with search for effective pharmacological agents, is development of vaccines, which hold great importance in preventing the spread of COVID-19. There are many approaches to constructing a vaccine for viruses like SARS-CoV-2, including inactivated vaccines, live-attenuated vaccines, S glycoprotein-based vaccines, vectored vaccines, and nucleic acid vaccines. Each approach has its own limitations, however. While inactivated vaccines pose less safety risks than live-attenuated vaccines, the elicited antibody-dependent enhancement effect still requires further investigation for its implications on immunity. There are existing candidates undergoing clinical trials for S glycoprotein-based vaccines and mRNA vaccines, including adenovirus type-5 (Ad5)-based vaccines and Moderna’s mRNA-1273 vaccine, respectively. Other types of nucleic acid vaccines are DNA vaccines, which require more development in a suitable delivery method. Future studies are warranted to find a suitable treatment modality for this deadly disease.