INTRODUCTION:

In late December 2019, the world woke up to a reality of pandemic of coronavirus disease which belong to the subfamily of orthocoronavirinae evoked by Severe Acute Respiratory syndrome 2 (SARS-CoV-2) and Middle east respiratory syndrome (MERS). This outbreak came to light when China informed the World Health Organisation (WHO) a cluster of cases of pneumonia of an unknown cause in Wuhan City in Hubei Province. Coronavirus are a group of RNA virus that cause disease in mammals and birds. In humans, these viruses cause respiratory tract infection that can range from mild to lethal. The orthocoronavirinae consist of 4 genera namely alpha coronavirus (α - CoV), Beta coronavirus (β - CoV), Gama coronavirus (γ - CoV) and delta coronavirus (δ - CoV). Both α and β CoV can infect mammals while δ and γ CoV are known to infect birds.

Human coronavirus was first characterised in 1960, are responsible for substantial proportion of upper respiratory tract infections in children. Since 2003, at least 5 new coronaviruses have been identified including severe acute respiratory syndrome coronavirus. The SARS emerged in animal coronavirus from Southern China and spread throughout the world very rapidly. During 2002-2003 outbreak SARS infection was reported in 29 countries in Northern America, South America, Europe and Asia. More than 8000 infected individuals were identified with 774 SARS related facilities^1,2. A small outbreak in 2004 involved only four more cases. MERS started in Saudi Arabia in 2012, killing nearly 858 people. According to experts the SARS-CoV which belongs to the β coronavirus genus originated in bats. SARS-CoV-2 made jump to humans at one of Wuhan’s open air wet market and can be transmitted from one human to another. As of now the 2019 novel coronavirus (2019 nCoV) has spread rapidly over 72 countries causing more than 90,000 deaths. Taking into consideration the global threat the WHO has declared COVID-19 a pandemic and public health emergency on March 11, 2020. Moreover, there are no vaccines against SARS-CoV-2 or no specific drugs to treat this virus. In this analysis we condense recent progress of SARS-CoV-2 and lay out a setup for prevention and treatment of 2019 novel coronavirus or COVID-19^3,4,5.

Structure of COVID-19:

The coronavirus spike protein is a multi-functional molecular machine that mediates coronavirus into the host cells. The SARS-CoV-2 genome is 30kb in size. It conceals a large, non-structural polyprotein which is further cleaved to generate 15/16 proteins, 4 structural protein and 5 accessory proteins (ORF3a, ORF6, ORF7, ORF8 and ORF9). The RNA is the largest known viral RNA and codes for large polyprotein. Moreover, the cleavage of the polyprotein by viral encoded protease to form

1. An RNA dependent RNA polymerase and an ATPase helicase.

2. A surface hemaglutinin-esterase protein present in OC43 and several other group II Coronavirus.

3. A large surface glycoprotein (S protein) which consist of S1 and S2 sub units that forms petal-shaped surface projections.

4. A small envelope protein (E protein)

5. A membrane glycoprotein (M protein)

6. A nucleocapsid protein (N protein) that forms complex with RNA.

The coronavirus surface spikes are homotrimers of S-proteins. The S1 subunit forms the head to the spike and has receptor binding domain. The S2 subunit forms the stem. It forms the stem which anchors the spike in the viral envelope and on the protease, activation enables fusion. The E and M protein are important in maintaining the structure. Inside the envelope there is a nucleocapsid which is formed by the multiple copies of nucleocapsid (N) protein. The lipid bilayer envelope, membrane protein and the nucleocapsid protect the virus when it is outside the host cells. Mutations are common in nature. In addition, coronavirus are capable of genetic combination of two viruses infect the same cell at the same time^2,4,5,6.

Etiology and Pathogenesis of SARS-CoV-2:

The SARS-CoV-2 is the seventh member of the coronavirus family that contaminate human. The emergence of SARS-CoV-2 has been marked as the 3^rd introduction of the highly pathogenic coronavirus after SARS-CoV and MERS-CoV in 21^st century. Patients with COVID-19 show clinical manifestations including fever, non-productive cough, dyspnoea, fatigue, normal or decreased leukocyte count and radiographic evidence pneumonia. Several groups of scientists in China have discovered that SARS-CoV-2 just like SARS-CoV require angiotensin-converting enzyme -2 (ACE-2) as receptor to enter the cells. The binding of the virus with the host cell receptors is a significant determinant for pathogenesis of infection. Dipeptidyl peptidase 4 (DPP4) was identified a functional receptor for MERS-CoV, because the receptor binding S1 domain of MERS-CoV spike protein was copurified with DPP4 specifically from lysates. Coronavirus S-protein has been reported as a significant determinant of the virus entry into the host cells. The envelope spike glycoprotein binds its cellular receptor ACE2 for SARS-CoV-2 and SARS-CoV, CD209L (a c-type lectin) for SARS-CoV and DPP4 for MERS-CoV. The entry of the virus into the cell initially identified to be accomplished by direct membrane fusion between virus and the plasma membrane. After the virus enters the cell, the viral RNA genome is released into the cytoplasm and is translated into two polyprotein and structural proteins after which the viral genome begins to replicate. The newly formed envelope glycoproteins are inserted into the membrane of the endoplasmic reticulum or Golgi and the nucleocapsid is formed by the combination of the genomic RNA and nucleocapsid protein^5,7,8. Then the viral particles germinate into the endoplasmic reticulum–Golgi intermediate (ERGIG). Lastly, the vesicles containing the virus particles fuse with plasma membrane to release virus by exocytosis. A report in Lancet shows Acute Respiratory Distress Syndrome (ARDS) is the main cause of death of COVID-19. Out of the 41 SARS-CoV-2 infected patients admitted in early stages of outbreak, 6 died of ARDS. ARDS is the common immunopathological events for SARS-CoV-2, SARS-CoV and MERS-CoV infections. One of the main mechanisms of the ARDS id the cytokine storm, the deadly uncontrolled systemic inflammatory response resulting from the release of large amount of pro inflammatory cytokines (IFN- α, IFN-γ, IL-1β, IL-6, TNF- α, TGF- β etc.) and chemokine (CCL2, CCL3, CCL5, CXCL9 etc.) by immune effector cells on SARS-CoV infection. This cytokine storm will trigger a violent attack by immune system to the body cause ARDS and multiple organ failure and finally lead to death in severe cases of SARS-CoV-2 infection just like SARS-CoV and MERS- CoV infection.

Immune response to coronavirus infection:

A host innate system detects viral infections by using pattern recognition receptors (PRRs) to recognize pathogen associated molecular patterns (PAMPs). PAMPs recognised by toll receptors (TLRs) include lipids, lipoproteins, proteins and nucleic acid of bacterial, viral, parasite and fungal origin. The recognition of PMAPs by TLRs also occurs in cell membrane, endosomes, lysosomes and endo cytolysomes etc. Moreover, different TLRs can induce different biological responses via subsequent activation of varied adapter proteins such as MyD88, TIRAP, TRIP AND TRAM and these proteins all share the Toll/ Interlukin-1-receptor structure. The function of TRAM and TIRAP is to recruit TRIF molecules to TLR4 receptor and MyD88 to TLR2 and TLRL4 receptor. Therefore, TLR signalling pathways are classified as MyD88 dependent pathway, which functions to activates immune – inflammatory factors and TRIF dependent pathway, which functions to activate type I interferon and inflammatory pathways^7,8,9,10.

Antigen presentation subsequently stimulates the body’s humoral and cellular immunitywhich are mediated by virus specific Band T cells. Similar to common acute viral infections the antibody profile against SARS-CoV virus has a typical pattern of IgM and IgG production. Comparing the humoral responses there are more researches on the cellular immunity of coronavirus. The latest report shows that the number of CD4+ and CD8+T cells in the peripheral blood of SARS-CoV-2 infected patients is significantly reduced, whereas its status is excessive activation has evidenced high proportions of HLA-DR (CD4 3.47%) and CD38 (CD8 39.4%) double positive fractions. CD4+T cells promote production of virus specific antibodies by activating T dependent B cells. However, CD8+T cells are cytotoxic and can kill viral infected cells. CD8+T cells account for about 80% of total infiltrative inflammatory cells in the pulmonary interstitium in SARS-CoV infected patients and play a vital role in clearing CoVs in infected cells and inducing immune injury. Moreover, CD4+T cells are more susceptible to MERS-CoV infection. MERS-CoV induces T-cell apoptosis by activating the intrinsic and extrinsic pathways. The reappearance of SARS-CoV is still a significant problem. SARS-CoV specific T-cells have been screened as SARS convalescent patients. All detected memory T-cells responses are directed at SARS-CoV structural proteins. According to the current research show that the T-cell response to the S proteins and other structural protein is long lasting and persistent. The body’s immune response to SARS-CoV and SARS-CoV-2 is also closely similar to those mediated by cytokines. This provides an evidence for the design of the SARS vaccine composed of the viral structural proteins, which can induce effective and long-term memory cell responses against the virus which has now become a global pandemic^7,10,11,12.

Treatment:

A present clinical management includes infection prevention, control measures and supportive care including supplementary oxygen and mechanical ventilation when indicated. Till date there have been a lot of studies published and trials done with different drugs and its combinations but till now no FDA approved concrete treatment plan or Vaccine has been confirmed. Few drugs and studies that have been done are as follows:

a. Infection prevention: Few steps that needs to be followed and guidelines given by WHO states that infection prevention can be done and is one of the best techniques in this COVID19 crisis. Which includes Quarantine of affected person, maintaining proper 1 metre distance between each person, more care in personal hygiene with regular hand washing and use of sanitizers or gloves/Masks.

b. Control measures: Random screening, screening of travellers, sanitizations of surroundings, and quarantine of test positive patients.

c. Supportive care:

Antiviral therapy:

1. Interferons (IFNs): these are low molecular weight glycoprotein cytokines produced by host cells in response to viral infections, TNFα, IL-1 and some other inducers. They have nonspecific antiviral as well as other complex effects on immunity and cell proliferation. Interferons attach to cell surface receptors and effect the cell in different levels of its life cycle. one of the important steps is its suppression of viral proteins synthesis.

2. Lopinavir/ritonavir: Both are Retroviral Protease Inhibitor and a potent CYP3A4 inhibitor. They interfere with virus replication and synthesis so leaves behind immature, non infectious virus cells that can be easily destroyed. A study done in south korea shows positive results by use of Lopinavir/ritonavir for SARS-CoV2, they found that most of the patient shows a substantial decrease of Coronavirus.

3. Ribavirin: It is a Purine Nucleoside analogue and has broad spectrum antiviral activity. It was mainly used for Herpes, Hepatitis C, Respiratory Syncytial Virus, and many DNA and Double stranded RNA viruses. It intercellularly inhibit GTP and Viral RNA synthesis.

4. HydroxyChloroquine/Chloroquine: Few studies that were conducted in China have shown promising results in use of Hydroxychloroquine//chloroquine in SARS-COV2 patients. A antimalarial drug, also used in Rheumatoid arthritis, lupus used in patients alone or in combination with Remdesivir were highly effective in control of 2019-nCov.

5. Remdesivir: The broad-spectrum antiviral nucleoside analogue inhibits SARS-CoV and MERS-CoV in vivo.it is been tested in different parts of India, U.S.A, Japan and have been approved in U.S.A and japan for patient with severe symptoms of SARS-CoV.

6. Various other anti-virals are currently being evaluated against infection. Nafamostat (a synthetic serine protease inhibitor. it is a short acting anticoagulant, also have some antiviral and anticancer properties), Nitazoxanide (a broad spectrum anti parasitic and broad-spectrum antiviral used in treatment of various helminthic, protozoal and viral infections), Penciclovir, Favipiravir, AAK1, Baricitinib, and Arbidol exhibited moderate results when tested against infection in patients and in-vitro clinical isolates.

Immunotherapy:

Monoclonal antibodies: From the studies it has been reported that development of monoclonal antibodies can be very helpful in the remission of the SARS-CoV-2 epidemic. Previous studies have identified a number of effective monoclonal antibodies that target the SARS-CoV spike protein to prevent the virus from entering host cells. The 193-amino acid (residues 318–510) receptor binding domain (RBD) of the spike protein is the key target of neutralising monoclonal antibodies. The SARS-CoV neutralising monoclonal antibodies CR3014 and CR3022 were found to bind non-competitively to the SARS-CoV RBD and neutralised the virus in a synergistic manner. Moreover, the epitope of CR3022 does not overlap with the binding site of ACE2 in the SARS-CoV-2 RBD. Thus, CR3022 could be a promising therapeutic candidate, alone or in combination with other neutralising monoclonal antibodies, for the treatment of COVID-19 pneumonia¹⁰.

Convalescent plasma therapy:

Convalescent plasma (CP) therapy is a well recognized adaptive immune therapy which has been used for century against infectious viral diseases such as influenza A (H5N1) and Ebola, poliomyelitis etc. The use of CP from recovered patients contains high neutralizing antibody (IgG, IgA, IgM, IgE and IgD) titer which can prevent and treat patients with COVID-19. Therefore, FDA has also approved the use of CP therapy for elimination of SARS –COV2 infection. It is evident from several studies in hong kong and Taiwan when infected patients were injected convalescent plasma, it showed positive results with speedy recovery, with a reduction of plasma viral load from ~105 copies/mL to undetectable levels in 24 h after plasma transfusion. It is found to be more effective when given at the earliest stage before the virus causes severe lung injury.

Cellular therapy:

Natural killer (NK): NK cells (also called as K cells, and killer cells) are components of innate immune system. They are the first line of protection against any invading viral pathogen as well as cancer cells. Specialized NK cells also provide protection against disease in the host. The killing property of the NK cells is dependent upon the balance between the signals coming from both stimulating and inhibitory receptors on the NK cell surface. The activating receptors recognise molecules that are expressed only on the surface of cancer cells and infected cells (but not in normal cells) and activate the cells that kills infected cells through a process called “antibody-dependent cellular cytotoxicity (ADCC)”. It is evident from certain studies that NK cells also have the ability to kill the Novel SARS-CoV infected cells. Recently An umbilical cord blood-derived NK cell therapy has been launched for the treatment of SARS-CoV-2 infection, as announced by Sorrento and Celularity, which is a clinical collaboration aiming at extending the use of an allogen CYNK-001. NK cell therapy is a promising feasible strategy for the prevention of novel SARS CoV 2 virus.

Mesenchymal stem cells (MSCs): It is a population of hematopoietic and non-hematopoietic stem postnatal stem cells, capable of renewing itself and possessing diverse differentiation potency into multi-lineages. Due to their biological significance and vast clinical applications, MSCs has turned out to be a promising area in the latest research field. These cells are well known for their anti-inflammatory and anti-apoptotic activity in threatened tissues as well as antimicrobial activity and immunomodulatory action by paracrine function. They can also stimulate neo-angiogenesis as well as activate other resident stem cells. These exceptional properties make MSCs an appropriate resource for the clinical treatment of some human diseases including acute/chronic lung injury and ARDS. Additionally, MSCs also contribute to enhancing tissue repair and reducing lung fibrosis. Therefore, to interrupt the disease progression and reduce mortality, along with the regular antiviral therapy MSCs can play an important role in the treatment of cytokine storm syndromes, ARDS and acute lung injury in patients with severe SARS CoV

Chinese medicine:

Glycyrrhizin, an active component of liquorice roots used in Chinese medicine, could effectively inhibit the replication of SARS- associated CoV in vitro. Furthermore, high doses of gly- cyrrhizin have been used in clinical trials and the compound was reported to be clinically effective for the treatment of SARS at that time. Recently, glycyrrhizin was predicted to have the ability to bind ACE2 with potential anti-COVID-19 effects. Hesperetin, a well-known traditional Chinese medicine, is a natu- ral predominant flavonoid found in citrus fruits. Hesperetin dose- dependently suppresses the cleavage activity of the 3C-like pro- tease (3CLpro) of SARS-CoV in cell-free and cell-based assays. Hesperetin was also reported to have the potential to inhibit ACE2 and therefore block infection with SARS-CoV-2. Baicalin, an- other traditional Chinese herbal medicine, is a flavone isolated from Scutellaria baicalensis. It has been shown that baicalin has antiviral activity against 10 clinical isolates of SARS-CoV by neu- tralisation tests. In addition, quercetin is a plant flavone that is widely used in traditional Chinese medicine and botani- cal medicine. Quercetin was reported to exert antiviral effects by inhibiting the 3CLpro of SARS-CoV and blocking the entry of SARS-CoV into host cells. Therefore, these studies suggest that Chinese medicine also plays a key role in the prevention and treatment of COVID-19 pneumonia.

CONCLUSIONS:

COVID-19 is a highly contagious disease caused by SARS-CoV-2. The disease may vary from asymptomatic cases, mild symptoms to life-threatening complications such as ARDS, multiorgan failure, sepsis, and death. It is causing more deaths and infections than SARS or MERS. Elderly patients are at the highest risk of fatality. The rapid spread of disease warrants intense surveillance and isolation protocols to prevent further transmission.

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Received on 28.11.2020 Modified on 31.01.2021

Asian Journal of Pharmaceutical Research. 2021; 11(2):117-121.

DOI: 10.52711/2231-5691.2021.00022