Genomic Variation and Treatment Strategies of COVID-19: A Descriptive Review

Coronavirus disease 2019 (COVID-19) was spread across China and affected more than 180 countries worldwide to date. SARS-CoV-2 is a beta coronavirus that shows genomic similarity with bat coronaviruses. The intermediate source in human viral transmission is caused by dromedary camels for MERS-CoV and civet cats for SARS-CoV. Transmission of the virus from human-to-human is achieved through close contact with infected persons. The genome of the coronavirus consists of four structural proteins, including Spike (S), Membrane (M), Envelop (E), and Nucleocapsid (N) proteins. These structural proteins are encoded within the genome 3' end. The spike protein is responsible for virus attachment to the host cell surface receptors (angiotensin converting enzyme-2 receptor), resulting in fusion and subsequently cell damage. The N protein, after binding, causes RNA genomic changes. The accessory proteins present in SARS-CoV open read frames (ORFs) are very similar to COVID 19. The COVID-19 infection triggered a number of deaths and even now affecting a number of confirmed cases. Coronavirus patients are characterized by pneumonia, cytokine storms, weakened lymphocytes, lymphocytopenia, and respiratory failure. However, the lack of antiviral vaccines permits emergency clinical trials since January 2020. Recently, several anti-viral drugs are being repositioned and restructured as part of an immediate investigation. In this review, we discussed the genomic sequence of SARS-CoV-2, its different features and current therapeutic strategies to combat this serious condition.


INTRODUCTION
Coronaviruses are in the family of coronaviridae. Coronavirus is a positive-sense single-stranded RNA (+ssRNA) with a much smaller size (65-125 nm in diameter). Coronaviruses are primarily subdivided into four classes, such as alpha, beta, gamma, and delta. Alpha and beta mainly infect people, while birds and mammals are infected with gamma and delta. Epidemics such as SARS-CoV in 2002-2003 East Respiratory Syndrome of Coronavirus (MERS-CoV) in 2012, Acute Lung Injuries (ALI), and Acute Respiratory Distress Syndrome (ARDS) in 2012 have occurred in the last two decades [1]. China informed World Health Organization (WHO) in December 2019 about one of the unfamiliar diseases that took over 1800 lives in the first 50 days. The SARS-CoV-2 situation was confirmed by the International Committee on Virus Taxonomy (ICTV) [2]. SARS-CoV (2002-2003 infected 8422 individuals with 916 deaths, with 11% mortality rate [3]. On the other side, COVID-19 infects individuals with a seven percent mortality rate [4]. The analysis clearly shows

CORONAVIRUS TRANSMISSION
The pandemic with an unknown etiology arose from the Chinese seafood industry for the first time. The source and transmission of the virus must be determined to develop potential therapeutics. As Bat-CoV is 96.2 percent, similar to human SARS-COV-2, the bat is reported to be the primary coronavirus reservoir. The person who has a history of visiting or contacting the infected area is reported to be infected with the virus. The National Health Commission of China notified the chances of transmission between health workers. One reason for transmission was the consumption of infected animals and direct contact with primary or secondary reservoirs. Asymptomatic infection can occur in persons with lower immune responses. The viral load found in asymptomatic patients has been found to be similar to the virus transmission capacity of symptomatic patients [25,26]. Fig. (1) shows the transmission of coronaviruses from animals to humans.

THE ENTRY MECHANISM OF THE HUMAN CORONAVIRUS
The four types of structural glycoproteins are contained in coronaviruses, including Spike (S), Membrane (M), Nucleocapsid (N), and Envelope (E). Spike glycoprotein is primarily responsible for interacting and entering the host organism. The Open-Reading Frame1 (ORF1) encodes structural proteins for all coronaviruses with unique genes [27]. Cryo-electron tomography has been shown to form an extra interior layer of the transmembrane protein in the carboxy region, thickening the viral membrane [28]. Coronaviruses are ingested according to various enzymes such as trypsin-like human Proteases in airways, cathepsins, and serine-2 Transmembrane proteases (TMPRSS2) responsible for the removal of glycoprotein. Spike (S) protein is composed of S1 and S2 subunits, responsible primarily for the binding to the host receptor and viral cell membrane fusion by forming a six-helical bundle, respectively [29,30]. The dipeptidyl peptidase-4 (DPP-4) was reported to act as a receptor for MERS-CoV, while ACE2 was shown to be the entry receptor for SARS-CoV [21].
The SARS-CoV-2 coronavirus structure is made up of glycoprotein fusion with implicit RNA polymerase, papain-like protease, helicase, and accessory proteins. SARS-CoV-2 spiked protein is mainly attached with van der waals forces to the receptor-binding domain [31 -33].  The glutamine residue 394 in SARS-CoV-2 Receptor-Binding Domain (RBD), which has a structural resemblance to 479 residues in SARS-CoV, can be identified by the essential lysine 31 on the human ACE2 receptor [34]. SARS-CoV-2 recognizes human ACE2 more prominently than SARS-CoV, responsible to increase the transmission rates from person to person [35]. N501T mutation in SARS-CoV-2 spike protein increased binding affinity to angiotensin-converting enzyme 2, causing pathogenic divergence from SARS-CoV [8].

GENOMIC VARIATIONS
Coronavirus genome consists of approximately 26000-32000 bases (SARS-CoV 29,712; SARS-Cov-2 ~30,000; MERS-CoV 30,119) including variability in Open Reading Frames (ORFs) [36]. The genomic sequence of SARS-CoV-2 was registered in the NCBI genome database (NC_045512.2), approximately 29.9 kb in size [37]. The genomic analysis of SARS-CoV-2 showed a similarity of 96.3%, 89% and 82% with bat CoV, SARS-like CoV, and SARS-CoV, respectively [38]. The genome of SARS-Cov-2 has 11 protein-coding genes with 12 expressed proteins. Basically, open reading frames are designed as replicase and protease (1a-1b), and major structural proteins are arranged from 5′ to 3′ order and preferred for drug targets [39]. The retrieved translated sequence of SARS-CoV-2 from GenBank showed that it encodes about 7096 long polyprotein residues with various structural and non-structural proteins [40]. The orf1ab gene in SARS-CoV-2 encodes pp1ab protein and 15 non-structural proteins (nsps), whereas the orf1a gene codes for pp1a protein and 10 non-structural proteins. The 15 nsps were categorised from nsp1 to nsp10 and nsp12 to nsp16. The orf1ab and orf1a genes are located at the 5' end and encode pp1ab and pp1a, respectively, and the 3' end of the genome contains four structural glycoproteins (S, E, M, N) and eight accessory proteins (3a, 3b, p6, 7a,7b 8b, 9b, and orf14) [31]. SARS-CoV has some differences in accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b) [41]. The accessory proteins help in virus transmission, initiates pathological events and produce pro-inflammatory cytokines and activate interferon signaling [38]. The genetic makeup of SARS-CoV-showed there are 380 amino acid changes from different protein to the proteins of recent SARS-CoV-2. For example, accessory proteins, S protein and N protein have 348, 27 and 5 amino acid changes, respectively [42]. The coronavirus phylogenetic tree revealed a structural similarity between SARS-CoV-2 and SARS-CoV [11,43]. The amino acid sequence of SARS-CoV-2 is quite similar to SARS-CoV, but there are differences in 8a and 8b proteins [31]. For example, 8a protein present is in SARS-CoV but not in SARS-CoV-2; 8b protein consists of 84 amino acids in SARS-CoV, whereas amino acids in SARS-CoV; 3b protein is composed of amino acids in SARS-CoV but only 22 amino acids in SARS-CoV-2 [44].

TREATMENT STRATEGY
At the moment, coronavirus cannot be fully cured by any therapy. The primary use of antibiotics and anti-viral medicines is to relieve loads of viral RNA [46]. The combination of lopinavir and ritonavir showed clinical effectiveness against SARS-CoV but not against 2019-nCoV [47]. Remdesivir blocked, in particular 2019-nCoV replication combined with chloroquine or immune interferon [8,48]. The results for newly-infected patients were successfully proved by isolated blood plasma from clinically treated COVID-19 patients.

Anti-viral Drugs
There are no successful anti-viral agents that can fight against COVID-19. Lopinavir is a protease inhibitor in only one in in vitro and pre-clinical studies. Anti-viral drug remdesivir has been shown to be effective against Ebola [49]. It shows efficacy against RNA viruses and can combat against RNA-dependent RNA-polymerase(RdRp) [50]. Lists of recent clinical trials of anti-viral drugs in COVID-19 patients are shown in Table 2. The data of recent clinical trials of antiviral is collected, separated and compiled from https://www.clinicaltrials.gov/ and http://www.chictr.org.cn/enindex.aspx

Antiparasite Drugs
The use of chloroquine as an antiviral agent is crucial for preventing malaria, autoimmune diseases, and amoebiosis infections [51]. The studies show that intravesicular-pH controls cell function and increases the pH-endosomal required to fuse the virus into a host organism, including glycosylation trimming. Chloroquine prevents vacuole and endocytosis from moving protozoans. Chloroquine is known to be useful either as prophylaxis or as a therapeutic agent. Chloroquine enables the inflow of responsible zinc to inhibit the in-vitro function of RNA polymerase [52 -54]. Hydroxychloroquine is less toxic than the analogue derivative of chloroquine. Hydroxychloroquine was reported to show cell culture activity during the SARS-CoV epidemic. The pharmacokinetic study showed that hydroxychloroquine was found to be as effective as chloroquine in the treatment of SARS-CoV-2 due to a lack of experimental evidence [56,57].
Ivermectin is a broad-spectrum FDA approved parasitic drug that shows activity against COVID-19 as a second-line drug. Ivermectin has a wide range of anti-viral activity against large numbers of viruses under in vitro conditions as it prevents viral replication. A single treatment with ivermectin reduced the virus to 5000 times in culture within 48 hours, but no further reduction to 72 hours. Ivermectin was known to inhibit the nuclear import of viruses and host proteins. It has been reported that the integrase protein (IN) of viruses and the importin (IMP5-007 / β1) heterodimer is responsible for IN nuclear import. As most RNA viruses rely on IMP / β1 during infection, ivermectin directly affects it and inhibits virus replication [58]. Several clinical trials to test therapeutic potency in 2019-nCOV started in different hospitals and universities. Several patient age groups were used to control adverse effects. The list of recent clinical trials of anti-parasitic drugs in COVID-19 patients is shown in Table 3. The data of recent clinical trials on anti-parasitic data is collected, separated and compiled from https://www.clinicaltrials.gov/ and http://www.chictr.org.cn/enindex.aspx

Corticosteroids
Corticosteroids are a group of steroid hormones that regulate various physiological processes. The protective effect of steroids in COVID-19 patients was seen in various clinical studies. Several studies have demonstrated the effectiveness of corticosteroids in alleviating adverse immune system reactions. A lab study of dexamethasone infected pigs showed that one or two doses of corticosteroids could reduce cytokine expression [59,60]. List of recent corticosteroid clinical trials in COVID-19 patients is shown in Table 4.

Antibodies
Monoclonal antibodies are mainly targeted at the spike glycoprotein virus that invades host organisms. There are two functional subunits of spike protein (S1 and S2), in which S1 is used to attach cells, and S2 is capable of fusing into the cells.
Monoclonal antibodies can only be monovalent, and only one antigen can be identified at the same time. Antibodies neutralizing coronavirus are frequently targeted at and make incompetent S1 binding receptor domains [62,63]. Some antibodies identify various epitopes in the domain of receptor bindings, such as SARS-CoV neutralizing the virus competency antibodies CR 3014 and CR 3022. Table 5 shows the list of recent clinical tests of antibodies in COVID-19 patients.

Hospital of Wuhan University
The data of recent clinical trials on corticosteroids data is collected, separated and compiled from https://www.clinicaltrials.gov/ and http://www.chictr.org.cn/enindex.aspx The data of recent clinical trials on antibodies data is collected, separated and compiled from https://www.clinicaltrials.gov/ and http://www.chictr.org.cn/enindex.aspx The data of recent clinical trials on plasma convalescent transfusion is collected, separated and compiled from https://www.clinicaltrials.gov/ and http://www.chictr.org.cn/enindex.aspx The data of recent clinical trials on vaccines is collected, separated and compiled from https://www.clinicaltrials.gov/ and http://www.chictr.org.cn/enindex.aspx

Transfusion of Convalescent Plasma
The administration of convalescent plasma to SARS-CoV-2 infected patients shows recovery from the virus's etiology and pooled mortality rates as significantly decreased compared with or without placebo [64 -66]. The health commissions of various backgrounds have asked recovered patients for donating their blood. Patients who received convalescent plasma reported a rapid recovery within 14 days, compared with other patients during the SARS CoV outbreak [67]. Table  6 shows the list of recent clinical trials of plasma therapies in patients with COVID-19.

Vaccines
The already revealed interaction among host receptors with coronavirus allows researchers to find a cure for nCoV 2019. In recent centuries, vaccination in severe diseases has been a significant defensive function. A clinic trial of six vaccines was carried out to test the efficacy of these vaccines, including mRNA1273(NCT042834461), S-protein adenoviral type 5 (NCT04313127), Chimpanzee adenoviral vector ChAdOx1 (NCT04324606), S-protein plasm encoding (NCT04336410), Lentiviral DCs modified (NCT04276896) and artificial antigen cells modified with lentiviral vector expression. The clinical trial without pre-clinical studies was concluded in a very short period because of the high and safe therapy potential of mRNA1279-COVID-19 (NCT04283461) encapsulated nanoparticles [68]. The safety profile of the mRNA vaccine is outstanding and has excellent immunological properties. mRNA vaccines are mostly induced by cellular and humoral immunity [69]. A list of recent clinical trials of vaccines in COVID-19 patients shown in Table 7 can be a game-changer for vaccine technologies.

CONCLUSION: FUTURE ASPECTS
Over the past two decades, coronavirus has shown worldwide health concerns. The disease is likely linked to hematological and respiratory problems. The spike protein of SARS-CoV-2 is more likely to reach the host compared to SARS-CoV's spike protein, which means the transfer rate in the SARS-CoV-2 is high. Asymptomatic patients also have a high transmission rate. The host immune response must be improved to fight against coronavirus. The intermediate reservoir of nCoV-2019 is still challenging for researchers. Coronaviruses are significantly attached to the ACE2 receptor of host organisms. The open reading frame of coronaviruses is responsible to distinguish between SARS-CoV-2 and SARS-CoV. Various clinical studies have started to identify potential therapies for eradicating this pandemic, and, until now, no effective nCoV-2019 drugs or vaccines are available. All drugs are based on the experience of SARS, MERS, and other strained viruses. Running clinical trials must be focused on quality data that can be used in possible prevention and treatments. In addition to medicines, techniques for respiratory support and modulation of immune status are highly required. Global resources with reasonable scientific justification are available for the planning of clinical trials. In recent clinical trials, the repurposing and repositioning of certain drugs have been processed. The repurposing of medicines has some barriers while repositioning clinical studies facilitate the discovery of new drugs. By this year, the way to find COVID-19 solutions should be through global cooperation with different clinical trial hospitals with a large number of patients. More work is required to find out exactly how this coronavirus is being approached.

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CONFLICT OF INTEREST
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