key: cord-343082-46lo7xtx
authors: Awasthi, Ankit; Vishwas, Sukriti; Corrie, Leander; Kumar, Rajesh; Khursheed, Rubiya; Kaur, Jaskiran; Kumar, Rajan; Arya, K.R.; Gulati, Monica; Kumar, Bimlesh; Singh, Sachin Kumar; Pandey, Narendra Kumar; Wadhwa, Sheetu; Kumar, Pardeep; Kapoor, Bhupinder; Gupta, Rajneesh Kumar; Kumar, Ankit
title: OUTBREAK of novel corona virus disease (COVID-19): Antecedence and aftermath
date: 2020-07-25
journal: Eur J Pharmacol
DOI: 10.1016/j.ejphar.2020.173381
sha: 
doc_id: 343082
cord_uid: 46lo7xtx

Outbreak of Coronavirus disease 2019 (COVID-19) started in mid of December 2019 and spread very rapidly across the globe within a month of its outbreak. Researchers all across the globe started working to find out its possible treatments. However, most of initiatives taken were based on various hypotheses and till date no successful treatments have been achieved. Some strategies adopted by China where existing antiviral therapy was initially used to treat COVID-19 have not given very successful results. Researchers from Thailand explored the use of combination of anti-influenza drugs such as Oseltamivir, Lopinavir and Ritonavir to treat it. In some cases, combination therapy of antiviral drugs with chloroquine showed better action against COVID-19. Some of the clinical studies showed very good effect of chloroquine and hydroxychloroquine against COVID-19, however, they were not recommended due to serious clinical toxicity. In some cases, use of rho kinase inhibitor, fasudil was found very effective.In some of the countries, antibody-based therapies have proved fairly successful. The use of BCG vaccines came in light; however, they were not found successful due to lack of full-proof mechanistic studies. In Israel as well as in other developed countries, pluristems allogeneic placental expanded cell therapy has been found successful. Some phytochemicals and nutraceuticals have also been explored to treat it. In a recent report, the use of dexamethasone was found very effective in patients suffering from COVID-19. Its effect was most striking among patients on ventilator. The research for vaccines that can prevent the diseaseis still going on. In light of the dynamic trends, present review focuses on etiopathogenesis, factors associated with spreading of the virus, and possible strategies to treat this deadly infection. In addition, it attempts to compile the recent updates on development of drugs and vaccines for the dreaded disease.

Thoughresearchoncoronavirus disease 2019 was going on at global levelsince last two decades,highly virulent transmission of COVID-19 came into existence as highly fatal human pathogen during June 2012 in Arabian Peninsula(https://www.businessstandard.com/article/international/china-suspends-public-transport-in-wuhan-confirms-571- and SARS-CoVare reported to have originated from bats while HKU1 and HCoV-OC43 have been found in rodents (Forni et al., 2017; Su et al., 2016) .

The components of SARS-CoV-2are spike glycoprotein (S), membrane protein (M), nucleocapsid protein (N) and envelope protein (E). The spikes present on the virusconsist of a single-pass transmembrane anchor,a large ectodomain and short intracellular tail. The ectodomain contains two subunits S1 and S2 (Li, 2016) .The homotrimers of S-protein help in building of spikes on the viral surface which play a key role in its attachment with host receptors (Beniac et al., 2006; Delmas and Laude, 1990) . The M glycoprotein performs three major functions i.e. itprovides shape to the virions, aids in promoting curvature of membrane,and facilitates binding to the nucleocapsid (Nal et al., 2005; Neuman et al., 2011) .The E-glycoprotein plays akey role in the assembly and pathogenesisof virus (DeDiego et al., 2007; Nieto-Torres et al., 2014) . The N-glycoprotein consists of two domains which bind to the RNA genome of the virion. It is also believed that N-glycoprotein bindstononstructural protein 3 (nsp-3) which, in turn helps in tying the genome to replicationtranscription complexes (RTCs) and helps in packaging of enfolded genome into virions (Chang et al., 2006; Fehr and Perlman, 2015; Hurst et al., 2009) . The structure of SARS-CoV-2 is shown in Fig. 1 .

The main symptoms of COVID-19 include runny nose, sneezing, common cold, cough, confusion, myalgia, diarrhea, vomiting, shortness of breath, wheezing and fever (Nassar et al., 2018; Tyrrell, 1996) .The virus enters the respiratory tract through nose and stays there for three days. Afterwards, it starts infecting upper respiratory tract (URT) with above-mentioned symptoms. COVID-19 causesURT illness including acute exacerbation of chronic obstructive pulmonary disease, bronchitis and pneumonia, with co-morbidities in digestive, cardiac (Zheng et al., 2020) , renal (Cheng et al., 2020) and circulatory systemsas well (Huang et al., 2020) .

From various studies, it has been found that SARS-CoV-2 enters the RBCs and interrupts their function of oxygen transport and causes multiple organ dysfunctioning. This occurs due to presence of viral proteins such as ORF8 and surface glycoprotein that bind with porphyrin and inhibit human heme metabolism. It has been also found that other viral proteins Orf 1ab, ORF10 and ORF 3a attack the 1-beta chain of hemoglobin and form porphyrins. This compromises the ability of Hb to carry oxygen (Wenzhong and Hualan, 2020).Using molecular docking studies, Wenzhong and Hualan found that chloroquine could relieve the symptoms of respiratory distress by preventing the attack of orf1ab, orf3a, and orf10 proteins on heme to form porphyrin. In addition to that it is reported to inhibit the binding of ORF8 9 transcribed viral DNA are then added. The added fragments attach to the target site of viral DNA if sample consists of virus. The added genetic material acts as a builder for DNA strands during amplification, while the labels added help in detecting the virus. The mixture is then placed under the RT-PCR machine. The machine cycles through heating and cooling of sample so that chemical reaction takes place and forms new copies of viral DNA. These cycles take place35 times, so that at the end of the cycle, about 35 billion identical copies of viral DNA formed. The fluorescence emission by sample is measuredby machine and helps in assessing presence or absence of virus (https://www.iaea.org/newscenter/news/how-is-thecovid-19-virus-detected-using-real-timert-pcr)

It has been found that chest CT imaging is more steady, practical and expeditious technique to diagnose and assess COVID-19. In some of the studies, it has been found that computed tomography is more sensitive tool than RT-PCR to assess COVID-19. The sensitivity of CT and RT-PCR was reported to be 98% and 71% respectively (https://www.itnonline.com/content/ct-provides-best-diagnosis-novel-coronavirus-covid-19).

Tao et. al. 2020, studied the comparison between RT-PCR and chest CT for the diagnosis of COVID-19. In the study total 1,014 patients underwent RT-PCR and chest CT. The results revealed that out of 1,014 patients, 601 (59%) showed positive results with RT-PCR while 888 (88%) patients showed positive CT scans results. So, it was concluded chest CT is more reliable technique for the diagnosis of COVID-19 (Ai et al., 2020; .

With the prevalence of COVID-19 reaching a new high every day, there is an immediate need to find safe and efficacious measures to diagnose, treat, mitigate and combat the disease.

Looking at the alarming dimensions that the disease is acquiring, treatment strategies among various systems of medicines are being investigated. Based on the treatments offered so far and clinical findings, the treatment strategies can be categorized into three classes.

Antibiotics, for obvious reasons,are not expected be effective in the treatment anda combination of antiviral drugsis being used. Studies also confirm that flu shots are not efficient in the fight against COVID-19 as the patients continue to suffer despite the treatment (https://www.wsj.com/articles/gilead-sciences-offers-experimental-drug-for-coronavirustreatments-testing-11580511519).In the meantime, Thai health officials claimed to have successfully handled the infection with acocktail of antiviral drugs that include lopinavir and ritonavir under the name "Kaetra" along with flu medication oseltamivir. However,a lot more studies need to be conducted to declare this combination as a treatment for COVID-19.

Randomized clinical trials using combination of antiviral drugsare already being conducted (Hung et al., 2020) . Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial. The possibility of successful treatment exists because the same combination was successfully used to treat SARS outbreak in 2002 andSARS-CoV-2is reported to be a strain similar to the earlier onewith the genomic sequence of COVID-19being about 75-80% similar to that of SARS.Lopinavir was used as major antiviral drug to treat during the SARS outbreak.Lopinavir has been reported to treat COVID-19 (https://www.antibodies-online.com/resources/18/5410/sars-cov-2-life-cycle-stages-andinhibitiontargets/),(https://clinicaltrials.gov/ct2/show/NCT03301090?term=NCT03301090#wrapper).

However, its efficiency to mitigate COVID-19 is yet to be fully established. To analyze antiviral effect of inferferon-α2b (IFN-α2b) and ribavirin, SARS-CoV-2was isolated from hCoV-Emc/2012 replication process by Vero and LLC-MK2 cells. Combination of IFN-α2b and ribavirin achieved comparable remissionin low concentration. As per Heinz Felmann et al. 2013, combination of IFN-α2b and ribavirin may also prove to be useful (Falzarano et al., 2013) . Anti-ebola and anti-HIV drugs combination (remdesivir+ galidesivir) showed significant action against the enzymes responsible for virus replication. These drugs have been able to alleviate the symptoms of COVID-19 similar to that in SARS and MERS(Li and De Clercq, 2020) . In an in-vitro cell line study, this drug also showed very good antiviral effect ).

The Gilead biotechnology company, USA reported preclinical trials of Remdesivir (a nucleotide analogue) which led to remission in animal models (Sheahan et al., 2020) . Later, it was reported to be effective in the treatment of COVID-19 patients also (Holshue et al., 2020) . Another study, conducted on 760 patientsin placebo-controlled trials also proved the effectiveness of remdesivir. This drug has now received emergency use authorization by USFDA on 1st May, 2020 (https://www.fda.gov/news-events/pressannouncements/coronavirus-covid-19-update-fda-issues-emergency-use-authorizationpotential-covid-19-treatment).

Frequently used antimalarial drug,Chloroquine (CQ)and Hydroxychloroquine (HCQ) have also been explored and found to be quite effective against COVID-19(Wang et al., 2020) . CQ and HCQ increase endosomal pH and interfere with the glycosylation of cellular receptor of SARS-CoV-2. Thereby they have the potential to block viral infection . Moreover, they change the pH of lysosomes and likely inhibit cathepsins, that leads to the formation of the autophagosome which cleaves SARS-CoV-2 spike protein. It is also reported that CQ and HCQ through the inhibition of MAP-kinase interfere with SARS-CoV-2 molecular crosstalk, besides altering the virion assembly, budding and interfering with the proteolytic processing of the M protein. It is reported that they interfere with ACE-2 receptor's glycosylation. Since, SARS-CoV-2 utilizes the similar surface receptor ACE-2, it is believed that CQ and HCQ can also thus prevent SARS-CoV-2 attachment to the target cells ( Zhou et al., 2016) . Some studies have also been initiated and showed very good effect of CQ and HCQ against SARS- CoV-2 (Gautret et al., 2020; Singh et al., 2020) ..; Gao et al., 2020; (Millán-Oñate et al., 2020) . However, due to reported potential clinical toxicity issues such as retinal toxicity, the use of CQ and HCQ is not recommended by WHO.

An antiviral drug favipiravir (Avigan), got approval in Japan in 2014. In 2016, this drug was used as an emergency aid for the Ebola virus outbreak.A clinical trial involving 80 participants (in Shenzhen city) demonstrated chest symptoms improvement in patients of COVID-19 treated with favipiravir. The drug was able to shorten the recovery time from 11 days to 4 days in mild and regular cases. Another trial showed that the drug shortened fever duration from an average of 4.2 days to 2.5 days. Favipiravirhas been reported to be effective, without any obvious side-effects, in helping coronavirus patients recover.In another study carried out in China, two mild and two severe COVID-19 associated pneumonia patients were treated with combined Western and Chinese medicine treatment (Lopinavir/ritonavir/arbidol/ShufengJiedu Capsule). Three of the four patients showed significant improvement in pneumoniaassociated symptoms. The remaining patient with severepneumonia showed signs of improvement; however, the efficacy of this combination treatment warrants further investigation (https://www.ncbi.nlm.nih.gov/pubmed/32037389).

In a recent study,Rho kinase (ROCK) inhibitor, Fasudil has been explored to treat COVID-19. In patients with COVID-19, activation of ROCK causes burst in inflammatory features, immune cell migration, apoptosis, coagulation, contraction, and cell adhesion in pulmonary endothelial cells, leading to endothelium barrier dysfunction and edema as hallmarks of lung injury. Fasudil attenuates this effect due to its excellent anti fibrotic activity (Abedi et al., 2020) . It has been found that angiotensin-converting enzyme-2 (ACE-2) is the receptor required for the cellular entry of SARS-CoV-2(Hoffmann et al., 2020).makes which is a negative regulator of the renin-angiotensin-aldosterone system, inactive. Since ACE-2 .opposes the actions of angiotensin II, it exerts beneficial effects against diseases such as lung injury, hypertension and cardiac remodeling.Envelope spike protein of SARS-CoV-2 mediates its attachment and fusion into the human cells through binding ACE-2 with superaffinity and efficiency. ACE-2 is widely expressed in alveolar epithelial cells and converts angiotensin 2 to angiotensin (1-7).Angiotensin II triggers a number of adverse effects like interstitial fibrosis, increased coagulation, interference with adaptive immunity by activating macrophages and other cells of the immune system, with consequent increased production of IL-6, TNFα and other inflammatory cytokines.ROCK inhibitors upregulate the axis of ACE-2, and are thereby found effective in treating COVID-19.

In one of the studies, a 53-year-old woman suffering from COVID-19 was treated with a combination of moxifloxacin and oseltamivir. She was treated with moxifloxacin 400 mg intravenously once a day for eight days and antiviral drug oseltamivir 75 mg orally twice a day for 5 days. After seven days treatment, patient's symptoms got reduced and she tested negative (Ding et al., 2020) .

In recent clinical studies the use of steroidal drug Dexamethasone has been very effective to treat patients suffering from COVID-19. It easily diffuses through the host cell membranes and bind to the glucocorticoid receptor in the cell cytoplasm. This receptor binding triggers a cascade of reactions that end up suppressing pro-inflammatory cytokines IL-1, IL-2, IL-6, IL-8, TNF, and IFN-gamma and reduce the severity of Covid-19. Dexamethasone also inhibits the overaction of macrophages in patients suffering from COVID-19. A British research team found that Dexamethasone's (2 mg tablet) effect was most striking among patients on ventilators. Those who were receiving oxygen therapy but were not on ventilators also saw improvement: their risk of dying was reduced by 20%. The steroid had no effect on people with less severe cases of COVID-19 -those not receiving oxygen or ventilation(Ledford, 2020). Table 2 summarizes various antiviral drugs which may have potential to treat COVID-19 with their mechanism of action. Table 3 summarizes possible drugs with dose to mitigate   COVID -19 and Table 4provides update about recent clinical trials on COVID-19 with possible targets. Lianhuaqingwen (LH) is a traditional Chinese medicine that has been used previously to combat SARS, influenza virus and enhance immunomodulatory effects. Runfeng et. al.

(2020), studied the antiviral and anti-inflammatory effect of LH against SARS-CoV-2. In this study African green monkey kidney epithelial cell (Vero E6 cells) was used as an in-vitro cell line. The cytopathic effect (CPE) and plaque reduction assay was used to assess the antiviral activity of LH in Vero E6 cells. The results revealed that the LH helped in inhibition of SARS-CoV-2 replication. It has been also found that LH showed significant reduction in pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrotic factor-α (TNF-α) and chemokine (C-C motif) ligand 2/monocyte chemo attractant protein 1 (CCL2/MCP-1).

In 1986 coronavirus. To target the virus, two peptide sequences from the nonstructural protein 4 of beta coronavirus (IRNTTNPSAR and PTDTYTSVYLGKFRG) were selected and were found to be potent T-cell epitopes. They were found to interact perfectly with epitope grooves of major histocompatibility complex (MHC) allelic protein (HLA-A*01:01 and HLA-DRB5*01:01) that formed a stable MHC complex. It can, therefore, be considered as potential peptide for development of peptide based corona virus vaccine (Basu et al., 2020) . In another study, computational approach along with bioinformatics tools was adopted for vaccine design. Based on the docking score as well as antigencity scores, natural inhibitors such as tanshinoneIia and methyl tanshinonate were identified as effective drugs and FVFLVLLPL (MHC class-I allele) and FVFLVLLPL(MHC class-II allele) were selected as best antigenic epitope respectively for vaccine design(Kumar, 2020). Recently US developed a vaccine and started first human trial (Phase 1 trial) in healthy human volunteers to monitor the desired responses in human immune system along with safety profile. The trial has enrolled 45 healthy volunteers for a period of 6 weeks. The vaccine is based on "messenger RNA vaccine platform technology". The experience on previous MERS vaccine development for targeting the surface protein of virus led to the idea of this vaccine (Roberts, 2020) .Out of the two vaccines, REGN 3048 and REGN 3051, manufactured by Regeneron, USA, were able to more keenly bind to viruses resulting in possibility of developing antibodies. Vaccine REGN 3048 was found to be ineffective againstCOVID-19, however the company is BacilleCalmette-Guérin (BCG) is a live attenuated strain of mycobacterium bovis used against tuberculosis and leprosy (https://www.who.int/biologicals/areas/vaccines/bcg/en/).

Vaccine repositioning indicated its potential use against multiple sclerosis, reducing blood sugar levels in type-1 diabetes and also in various types of cancer like non-Hodgkin's lymphoma and bladder cancer. It demonstrates a non-specific effect and thus its use may prove to be favorable against viral pathogens as well (Moorlag et al., 2019) . A study reported that oral zinc sulphate being an effective immunomodulator can be combined with BCG vaccine to provide protection against COVID-19 (Sharquie, 2020). The outcomes of the ongoing randomized trials (phase III) of BCG vaccine are eagerly anticipated (https://clinicaltrials.gov/ct2/show/NCT04328441?term=BCG+vaccine&cond=COVID-19&draw=2&rank=1);(https://clinicaltrials.gov/ct2/show/NCT04327206?term=BCG+vaccine &cond=COVID-19&draw=1&rank=2). Table 5 compiles the patents on vaccines having therapeutic potential to treat COVID-19 whileTable 6 summarizes the trials going on across the globe for the development of drugs and vaccines for the treatment of COVID-19.

Certain nutraceuticals have also shown efficacy in combating COVID-19. Their mechanisms are discussed here. Viral toll-like receptor (TLR7) of COVID-19 is responsible to trigger hydrogen peroxide generation within the alveolar macrophages via nicotinamide adenine dinucleotide phosphate hydrogen oxidase 2 (NOX2) activation which oxidizes Cys98 on TLR7. Such oxidation blocks receptor potential to conduct signals for type 1 interferon production. Nutraceuticals having potential to inhibit NOX2, superoxide generation or preventing oxidation of Cys98 in TLR7 have potential to evoke TLR7 mediated type 1 interferon production towards RNA virus infections including COVID-19. RNA virus infections have been shown to induce O-GlcNacylation of mitochondrial antiviral signalling protein (MAVS) at multiple sites which prohibits its susceptibity for K63-linked ubiquitination and further interferon regulatory factor 3 (IRF3) activation. Glucosamine supplementation has been investigated to upregulate MAVS to activate IRF3 in response to viral infections. Currently, vitamin C infusion is under clinical trial for the treatment of severe COVID-19 virus infected pneumonia as it plays important role in reducing inflammatory response and has antioxidant property. Vitamin C has earlier been reported to prevent neutrophil accumulation, alveolar fluid and cytokine surge caused by sepsis (Peng, 2020) .

The US-FDA has recently issued certain guidelines pertaining to the ongoing clinical trials for the development of medicinal products. Impact on the conduct of clinical trials of medical products is anticipated since challenges may arise from quarantines, site closures, travel limitations, interruptions to the supply chain for the investigational product, or other considerations if site personnel or trial subjects become infected with COVID-19 (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/fda-guidanceconduct-clinical-trials-medical-products-during-covid-19-pandemic).

Plasma therapy can reduce the mortality rate of COVID-19 (da Silva, 2020) . In this therapy, 

Placenta expanded cells are obtained from the placenta and are designed in such a way that theycan be administered to the patients without tissue or genetic matching. The main function of these cells is to release biomolecules such as growth factors, cytokines and chemokines.

These cells act in a endocrine and paracrine manner and help the body to stimulate its defense mechanism and promote healing (https://www.pluristem.com/placental-expanded-plxproducts/). The Israel biotech company, Pluristem Therapeutics Inc. reported that the patients who had n2019-CoVsSARS-CoV-2 and were at a higher risk of death due to respiratory collapse and multiorgan failiure such as kidney and heart failure recovered after receiving this therapy. This happens due to the immunomodulatory effect of the pluripotent plasma cells (PLX). During treatment the 15 milliliter doses of PLX cells is administered to patients by inter-muscular route (https://theindiantelegraph.com.au/israel-has-found-a-possible-100cure-for-coronavirus/).

Melatonin is a N-acetyl-5-methoxytryptamine and having many health benefits such as remission from sleeping disorders, viral infections, delirium, respiratory disease and atherosclerosis . Recent studies on COVID-19 revealed that the main cause of COVID-19 pathology is exaggerated immune response, oxidation and inflammation.

All these factors lead to cytokine storm and give rise to Acute Respiratory Distress Syndrome Table 7 .

The worldwide spread of COVID-19 has become a big challenge to control. It has already been declared as pandemic with more than 10 922 324 peoples affected across 195 countries till July 4 th 2020. An aggressive approach is required to take care of critically compromised patients in addition to sincere efforts to stop the transmission of disease. Currently many government agencies and pharmaceutical companies are working towards development of effective medicines and vaccines. Also, the available treatment strategies have been adopted to benefit the affected people, however, major step still remains to stop the transmission and alleviate the symptoms of affected people. Use of hydroxychloroquine as well as antiviral drugs are found effective against COVID-19, however, detailed clinical studies are required.

Some biotechnology-basedtechniques such as antibodies, cell and RNA based therapies have also been found to be very effective. It is expected that dexamethasone could bring some hope to treat this disease.However, it will be too early to give conclusive remarks on the currently available treatments since more evidence-based data is required to be generated.

Government agencies are working on their part; however, a coordinated effort is needed globally to help prepare the healthcare framework cope up with the unprecedented challenge of COVID-19.

This review did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors declare no conflict of interest. 

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Treatment of Ebola and other infectious diseases: melatonin "goes viral

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Inside the cell, SARS-CoV-2 forms a layer called capsid vesicle. Microtubules transport viruses to the cytoplasm. Viral RNA is involved in transcription process in the cytoplasm and conversion into viral genome mRNA. It also affects translation processes and develops viral protein. Viral RNA and viral proteins form new viruses. Thus virus multiplies and forms capsulated nucleoprotein. The multiplied viruses cause apoptosis in the cell and affect other cells also