9.5 Viral entry- triggers cascade of immunological responses
SARS COV binding to ACE2 receptors of pneumocytes in lungs triggers inflammation in lower respiratory tract.71 It has been shown that when SARS spike proteins bind to the ACE2 receptors the complex thus formed is proteolytically processed by TMPRSS2 (Type II transmemberane protease) which leads to cleavage of ACE2 and spike proteins are activated (Figure 7b).72,73 It has been suggested that cells in which ACE2 and TMPRSS2 co-exists, such cells are more susceptible to SARS COV infections.74 Clinical presentation suggest that SARS COV-2 also requires ACE2 and TMPRSS2 for entry into the host cells.51 Entry of virus and cell infection triggers the immune response and antigen- presenting cells (APC) initiate a set of Immunological reactions. Primarily APC perform two functions (1) CD4 +- T- helper cells (Th 1) combat foreign antigen particle (2) secreting inter-leukin-12 for further stimulation of Th1 cells. CD8+-T-killer cells (TK) are stimulated by Th1 cells that will target any of the cell containing foreign antigen and eventually activated Th 1 cells trigger B- cells to generate antigen specific antibodies.75
9.6 Potent receptor for SARS COV-2- ACE2
Coronaviruses used to enter host cell by two distinct routes,73 the first possible route is cell mediated endocytosis. The surface spike S glycoproteins has been accepted as key determinant, for coronaviruses entry, with S1 domain mediating the binding of receptor and domain S2 is responsible for membrane fusion of cell.58 The second possible route is that spike protein S is being cleaved by S-activating protease co-expressed with the host cell receptor, thereby inducing the direct fusion of cellular and viral membrane.67

10 TRANSMEMBRANE SPIKE (S) GLYCOPROTEINS

10.1 Structure
Spike proteins S are present on surface of coronaviruses and facilitate in viral entry for infection.76 The entry of cronavirus into the host cells is mediated by spike glycoproteins (S) that form homo-trimers that extend above the surface of virus77 (Figure 8a). S consists of two functional subunits which are present for attachment with the receptor of host cell (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit). For most CoVs, at the boundary between the S1 and S2 subunits, S is cleaved which remain non-covalently bound in the prefusion conformation.52,54,62,70,78-80 The diatal S1 unit consists of receptor binding domains and participates in the stability of the prefusion state of the membrane-anchored S2 subunit which consists of the fusion machinery52,54,55,58,60,81,82 (Figure 8b).
Trimers are being formed by spike molecules, which must be cleaved by cellular proteases, so that fusion peptide can aid in fusion of viral membrane with infected cells. The proteases produce two subunits S1 and S2 from the spike molecule, and S1 subunit consists of critical RBD receptor binding domains to bind with ACE2 of host cells. The receptor binding motif which is present in receptor binding domain forms direct contact with ACE2. Several others critical structures of spike proteins including Central Helix (CH) and heptad repeat 1 and 2 (HR1 and HR2) domains also help in fusion of virus with host cells51 (Figure 9).

10.2 Spike proteins and conformational changes

For all CoVs, the so-called S2 site located immediately upstream of the fusion peptide is being used for further cleavage of S.62,83 This cleavage has been reported to trigger conformational changes that activate the proteins for membrane fusion via extensive irreversible changes.60-62,70 Consequently, entry of coronavirus into susceptible cells is a complicated activity that requires coherent action of receptor binding and proteolytic processing of the S protein to enhance virus-cell fusion. After getting entry into the host cell, usually through contact with contaminated surfaces and aerosolized viral particles, the virus needs to undergo biological cycle. Spike proteins which are encoded by S gene on open reading frame on viral genome need to interact with viral receptors on the surface of host cells. Coronavirus spike binds with angiotensin-converting enzyme-2 (ACE-2), which are present in epithelial cells of the lungs.84,85 This is one of the major reasons why coronaviruses often cause respiratory disorders. ACE-2 receptors may also be highly expressed in intestinal tissues leading to diarrhea, as detected in few cases of SARS COV-2 patients.86 After binding to ACE-2 receptors via the RBD of the S1 and S2 domains of the spike protein, the viral envelope fuses with the host cell membrane and is further internalized. Genetic material approximately of the size 20-32 kb, is released into the cytoplasm for replication51 (Figure 10).
10.3 Escaping from host immune response
It has been suggested previously that coronaviruses use glycan shielding and conformational masking to prevent identification by the immunity of host cells.53,87,88

10.4 Structural proteins S are important considerations for anti-viral drug development

Structural proteins are one of the most important targets for vaccines or anti-viral drug development because of their functional role in fusion and entry of virus in host cells.89 Because coronavirus spike glycoproteins are exposed on surface and mediate the virus entry into host cells, it is the main focus of vaccine design by neutralizing antibodies upon infection and proper folding is accomplished by N-linked glycans that are present on S trimmers,90 and for modulating accessibility to host proteases and neutralizing antibodies.53,87,88,91

11 TREATMENT

To date, there is no well-known, effective & pharmacologically proved treatment of SARS-CoV-2. Different antiviral drugs were administered for 90 % of COVID-19 patients in different reports.17,92,93 Although, major antiviral treatments that were proposed, include lopinavir/ritonavir,17,92-95 oseltamivir, ganciclovir,17,92,93 Arbidol (umifenovir), remdesivir, favipiravir, ribavirin,96 chloroquine,96-100 hydroxychloroquine,97,100,101 and alpha-interferon.96 Plasma transfusion was also reported for the treatment of corona virus.92 This literature review is subjected to compare the efficacy of major antiviral drugs against the treatment of COVID-19 and choose the most appropriate one to treat SARS-CoV-2.
11.1 Treatment through convalescent plasma
Immunoglobulins or convalescent plasma had been used as a last weapon to treat viral infections in past. Studies have revealed that the use of convalescent plasma improves the health of patients as well as lowers the rate of mortality than those who are not treated with immunoglobulins.102-104 The use of convalescent plasma was administered in 2014 by WHO for the treatment of Ebola-virus disease.105 In 2015, immunoglobulins were also used for the treatment of MERS-CoV.106 By using immunoglobulins, immune response is developed in patients by 10 to 14 days.102 For the treatment of SARS-CoV-2 pneumonia, convalescent plasma was taken from recovered patients of COVID-19 and transfused in infected patients of SARS-CoV-2.92
11.2 Treatment through drugs
Ritonavir/lopinavir is a drug used against HIV infection.107 Lim et al. (2020) reported that ritonavir/lopinavir was found effective against the SARS-CoV-2 pneumonia.95 In clinical experiments as well as in vitro studies, ritonavir/lopinavir found to have anti-SARS-CoV activity. Another antiviral drug ribavirin was found effective by mono-therapy against SARS. Combined therapy with ribavirin and ritonavir/lopinavir; lowers the risk of ARDS and even death.108 While on the other hand, Cao et al. (2020) reported that no significant benefits were observed by the use of ritonavir/lopinavir with standard care in adult patients having acute COVID-19 infection.94
RNA-dependent RNA polymerase inhibitor known as favipiravir was suggested for the treatment of nCoV-19 on February 15, 2020 in China. It has anti-viral activity against influenza virus. It also has potential action against SARS-CoV-2 that is RNA virus like influenza virus. Favipiravir blocks the replication process of various viruses by inhibiting RNA polymerase activity.109 It inhibits replication of alpha-, arena-, flavi-, filo-, noro-, bunya-, and several other RNA-viruses.110 Now favipiravir is under the clinical trials to treat SARS-CoV-2 pneumonia by the Clinical Medical Research Center of the National Infectious Diseases and the Third People's Hospital of Shenzhen on February 14, 2020. Now results from a group of 80 patients in China (including control and experimental groups) revealed that anti-viral action of favipiravir was better than that of ritonavir/lopinavir. From clinical trials till now, favipiravir showed no or fewer adverse reactions in treatment group as compared to ritonavir/lopinavir group.111
Another broad-spectrum antiviral drug alpha-interferon (IFN-α) is used for the treatment of hepatitis.112 IFN-α can be used to stop the replication of SARS-CoV as it gave positive results in vitro.96 Influenza antiviral drug, arbidol effectively retards the SARS-CoV-2 infection in vitro.113
Remdesivir, like IFN-α is also an anti-viral drug that was found to inhibit RNA polymerase activity in vitro against various RNA viruses.114 Sheahan et al. (2020) reported that animal research on mice showed that remdesivir was effective to lessen the viral attack on lung tissues of mice having MERS-associated-CoV infection.115 It also reduces pathological damage to lungs and improves the functioning of lungs. It was revealed that anti-viral drug remdesivir potentially stops the SARS-CoV-2 infection.116,117 It is also reported that combined therapy of chloroquine and remdesivir gives high effectiveness to control nCoV-19 in vitro.118 Studies on rhesus macaque model also showed positive results against MERS-CoV infection.119 But further pre-clinical trials should be conducted for effective and safe use of remdesivir to treat COVID-19 infection.96,120
Chloroquine and hydroxychloroquine (analogue drug of chloroquine), both have anti-malarial as well as broad spectrum anti-viral characteristics.99,100,121 These drugs have good safety profile for clinical use as well as economically cheap.97,99 The in vitro anti-viral activity of chloroquine and hydroxychloroquine has been reported in different publications.118,122-125 Both hydroxychloroquine and chloroquine were found to inhibit the growth of several viruses (including the SARS-associated coronavirus and MERS associated coronavirus) in cell culture. This inhibition is achieved by interfering the process of glycosylation of SARS-CoV receptors present on cell surface as well as reducing the replication of virus.97,121,126,127-129 In the light of previous knowledge about these dual nature drugs (anti-malarial as well as anti-viral), scientists are compelled to work on their efficacy against SARS-CoV-2.97,99
As this virus emerged from China so most of the work is reported from patients in China. It was found that chloroquine had potential benefits against SARS-CoV-2 in Chinese patients to treat the viral infection.99 Wang et al. (2020) also reported that SARS-associated-CoV-2 infection was found blocked, by the use of chloroquine in vitro.116 Combined therapy with chloroquine has significant role in antiviral treatments.127 It is also observed that hydroxychloroquine has excellent safety profile than that of chloroquine.130 French scientists confirmed that combined therapy of hydroxychloroquine and azithromycin proved more efficient for the elimination of SARS-CoV-2.101 The toxic and therapeutic dose of hydroxychloroquine and chloroquine has a very narrow margin and slight over-dose may cause cardiovascular problems.131 Therefore, the use of hydroxychloroquine and chloroquine should be under strict rules and there should be no recommendations for self-treatment. Two types of salts in chloroquine (phosphate and sulfate) play major role in its antiviral activity.100 Results of more than 100 COVID-19 patients from different hospitals of China were collected and it was found that chloroquine phosphate plays a crucial role in the treatment of patients according to the news briefing. In the conference of regulatory and government authorities held on February 15, 2020, there was an agreement taken that chloroquine phosphate has a significant role in the treatment of COVID-19.
Recently, Gao et al. (2020) and Multicenter collaboration group of Department of Science and Technology of Guangdong Province and Health Commission of Guangdong reported that 500mg twice daily administration of chloroquine for the treatment of SARS-CoV-2 pneumonia cause reduction in the duration of stay in hospital for treatment. So, chloroquine was included in the guidelines to treat and prevent this life threatening SARS-CoV-2 pneumonia.99 To meet the urgent need of treatment for life threatening corona virus-19, hydroxychloroquine and chloroquine are the drugs that would be recommended to prevent SARS-CoV-2 pneumonia.97,99 In the light of previous experiments in respect to antiviral research with chloroquine and hydroxychloroquine, the scientific community of the whole world should consider this useful information to prevent COVID-19.
11.3 Care
Risk of infection from above described virus can be reduced to maximum by care and adopting following precautionary measures. Wash hands with soap and use sanitizer regularly. Avoid handshake and close contact with infected person and don’t visit outside, crowded places and gatherings. Cover your mouth with mask. Cough and sneeze under elbow to protect others.
12 SUMMARY
This review sum-ups the current and actual findings of COVID-19 infection along with pathogen structure, transmission, characteristics, antigenicity, treatments and extensive care. The most common symptoms and clinical features of this infection were also explained. Only biopsy report for the pathological characteristics of this infection was studied and reported in this review but autopsy report will be added in future work by further analysis. As this infection is spreading all over the world so rapidly. WHO issued health emergency for public and reported current confirmed cases, overall death rate regarding this infection minute by minute. Currently no vaccine and specific treatments have been discovered for this dangerous infection. But the best strategy to overcome this infection is to control the source of infection, protecting people, decreasing the transmission by avoiding gatherings and following social distancing instructions. Infected people should be quickly traced, isolated from others and treated timely. People who were in close contact with the infected ones should be suggested for quarantine. Healthy people should be alert and sensible to protect themselves by staying at home and follow precautionary measures as well. Authorities should strictly address people to stay at home, avoid close contact and gatherings. Institutions should be closed further. These precautionary measures will be very helpful to overcome this worldwide pandemic. Further research will be focused on the alternative treatments and tests for early diagnostics, development of vaccine and useful drugs to control this pandemic rapidly.
CONFLICT OF INTEREST
All the authors are volunteer for this review without any payment from any organization.