Pathogenesis, Diagnosis and Possible Therapeutic Options for COVID-19

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread so rapidly and severely affected the people of almost every country in the world. The highly contagious nature of this virus makes it difficult to take control of the present pandemic situation. With no specific treatment available, the coronavirus disease 2019 (COVID-19) presents a threat to people of all ages including the elderly people and people with other medical complications as a vulnerable group to this disease. Better understanding of viral pathogenesis, appropriate preventive measures, early diagnosis and supportive treatments of the infected patients are now the general solutions to fight against this viral transmission. But, as an emerging disease, most about it remains still poorly understood. This article holds an overview on the origin and structure, pathogenesis, diagnosis and possible therapeutic options for the causative agent, SARS-CoV-2 and disease, COVID-19. However, few therapeutic options, laboratory experiments and other strategies proposed here need to be further clinically tested.


Introduction
The severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) known to cause coronavirus disease 2019 , is rapidly spreading from its origin in Wuhan City of Hubei Province of China to the rest of the world [1]. Over the past few decades, a large number of people have been affected with the 3 epidemics caused by coronavirus family in the world. In the previous epidemics, initial hotspots of diseases were Middle East, Saudi Arabia (Middle East Respiratory Syndrome coronavirus (MERS-CoV) and China (SARS-CoV) where animal to human, and then human to human transmissions of pathogens were reported in other countries [2] [3]. For COVID-19, as suggested by epidemiological evidence in China, this outbreak began from a seafood and live animal shopping center in Wuhan, Hubei Province on December 12, 2019 [4]. Number of COVID-19 cases has risen substantially in the world compared to SARS and MERS, and it would probably take longer to halve the disease cases; meaning that control measures would have to be in place for a longer period of time [5]. On 12 March, 2020, WHO declared COVID-19 as pandemic pointing the spread of the virus in more than hundred countries [6]. Coronaviruses are a group of highly diverse, enveloped, positive-sense, single-stranded RNA viruses. They cause several diseases of respiratory, enteric, hepatic and neurological systems with varying severity among humans and animals [7][8]. Human coronavirus (CoV) infections have traditionally caused a low percentage of annual respiratory infections. According to serology and genome phylogeny, coronaviruses are classified into four genera termed Alpha, Beta, Gamma and Delta coronavirus. So far, seven human coronaviruses have been determined, containing two alpha CoVs (HCoV-229E and HCoV-NL63) and five beta CoVs (HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV [9]. Over the past two decades, three coronaviruses, SARS-CoV,MERS-CoVand SARS-CoV-2 have emerged and caused severe human diseases [10] [11].There are some overlapping and discrete aspects of the pathology and pathogenesis of these coronaviruses which cause severe lung diseases in humans owing to death in critical case [12]. As of April 13, 2020, SARS-CoV-2 has spread in 210 countries and territories around the world and 2 international conveyances and taken more than one hundred thousand lives with almost 1.8 million more infected cases [13]. Understanding the pathogenesis of SARS-CoV-2 infection, early diagnosis and supportive treatments are crucial to combat the outbreak of this highly contagious virus. This article includes an overview on molecular pathogenesis, diagnosis and possible therapeutic options for COVID-19 which should hold a scientific understanding necessary to prevent the viral transmission.
Some primary reports state that two species of snakes also could be probable reservoir of disease COVID-19. However, there exists no strong evidence of coronavirus reservoirs other than mammals and birds. Again, genomic analysis predicts that there is another source which is significantly identical with the viral RNA sequence. Pangolin-CoV and SARS-CoV-2 have been proved to have 99% similarity in the receptor -binding domain of the S protein [21][24] [39].
Inspite of all these, thesimilarity of pangolin-Cov to SARS-CoV-2 is not as much as the analogy of RaTG13 to SARS-CoV-2 [40]. That is because pangolin-CoV shares only 92% of their complete genomes with SARS-CoV-2. Hence, it is inadequate to prove the effectiveness of pangolin as an intermediate host. A metagenomic study of 2019 depicts that,SARS-CoV was the most widely distributed coronavirus among a sample of Sunda pangolins [41]. Thus, they might act as an intermediate host and went through recombination and then infected humans. proteins. At the 5′-terminus of the genome, two genes are located namely: orf1ab (encodes pp1ab protein) and orf1a (encodes for pp1a protein). Altogether, they constitute 16 non-structural proteins (nsps) (nsp1-nsp10 and nsp12-nsp16) (Figure 01). Genes at the 3' terminus encode for structural proteins including Hemagglutinin Esterase (HE) (found in beta-CoVs), Spike (S), Small Membrane (E), Membrane (M), Nucleocapsid (N) and Internal (I) protein. Nucleocapsid protein complexes and viral RNA and together develop a helical capsid shape. Formation of peplomers integrated in the envelope and finally giving it a corona or crown shape peplomers which is done by Spike protein trimers [38] [42]. In some cases, trasmembrane protein HE forms small spikes. "M" and "E" protein work for virus assembly. Structural proteins also include eight accessory proteins: 3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14. If compared at amino acid level, the sequence of SARS-CoV-2 is almost same as that of SARS-CoV. Still, some significant dissimilarities between them were found. For instance: The 8a protein is common for SARS-CoV, but in 2019-nCoV, it is absent. The 8b protein of SARS-CoV has 84 amino acids, whereas, that of SARS-CoV-2 is much longer(121 amino acids). The 3b protein of SARS-CoV comprises of 154 amino acid, but in case of 2019-nCoV, it is very much shorter(only 22 amino acids).
Through further studies, it is possible to determine how the differences influence the activity and degree of pathogenicity of SARS-CoV-2 [42] [43].

Mode of SARS-CoV-2 Transmission and Viral Entry Inside the Cell
Wild animals including bats are the possible hosts and reservoirs of the SARS-CoV-2. The human-to-human transmission of SARS-CoV-2 is achieved mainly via respiratory droplets of an infected individual. However, the virus can also be transmitted through the aerial droplets and contact even with an asymptomatic COVID-19 patient [44]- [46]. The presence of this virus has also been reported in the feces of the COVID-19 patient but whether the virus from feces can cause the disease or not is still poorly understood [47].
After entering the human body, SARS-CoV-2 first enters the cells of the host before replication.
The first step in viral entry to human cell is the binding of a viral trimeric protein called spike protein with the human receptor angiotensin converting enzyme 2 (ACE2) (Figure 02). These spike proteins protruding from the membrane of the virus are responsible for the characteristic shape of the virus. TheACE2 receptor is responsible for the entry of both SARS-CoV-2 and SARS-CoV inside the cells of human body [48]. The viral spike proteins mediate the van der Waals interaction with ACE2 receptors during the viral entry which is a critical step in the manifestation of viral infection. Another type of proteins called transmembrane protease serine 2 (TMPRSS2) is required for initial priming of the spike protein with the ACE2 receptor [49].
After the receptor binding and processing by TMPRSS2 the virus enters the host cell via endocytosis.The SARS-CoV-2 and SARS-CoV spike proteins have almost 77% sequence identity and importantly a high degree of homology [50]. Recent study suggests that, the SARS-CoV-2 spike protein can bind with ACE2 receptors more effectively than that of SARS-CoV and hence the SARS-CoV-2 might be more effective in invading the human cells than SARS-CoV [51]. After entering the cell, the virus triggers replication to produce multiple copies of viral materials and after assembly it lyses the infected cells to get out in multiple copies and continue the infection of more healthy cells.

Replication of Coronavirus inside Human Body
The

Infection
As Transforming growth factor β (TGFβ) and chemokines i.e., CCL2, CXCL10, CXCL9, and IL-8 were also found in elevated amounts in severe SARS disease patient compared to mild patient.
A recent study has found increased expressions of cytokines i.e., IL-6, IL-10, and TNFα, lymphopenia in both types of T cellsbutdecreased expression of IFN-γ in patients with severe COVID-19 [71]. Another study has found significantcorrelation between elevated level of

Diagnosis of COVID-19 Patient
Initially the COVID-19 patients are identified on the basis of presence of clinical symptoms associated with primary stage of disease progression. But confirmatory test of the COVID-19 patient can be achieved by variety of methods i.e., nucleic acid-based identification, computed tomography scan (CT scan), immune-reaction based techniques in the laboratory (Table 02).

Real-time Quantitative Polymerase Chain Reaction (RT-qPCR)
RT The amplification of any of these gene can be achieved by providing appropriate forward and reverse primers. A recent study has found 95% sensitivity of the PCR amplification of E and RdRp gene rom SARS coronavirus [76]. Another study suggested that, the presence of SARS-CoV-2 virus can be detected from the stool of the infected patient using the nucleic-acid based detection techniques. Again, the same study found negative results with both nucleic acid-based tests and test kits from the urine and blood sample of the patient. This is might be due to the low concentration of the virus in those samples [77]. Another study found 91.7% positive rates (11/12 patients) in RT-qPCR test using saliva sample from COVID-19 patients, suggesting that saliva could be a non-invasive sample from the infected patient [78]. However, although the RT-qPCR is an effective technique for the identification of SARS-CoV-2 virus from COVID-19 patients but it may come out with false positive results sometime and the technique itself is highly laborious and involves the management of biohazards.

CT Scan Imaging
Although the RT-qPCR is a specificSARS-CoV-2 detection technique but the chance of false positive results can not be ignored. Therefore, many clinicians offer the use of CT scan but use of the combination of both techniques is more helpful and thus CT scan can assist the mass identification in a heavilyinfected area. A recent study suggested that, CT scan is more sensitive in the detection of SARS-CoV-2 virus from COVID-19 patient than the PCR-based method [79]. But other lung abnormalities i.e., lung cavitation, discrete pulmonary nodules, pleural effusions, and lymphadenopathy were absent [83] [84]. Although, these findings suggest CT scan to be an effective identification method of SARS-CoV-2 but it also has few disadvantages i.e., incapability to discriminate between the abnormalities caused by pneumonia and other lung diseases and COVID-19 [85].

Immune Reaction-based Test Kits
In addition to the above described techniques, antigen-antibody (Ag-Ab) immune-reaction based kits provide more cost-effective and rapid diagnosis method. A recent study has proposed a detection method with proven 88.66% sensitivity and 90.63% specificity in laboratory experiment. The technique utilizes immune reaction of combined human antibodies i.e., immunoglobulin M (IgM) and IgG with SARS-CoV-2 coronavirus spike proteins from the blood sample of COVID-19 patient. This technique also showed consistent result for serum and plasma from venous blood and fingerstick blood sample [86]. Enzyme-linked immunosorbent assay (ELISA) based on the immune reaction can also be used for the detection of SARS-CoV-2. A study has suggested that the ELISA technique is capable of detecting the virus using recombinant nucleocapsid and spike viral proteins with positive rates of 80.4% and 82.2%, respectively.
However, the positive rate of the experiment was less (60%) during the initial stage (0 to 10 days) of the infection which might be due to the low concentration of antibodies in blood sample [87]. Thus, these techniques could involve false negative results in some cases.

Major Drug Targets against SARS-CoV-2
Different proteins involved in different stage of vial life cycle of SARS-CoV-2 can serve as potential therapeutic targets for designing antiviral drugs ( Table 03). The best possible therapeutic options for SARS-CoV-2 are 3-chymotrypsin-like protease, papain-like protease, helicase, and RNA-dependent RNA polymerase, structural proteins i.e., spike glycoprotein.
These proteins are essential for the maintenance of the virus life cycle and hence recognized as most attractive drug targets against SARS-CoV-2 [88]. Analysis of the genome sequences of the catalytic sites and key-drug binding sites of these enzymes suggest high degrees of sequence similarity with those from SARS and MERS coronaviruses. And thus, the known inhibitors of these enzymes from SARS and MERS coronaviruses could be repurposed for the possible treatments of COVID-19 [89]. Among these, the Spike protein of the coronavirus attaches to the angiotensin-converting enzyme 2 (ACE2) host receptor which facilitates the viral fusion and entry inside the host cell. So, the blocking of the interaction of the spike protein with the ACE2 receptor with specific inhibitor provides a strategy for potential antiviral development [90].   μM, respectively in the same experiment [100]. Ivermectin, another FDA-approved drug is capable to inhibit the SARS-CoV-2 viral replication effectively [101]. Another recent study has shown that the ribonucleoside analog β-D-N 4 -hydroxycytidine has antiviral activity not only against SARS-CoV-2 but also on other multiple bat coronaviruses including SARS and MERS coronaviruses. This analog is orally bioavailable and has increased potency against a coronavirus bearing resistance mutations to the nucleoside analog inhibitor Remdesivir [102].

SARS-CoV-2 infection of lung cells depends on cell membrane protease and Camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells. This inhibitor is also
effective to inhibit the SARS and MERS coronavirus entry inside the lung cell [103]. A clinical study suggested that the combination of Lopinavir and Ritonavir showed notable therapeutic benefits to COVID-19 patients compared to using pneumonia-associated adjuvant drug alone [104]. These two drugs are also being tested in clinical trials now although, another clinical experiment claimed that no benefits were observed in hospitalized COVID-19 patient beyond the standard care using the combined medication [ In vitro experiment. [100]
In vitro experiment.
Alleviates the COVID-19 condition in combination with Lopinavir.
In vitro and clinical experiment (Under trial) [103][104] [107] Table 04: Different drugs that were found to act against SARS-CoV-2 in different laboratory and clinical experiments. General information was retrieved from DrugBank [108].

Repurposing Options of Drugs and Other Candidates for SARS-CoV-2
Drug repurposing is an effective drug discovery strategy from existing drugs, which could shorten the time and reduce the cost compared to de novo drug discovery.Since, different structural proteins and overall genome sequences of all three SARS-related coronaviruses have significant similarities and hence, it is logical that the drugs which work on previous two SARSrelated coronaviruses might work on novel coronavirus as well [109].
Another laboratory experiment on a series of flavonoid derivatives showed different Biflavonoids can have potential inhibitory effects on 3CL pro of SARS-Cov-2. Among different Biflavonoids, Amentoflavone was reported to have better 3CL pro inhibitory activity with IC50 value of 8.3 μM [112].
Helicase is the enzyme responsible for the unwinding of double stranded nucleic acid complex during replication and the inhibition of this enzyme prevents the viral reproduction cycle.
Bannanin and its different derivatives (Iodobananin, Vanillinbananin and Eubananin) can effectively inhibit the SARS-CoV helicase activity [113]. Nucleoside analogs incorporate inside the extending DNA strand during the replication and terminates the replication process and thus inhibits the viral replication cycle. A nucleoside analog, β-D-N 4 -hydroxycytidine was shown to inhibit the SARS-CoV viral replication with an approximatedEC50 of 10 µM in a laboratory experiment [114].
PL pro is an enzyme which cleaves the initial polyprotein complex is a potential drug target for SARS-CoV-2. Different Tomentin derivatives were shown to inhibit the PL pro enzyme with IC50 value ranging between 5.0 and 14.4 μM [115].

Therapeutic Monoclonal Antibody Preparation
Native virions present the most poten immunogen or antigen to be used in immunization or monoclonal antibody preparation [116]. Coronavirus spike protein is the most antigenic protein which interacts with ACE2 receptor and this interaction mediates the viral entry inside the host cell as mentioned earlier. Monoclonal antibodies (mAbs) provide excellent therapeutic option to prevent the SARS-CoV viral entry inside the host cell by interfering with the ACE2-Spike protein interaction [117].
A SARS-CoV-specific human monoclonal antibody, CR3022, has been proven to bind the receptor binding domain (RBD) of SARS-CoV-2 spike protein in a recent laboratory experiment.
This finding suggests that CR3022 alone or in combination with other antibodies could be potential therapeutic option for COVID-19 treatment [118]. F26G18 and F26G19 are two different antibodies which can effectively bind with RBD of SARS-CoV spike protein in vitro [119]. However, the RBD shows high rates of mutations and therefore, might escape the mAbsmediated neutralization in some cases. Therefore, other antibodies which are specific todomains except RBD i.e., two conserved heptad repeats (i.e. HR1 and HR2) of Spike protein can provide better therapeutic benefits [120]. Moreover, S230 is another monoclonalantibody which has recently been shown to block the SARS-CoV viral entry inside the cell by blocking the interaction with S1 subunit of spike protein with the cell membrane receptor ACE2 [121].
Beside these, immune sera from convalescent patients have been shown to be effective in the treatment of patients infected with SARS-CoV-2 making passive immune therapy with human monoclonal antibodies an attractive treatment option for COVID-19. So, the simplest and most direct way based on mAbs therapeutic interventions to treat the COVID-19 patientwould be to use the plasma from convalescent patients [122]. Thus, different mAbs could be used to alleviate the COVID-19 condition.

Therapeutic Intervention with Interferon
Recombinant human interferons were shown to be effective in treating SARS-CoV patient immediately after the SARS-CoV outbreak. IFN-β was shown to be more effective in a laboratory experiment followed by IFN-γ and interferon IFN-α in inhibiting viral replication in different cell lines [123]. In yet another in vitrostudy, IFN-β 1a was proven to inhibit the SARS-CoV replication in Vero E-6 cell line [124].
Falzarano et al. reported the efficacy of a combination containing IFN-α2β and Ribavirin on a novel β coronavirus, nCoV, as the causative agent of severe respiratory illness in humans originating in Saudi Arabia, Qatar and Jordan. They found both interferon-α2b and Ribavirin alone and the combination is effective against nCoV. However, the combination of interferon-α2b and Ribavirin together showed slightly better effect [125]. Conversely, SARS-CoV ORF 3b, ORF 6, and nucleocapsid proteins were reported to inhibit a key protein required to express IFN-β, IFN regulatory factor 3 (IRF-3) in host cell in a distinct in vitro study [126].
A recent study has suggested interferon β as the most suited option for the treatment of COVID-19 and also SARS-CoV-2 might have more sensitivity towards IFN than other SARS-related coronaviruses [127]. Another in vitro study about the effects of interferon on SARS-CoV and SARS-CoV-2 has found that the pretreatment of viral infected cells with type I interferon (IFN-I) resulted in much higher decrease in SARS-CoV-2 virus titer than that of SARS-CoV titer in 48 hours. A deficit in the viral nucleocapsid protein was observed after the treatment with IFN-I.
The experiment also suggested that SARS-CoV-2 might be more sensitive to IFN-Itreatment and this is might be due to the change in viral protein [128].

RNA Interference
RNA interference (RNAi) is a conserved biological response to double stranded RNA that is commonly used in post-transcriptional gene silencing [129]. This technique utilizes 19-23 base pair double-stranded RNA to degrade targeted RNA in a specific and stepwise manner [130].
RNAi is commonly used to generate gene-knockouts and study gene function in different organisms. RNAi assisted inhibition of replication has been reported in many viruses including those that infect human in different studies [131].
RNAi technique was also studied extensively to inhibit different viral proteins of SARS-CoV in different laboratory experiments. It is capable of inhibiting the SARS-CoV spike protein encoding gene expression in HEK 293T cells as proven in in vitro experiment [132]. In another distinct laboratory experiment, designed small interfering RNA (siRNA) was directed against three subgenome RNA of SARS-CoV. Significant reduction in the expression of the targeted RNAs was observed without interfering the expression of other RNAs [133]. The SARS-CoV viral replication can be effectively reduced as evident with 85-90% reduction in viral genome RNA using siRNA-based RNAi technology in vero E6 cells in laboratory experiment [134].
Moreover, a previous study carried out on animal model using rhesus macaque (Macaca mulatta) showed significant improvement in SARS-severity in the subject animals rather than the controls upon siRNA-based therapy [135].

Vaccine
Vaccination is one of the most efficient ways to prevent viral diseases. A vaccine helps the body's immune system to recognize and fight pathogens like viruses or bacteria, which then keeps us safe from the diseases they cause [136]. With no specific antiviral drug and specific vaccine for COVID-19, the development of such one is urgently required but it would take a significant period of time. Moreover, there are significant hurdles to overcome during the development of vaccine against SARS-CoV i.e., immunopotentiation in the form of eosinophilic infiltration or increased infectivity, target population on which the vaccine will work and the mutation rate of the virus.
There are different types of vaccines i.e., live vaccines, attenuated vaccines, Inactivated/killed vaccines, Subunit and conjugate vaccines, Toxoid vaccines [137]. In the case of COVID- 19,79 vaccine candidates are in active development (confirmed as of early April 2020), 74 were not yet in human evaluation with only five in phase I clinical trial.
As reported by Coalition for Epidemic Preparedness Innovations (CEPI) scientists in April, 2020, 115 total vaccine candidates are in early stages of development, with 78 confirmed as active projects (79, according to the Milken Institute), and 37 others announced, but with little public information available (presumed to be in planning or being designed) [138][139].
In April after the CEPI report was published, Phase I-II randomized, interventional trials for dosing and assessment for side effects began in Wuhan, China on the candidate vaccine, Ad5-nCoV (CanSino Biologics) [140], and in England on the candidate, ChAdOx1 nCoV-19 [141]. Table 05 and 06 enlist most updated information about the vaccine development progresses in clinical and preclinical phases in different companies and industries.
Phase I trials test primarily for safety and preliminary dosing in a few dozen healthy subjects, while Phase II trials, following success in Phase I, evaluate effective dose levels and side effects of the candidate vaccine in dozens-to-hundreds of people either with the targeted disease or in healthy subjects. A Phase I-II trial conducts preliminary safety and dosing testing, is typically randomized, placebo-controlled, and at multiple sites, while determining more precise, effective doses [142]. Therefore, due to the extended period of clinical phases and additional manufacturing steps, it will certainly take a while for the vaccines to be available in the market.

Concluding Remarks
After CoV-2 but these are not clinically proven and hence need further evaluation. Of course, a definitive cure which can help patients recover will be available soon.

Conflict of Interest
Authors declare no conflict of interest regarding the publication of the manuscript.