Current Understanding of Novel Coronavirus: Molecular Pathogenesis, Diagnosis, and Treatment Approaches

: An outbreak of “Pneumonia of Unknown Etiology” occurred in Wuhan, China, in late December 2019. Later, the agent factor was identiﬁed and coined as SARS-CoV-2, and the disease was named coronavirus disease 2019 (COVID-19). In a shorter period, this newly emergent infection brought the world to a standstill. On 11 March 2020, the WHO declared COVID-19 as a pandemic. Researchers across the globe have joined their hands to investigate SARS-CoV-2 in terms of pathogenicity, transmissibility, and deduce therapeutics to subjugate this infection. The researchers and scholars practicing different arts of medicine are on an extensive quest to come up with safer ways to curb the pathological implications of this viral infection. A huge number of clinical trials are underway from the branch of allopathy and naturopathy. Besides, therapy and nano-medicine protocols has to be optimized for better clinical and functional outcomes of COVID-19-affected individuals. This article unveils a comprehensive review of the pathogenesis mode of spread, and various treatment modalities to combat COVID-19 disease. expression of ACE2, and TMPRSS2 assists in S protein priming, leading to enhanced binding of SARS-CoV-2 and higher proinﬂammatory cytokine release. The higher expression of ACE2 is reported to affect SRC and RPS3, the two crucial genes engaged in inﬂammatory responses and viral replication. Further, the expression of ACE2, at the same time, is enhanced by the infection. It is also evident that at the same time, SARS-CoV-2 may downregulate ACE2 expression, which further contributes to an increase in angiotensin-2-induced lung injury. The ACE2-mediated negative regulatory activity is curtailed by SARS-CoV-2 and contributes to aggravating the severity of illness.


Introduction
The city of Wuhan in China witnessed an outbreak of pneumonia caused by a novel coronavirus in the late winter of December 2019, and with a rampant spread throughout China, it periled the world to emerge as a pandemic [1]. The etiological agent of the disease was originally called as 2019 novel coronavirus (2019-nCoV), but later it was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the disease process as Coronavirus Disease-2019 (COVID-19) by the WHO [2]. Later, SARS-CoV-2 was declared an International Public Health Emergency by the WHO on 30th January 2020. As of 14th March 2021, the world has encountered 120 million confirmed cases and 2.6 million deaths reported among them (source: Worldometer, www.worldometers.info). The transmitting capacity of SARS-CoV-2 is beyond that of its ancestral SARS-CoV that caused an outbreak of SARS in 2003 [3]. The escalation in the number of confirmed COVID-19 cases forecasts the prevailing scenarios gloomily and warrants stringent preventive and control measures. The clinical spectrum and manifestations involve the respiratory system undergoing the major brunt of COVID-19 infection; however, damage to the cardiovascular system has been reported by some patients [4]. Some patients with existing cardiovascular diseases (CVDs) may have a higher risk of associated mortality. Therefore, it is equivalently important to understand the mechanisms and extent of damage to the cardiovascular system by SARS-CoV-2 infectivity and come up with an effective treatment for these patients while minimizing the associated mortality.
Currently, there is no definite antiviral therapy developed for treating SARS-CoV-2 infection. The mainstay of treatment is rendering supportive care, as per the presenting complaints of the patient. Even treating with a combination of recombinant Interferon (IFN) and Ribavirin has been reported to show limited response against SARS-CoV-2 infection [5,6]. Following the episodes of SARS and MERS epidemics, persistent efforts have been made in streamlining newer antiviral agents to target the viral proteases, polymerases, MTases, and entry proteins respectively. However, none proved to be fruitful in terms of effectivity during the clinical trials [7][8][9]. With the flattening of the COVID-19 curve of infectivity, immunotherapies involving plasma and antibodies procured from convalescent patients have been proposed to address the present circumstances [10]. Besides, various vaccine strategies involving utilization of inactivated viruses, live-attenuated viruses, viral vector-based vaccines, subunit vaccines, recombinant proteins, and DNA vaccines have been evaluated in animals and are emerging as a preventable strategy [11,12]. Due to the lack of traditional licensed treatment or vaccine, the infection mandates implementation of infection control measures with establishing an early diagnosis, reporting, isolation, and rendering supportive treatments effectively. Individualized efforts by inculcating the healthy practice of hand hygiene, respiratory hygiene and cough etiquette, wearing well-fitted face masks, and avoiding crowded places will supplement for the same.
Various therapeutic strategies have been used to treat this viral infection, but there have been unsuccessful reports recorded so far. However, natural products including plant-derived compounds and drugs can be a potential therapeutic approach to combat its growth and spreading capacity in the future. In this review, we have discussed the various pathological aspects including synthesis, maturation, infectivity, and diagnostic prospects of COVID-19. Additionally, we have also highlighted the recently used drugs and vaccines in combating SARS-CoV-2 infection.

Classification, Origin, Primary Reservoirs, and Hosts of Coronavirus
Adetailed outline of the source and transmission is important for developing preventive strategies for containing the spread of the infection. SARS-CoV-2 belongs to the order Nidovirales and family Cornaviridae. The family comprises two subfamilies, i.e., Torovirinae and Coronavirae. Subsequently, the members of the sub-family Coronaviridae are further divided into four genera: (i) alpha coronavirus, 229E, and NL63 are the human coronaviruses that are responsible for croup and the common cold, (ii) In comparison, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, and SARS-CoV are classified as beta coronaviruses, (iii) Gamma coronavirus includes viruses of whales and birds, and (iv) Delta coronavirus comprises viruses isolated from birds and pigs [13]. These viruses infect a wide variety of host species, and diversity among these viruses leads to different pathological outcomes. The coronaviruses with pathological manifestations have been enumerated in Table 1 for a better understanding of the same. SARS-CoV-2 belongs to the beta coronavirus family as that of extremely pathogenic viruses such as SARS-CoV and MERS-CoV. It is a positive-sense single-stranded RNA (+ssRNA) and an enveloped virus. SARS-CoV-2 is regarded as a novel beta coronavirus infecting humans [14]. Indications have been received from the phylogenetic analysis of the SARS-CoV-2 genome suggesting its close relation (with 88% identity) to two coronaviruses that were bat-derived, SARS-like, and were collected in eastern China in 2018 (bat-SL-point of exposure for infected individuals: a seafood market in Wuhan, Hubei Province, China. The restaurants associated with this market are famous for providing diverse kinds of wild animals for consumption [29]. The role played by the Hunan Seafood Wholesale Market in spreading the disease is not clear yet. However, the Huanan South China Seafood market also trades in live animals, including bats, poultry, marmots, bats, and snakes [25]. This might be a point of zoonotic transmission [29]. Albeit, SARS-CoV-2 is alleged to have zoonotic origination with further human-to-human transmission, but the likelihood of its fecal-oral transmission mandates to rule out the same with additional epidemiological investigations [30]. Though evidence has been provided through a genomic study that the introduction of the virus into the Hunan Seafood Market took place from a yet unknown location, and from there on, it started spreading extremely quickly, but the human-tohuman transmission might have started earlier [31]. In addition, it might transmit through direct contact as in other respiratory viruses, such as by shaking contaminated hands or exposure to contaminated surfaces (fomite transmission). Still, other potential routes of SARS-CoV-2 transmission such as blood transfusion, organ transplantation [32], and trans-placental and perinatal transmission need to be adduced more concretely.
The clusters of infected medical personnel and family members have confirmed the occurrence of person-to-person transmission [26]. The aforementioned transmission seemingly occurs among close contacts majorly through respiratory droplets produced upon coughing or sneezing by an infected person. The findings regarding the persistence of SARS-CoV-2 on surfaces for up to 96 h and other coronaviruses for up to 9 days account for a larger aspect of fomite transmission of infection [33,34]. However, the asymptomatic transmission of the infection remains controversial. An initial study that was published on 30 January 2020 reported on asymptomatic transmission, but it has been subsequently found that the researchers failed to direct interview the patient who certainly showed symptoms before disease transmission [35,36]. Another recent study, published on 21 February, also mentioned asymptomatic transmission; however, such studies can be limited due to errors in self-reported symptoms or unknowingly coming in contact with other fomites and cases [37]. The findings regarding the characteristics of the disease have been changing rapidly, and it is important to note that all are subjected to a selection bias. The average incubation period has been pegged at 5.2 days with a 95% confidence interval (95% CI) of 4.1-7.0 days, while the other case studies have reported around 19 or 24 days respectively [38][39][40]. However, the definition of the case largely relies on a window of 14 days [41]. Estimation of the basic reproductive number (R 0 ) has been done with varying interpretations and results. The average number of infections that can spread from a single infected individual in a fully susceptible population is measured by R 0 [42]. R 0 was found to be 2.7 for SARS from studies conducted on prior outbreaks and 2.4 for pandemic H1N1 influenza in 2009 [43,44]. Another study estimated the R 0 , was 2.2 (95% CI: 1.4-3.9) [40]. However, on further analysis of other 12 available studies, the R 0 was found out to be 3.28 [45]. Though it must be taken into consideration that R 0 only represents an average value, the role of super-spreaders cannot be neglected, as they can be significantly responsible for an outbreak within large clusters, and they would otherwise not affect the value of R 0 significantly [46]. Moreover, R 0 might be unstable during the acute phase of the pandemic [42]. Notably, the comparison between epidemiological profiles of viral infections is enumerated in Figure 1. A study conducted on nine pregnant women who had developed COVID-19 during the later stages of their pregnancy suggested that the development of symptoms in pregnant persons is not any worse than that in nonpregnant persons, and there is no corroborating evidence for vertical transmission causing intrauterine infection [47]. A hospital-based study comprising 138 COVID-19 patients suggested that 41% of patients had a hospital-associated transmission of SARS-CoV-2 infection. Another study suggested a gradual increase in the percentage of cases among health care workers with due course of time [40]. The exposure to a higher concentration of virus due to prolonged contact in proximity is reflected from these case studies. causing intrauterine infection [47]. A hospital-based study comprising 138 COVID-19 patients suggested that 41% of patients had a hospital-associated transmission of SARS-CoV-2 infection. Another study suggested a gradual increase in the percentage of cases among health care workers with due course of time [40]. The exposure to a higher concentration of virus due to prolonged contact in proximity is reflected from these case studies.

Genome Structure and Life Cycle
The incorporation of three major structural proteins defines the complex structure of the virus. The three proteins accounting for its structure are nucleocapsid protein N that is internally phosphorylated; glycoprotein S, representing the spike; and glycoprotein M, which is an unusual transmembrane protein [48]. The bulky spike, which is found in the viral envelope with peplomers ranging between 15 nm to 20 nm and is represented by the glycoprotein S of 200K [49]. Moreover, the minor transmembrane protein E is also present in the structural region. An envelope protein that performs both the functions of hemagglutination and esterase (HE) is found in several species of coronaviruses [50]. Possessing a genome size of 30 kb, these are single-stranded RNA viruses with a positive-sense RNA [51]. While the 5' end is capped, the 3' terminus is reportedly infectious and is polyadenylated. The expression of individual genes takes place through a complicated process, due to its bigger size, where at the 5' end sequence, the release of the sets of nested mRNAs occurs. Extensive rearrangements facilitate the recombination of heterologous RNA. The leader RNA, which contains 65 to 98 nucleotides in an untranslated (UTR) sequence,occupies the 5' end of the genome along with the 5' end of the remaining subgenomic mRNAs. At the 3' end of the genome, the poly-A tail incorporates and follows another UTR region comprising 200 to 500 nucleotides. These two UTR regions are accountable for regulating the RNA transcription and replication processes. The genome of the coronavirus contains 7 to 14 open reading frames (ORFs). The beginning portion of the genome has gene one, which is 20-22 kb in length, and spreads across two-thirds of the genome. There are two ORFs, i.e., 1a and 1b, present in this portion, and these ORFs overlap and collectively function as the viral RNA polymerase (Pol). In the genome in this series, four significant structural proteins are incorporated, i.e., 5'-S (spike)-E (envelope)-

Genome Structure and Life Cycle
The incorporation of three major structural proteins defines the complex structure of the virus. The three proteins accounting for its structure are nucleocapsid protein N that is internally phosphorylated; glycoprotein S, representing the spike; and glycoprotein M, which is an unusual transmembrane protein [48]. The bulky spike, which is found in the viral envelope with peplomers ranging between 15 nm to 20 nm and is represented by the glycoprotein S of 200K [49]. Moreover, the minor transmembrane protein E is also present in the structural region. An envelope protein that performs both the functions of hemagglutination and esterase (HE) is found in several species of coronaviruses [50]. Possessing a genome size of 30 kb, these are single-stranded RNA viruses with a positivesense RNA [51]. While the 5' end is capped, the 3' terminus is reportedly infectious and is polyadenylated. The expression of individual genes takes place through a complicated process, due to its bigger size, where at the 5' end sequence, the release of the sets of nested mRNAs occurs. Extensive rearrangements facilitate the recombination of heterologous RNA. The leader RNA, which contains 65 to 98 nucleotides in an untranslated (UTR) sequence, occupies the 5' end of the genome along with the 5' end of the remaining sub-genomic mRNAs. At the 3' end of the genome, the poly-A tail incorporates and follows another UTR region comprising 200 to 500 nucleotides. These two UTR regions are accountable for regulating the RNA transcription and replication processes. The genome of the coronavirus contains 7 to 14 open reading frames (ORFs). The beginning portion of the genome has gene one, which is 20-22 kb in length, and spreads across two-thirds of the genome. There are two ORFs, i.e., 1a and 1b, present in this portion, and these ORFs overlap and collectively function as the viral RNA polymerase (Pol). In the genome in this series, four significant structural proteins are incorporated, i.e., 5'-S (spike)-E (envelope)-M (membrane)-N (nucleocapsid)-3' (Figure 2) [52]. The structural proteins are the key player in the virion'sassembly and defining the viral infectivity. The homotrimers of S proteins constitute the spikes on the viral surface and facilitate attachment to the host receptors [53,54]. There are three transmembrane domains in the M protein that play a key role in shaping the virions, promoting membrane curvature, and binding to the nucleocapsid [55,56]. The E protein takes part in the process of assembly and release of the virus along with its involvement in the viral pathogenesis [57,58]. The N protein has two domains with the potentiality of binding the virus RNA genomic material through different mechanisms. It has been reported that N protein can bind to nsp3 protein to tether the genome to the replicase-transcriptase complex (RTC) and packaging the encapsidated genome into virions, respectively [59][60][61]. This N protein has also been found as an antagonist of the interferon (IFN) and the viral encoded repressor of RNA interference to perquisite the process of viral replication [62]. Within these genes, there are various other ORFs coding for other non-structural proteins such as HE glycoprotein. Depending on the features of the gene order, method of expression, and nucleotide sequence, the marking of each gene in coronavirus is distinct, though all of them are conserved amongst the same serogroup. As other coronaviruses encode smaller ORFs in these regions, the SARS-CoV-2 differs from them in the 3' region of the genome regarding the expression of 8 novel proteins that have been marked as accessory proteins, and these ORFs are presently investigated. Both the ORFs, i.e., 1a and 1b, are identically translated into two poly-proteins at the N-terminus while there is no production of the C-terminal identical polyproteins due to frame-shifting. ORF-1b encodes for a multifunctional helicase protein and the viral RNAdependent RNA polymerase (RdRp). This protein holds the 5' triphosphatase, NTPase, and dNTPase activities. While all the structural proteins are not essentially required for the process of viral replication, inactivation of the viral function results from the deletion of one or more structural proteins. ORF-3a has a structural protein as one of its products has a role in the viral biogenesis as it is O-glycosylated, possessing the capability to bind S, N, M glycoproteins together, and is triple-membrane spanning in nature [63].
Coronaviruses encode for Mpro (main protease), also known as 3CLpro, which is a chymotrypsin-like protease and has similarities with the 3C protease of picornaviruses [64]. Sixteen non-structural proteins (nsps) are produced due to the remaining polyprotein being further processed by this protease. Among the nsp 1-16, most of the nsps have been found to have a role in the replication of these viruses. However, the functionality of some nsps is yet to be understood. The known functions of the 16 nsps have been summarized in Table 2. SARS-CoV species of coronavirus have the maximum presence of nsps. Nsp3 is one such nsp that has multiplefunctions and contains both protease and ADP-ribose 1" phosphatase activity [65]. The two proteins nsp7 and nsp8 formulate a cylindrical structure and play a crucial role in synthesizing RNA for coronavirus and single-strand RNA binding protein (nsp9), respectively [66].
The life cycle of these viruses is a multistep event. This includes five steps in the lifecycle of the virus within the host, i.e., attachment, penetration, biosynthesis, maturation, and release [59]. First, the viral S protein attaches to the Angiotensin-Converting Enzyme-2 (ACE2) receptor of the host, then it enters the host cells through membrane fusion or endocytosis. Following the entry, there is aproteolytic cleavage of the virus envelope resulting in the release of genomic RNA in the cytoplasm, and smaller RNAs (sub-genomic mRNAs) are made. The viral RNA enters intothe nucleus for replication once the viral contents enter the host cells. The viral mRNA is used in the biosynthesis procedure for producing viral proteins. The mRNAs further undergo translation procedures to produce several proteins (S, M, N, etc.), which are essentially required for viral assembly. The S, E, and M proteins enter the ER and result in the formation of nucleoprotein complex by combining N protein and genomic RNA (+strand). It is the ER-Golgi apparatus compartment wherein the complete virus particle (proteins and genomic RNA assembly) is formed. Thereafter, the latest viral particles are made and set off through the formation of vesicles and exocytosis, i.e., maturation ( Figure 2). Specific genes are present in all the coronaviruses in ORF1 downstream regions, which encode the proteins for nucleocapsid, viral replication, and spikes formation [102]. On the outer surface of the coronaviruses, there are glycoprotein spikes that facilitate the entry and attachment of the virus to the host cells. The virus can infect multiple hosts due to loose attachment of the receptor-binding domain (RBD) among the virus [103,104]. Other coronaviruses recognize carbohydrates or aminopeptidases as the key receptor for entry into human cells, while SARS-CoV and MERS-CoV recognize exopeptidases [105]. Cellular proteases determine the entry mechanism of a coronavirus which includes cathepsins and transmembrane protease serine 2 (TMPRSS2) and human airway trypsin-like protease (HAT), that split the spike protein and establish further penetration changes [106,107]. As a key receptor, MERS coronavirus employs dipeptidyl peptidase 4 (DPP4), whileACE2 is required as a key receptor by SARScoronavirus and HCoV-NL63 [101,103]. A typical coronavirus structure with spike protein is possessed by SARS-CoV-2, and it expresses nucleoproteins, polyproteins, and membrane proteins such as papain-like protease, helicase, RNA polymerase, glycoprotein, 3-chymotrypsin-like protease, and accessory proteins [15,108]. To maintain the van der Waals forces, the SARS-CoV-2 spike protein comprisesa 3-D structure in the RBD region [109]. The critical lysine 31 residue on the human ACE2 receptor recognizes the 394 glutamine residue present in the SARS-CoV-2 RBD region [110]. The complete mechanism of SARS-CoV-2 pathogenicity, from attachment to replication, has been elaborated in Figures 2 and 3. tease, helicase, RNA polymerase, glycoprotein, 3-chymotrypsin-like protease, and accessory proteins [15,108]. To maintain the van der Waals forces, the SARS-CoV-2 spike protein comprisesa 3-D structure in the RBD region [109]. The critical lysine 31 residue on the human ACE2 receptor recognizes the 394 glutamine residue present in the SARS-CoV-2 RBD region [110]. The complete mechanism of SARS-CoV-2 pathogenicity, from attachment to replication, has been elaborated in Figures 2 and 3

Risk Factors of Coronavirus
Most incidences of SARS-CoV-2 infection are largely observed in adult male patients wherein the patient's median age was between 34 years and 59 years [37,38,111,112]. People suffering from chronic comorbidities like cerebrovascular diseases, cardiovascular ailments, and diabetes are more likely to be infected by the SARS-CoV-2 [113]. In adults 60 years and above, there has been the highest proportion of severe cases along with individuals with existing comorbidities such as cerebrovascular diseases, cardiovascular ailments, and diabetes [37,111]. Co-infections caused by fungi and bacteria may also be associated with severe manifestations [113]. Interestingly, fewer cases of SARS-CoV-2 infection have been reported among children less than 15 years of age [37,38,111,112]. As per a study published on 29th January and conducted in Wuhan on 425 COVID-19 patients, no cases were reported in children under the age of 15 years [40,114]. However, as of March 14th, 2021 according to the American Academy of Pediatrics, the USA alone hosts 3,231,836 pediatric COVID-19 cases, and these cases contribute 13.2% (3,231,836/24,487,634) of all cases. Overall, there are 4294 cases per every 100,000 children in the entire USA population (data retrieved from https://services.aap.org/en/pages/2019novel-coronavirus-covid-19-infections/children-and-covid-19-state-level-data-report/). At a glance, most infected patients showed mild symptoms without fever or pneumonia, having a good prognosis [115]. Notably, a study discovered that a child was asymptomatic despite suffering from radiological ground-glass lung opacities [26]. In a nutshell, children can be less likely to get infected, or even if they are infected, they exhibit milder manifestations as compared to adults. It is more likely that the parents or guardians of such children might not seek treatment, which can lead to underestimation of the instances of the disease in this age group.

Risk Factors of Coronavirus
Most incidences of SARS-CoV-2 infection are largely observed in adult male patients wherein the patient's median age was between 34 years and 59 years [37,38,111,112]. People suffering from chronic comorbidities like cerebrovascular diseases, cardiovascular ailments, and diabetes are more likely to be infected by the SARS-CoV-2 [113]. In adults 60 years and above, there has been the highest proportion of severe cases along with individuals with existing comorbidities such as cerebrovascular diseases, cardiovascular ailments, and diabetes [37,111]. Co-infections caused by fungi and bacteria may also be associated with severe manifestations [113]. Interestingly, fewer cases of SARS-CoV-2 infection have been reported among children less than 15 years of age [37,38,111,112]. As per a study published on 29th January and conducted in Wuhan on 425 COVID-19 patients, no cases were reported in children under the age of 15 years [40,114]. However, as of March 14th, 2021 according to the American Academy of Pediatrics, the USA alone hosts 3,231,836 pediatric COVID-19 cases, and these cases contribute 13.2% (3,231,836/24,487,634) of all cases. Overall, there are 4294 cases per every 100,000 children in the entire USA population (data retrieved from https://services.aap.org/en/pages/2019-novel-coronavirus-covid-19 -infections/children-and-covid-19-state-level-data-report/, accessed on 14 March 2021).
At a glance, most infected patients showed mild symptoms without fever or pneumonia, having a good prognosis [115]. Notably, a study discovered that a child was asymptomatic despite suffering from radiological ground-glass lung opacities [26]. In a nutshell, children can be less likely to get infected, or even if they are infected, they exhibit milder manifestations as compared to adults. It is more likely that the parents or guardians of such children might not seek treatment, which can lead to underestimation of the instances of the disease in this age group.

The Clinical Manifestations of COVID-19
Patients infected with SARS-CoV-2 exhibit clinical manifestations ranging from nonspecific mild symptoms to severe pneumonia and damage of organ functions. Most common symptoms are cough (59.4-81.8%), dyspnea (3.2-55.0%), fever (77.4-98.6%), fatigue (38.1-69.6%), sputum production (28.2-56.5%), myalgia (11.1-34.8%), and headache (6.5-33.9%) [109,116]. Other lesser common symptoms include chest pain, hemoptysis, rhinorrhea, nausea, vomiting, sore throat, diarrhea, and conjunctive congestion. Though according to one study, out of the 140 confirmed COVID-19 patients, 39.6% of individuals exhibited gastrointestinal symptoms [117], and 10.1% of patients infected with COVID-19 exhibited gastrointestinal discomfort at its onset [111]. Though many patients developed a fever after hospitalization when they did not have any fever at the onset of the infection [116], and several patients with severe infection did not have a fever at all. Several clinical manifestations such as dry cough, headache, fever, sore throat, and dyspnea are common symptoms caused by all three viruses, i.e., SARS-CoV, MERS-CoV, and SARS-CoV-2. Though, COVID-19 patients exhibit lesser gastrointestinal involvement as compared to patients suffering from MERS and SARS [118]. Patients infected with MERS have a higher occurrence of renal failure, which, even though is characteristic, is not commonly found in other types of coronavirus infections in humans [119,120].

Effect of ACE-2 on SARS-CoV-2 Infection
A transmembrane trimetric glycoprotein that protrudes from the viral surface makes up the spike and determines the host tropism and the diversity of the coronaviruses. There are two functional sub-units of a spike where subunit S1 performs the function of binding to host cell receptors, and subunit S2 works for the fusion of cellular and viral membranes. The functional receptor for SARS-CoV has been identified as the ACE2 [121]. It has been shown through structural and functional analysis that ACE2 binds to spike for SARS-CoV-2 [122][123][124]. The expression for ACE2 was on the higher side in the heart, kidney lung, ileum, and bladder [125]. Inside the lung, lung epithelial cells had a higher expression of ACE2. This is associated with heart function and the development of systemic diseases such as diabetes mellitus, and hypertension. Besides the above, it has been earmarked as the main receptor for coronaviruses, such as SARS-CoV and SARS-CoV-2. SARS-CoV-2 (causing COVID-19 infection) is activated by binding the S glycoprotein to ACE2, which is expressed in the lungs and heart [126]. The virus SARS-CoV-2 has been reported to primarily attack alveolar epithelial cells, which results in severe respiratory symptoms ( Figure 4). These symptoms have been reported to have a severe exhibition in cardiovascular disease patients, and this might be linked with increased secretion of ACE2 among these patients compared with healthy individuals. It can be noted that ACE2 levels can be upregulated by using renin-angiotensin-aldosterone system inhibitors [127]. Since ACE2 has been found as the functional receptor for SARS-CoV-2 infection, it is important to be vigilant for the potential effects of antihypertensive treatment using ACE inhibitors or angiotensinreceptor blockers (ARBs) in patients with COVID-19. The switching of the antihypertensive agent from ACE inhibitors/ARBs in COVID-19-infected individuals with hypertension as an underlying disease to other treating agents is seemingly controversial and requires adducing solidly for the same [128].
cleavage site at the S1/S2 site, is a characteristic that is unique to SARS-CoV-2. During the biosynthesis, the S1/S2 site of the SARS-CoV-2 is completely subjected to cleavage, which is in drastic contrast to the SARS-CoV spike that has been included in the assembly without cleavage [123]. The cleavage was subjected upon the S1/D2 site through varied proteases such as cathepsin L and TMPRSS2, and the virus is made extremely pathogenic by the ubiquitous expression of furin [131,133].  . This schematic describes the proposed mechanistic role of ACE2 on SARS-CoV-2 infection. The SARS-CoV-2 virus uses the ACE2 receptor to gain entry into the cell (airway epithelial cells), leading to an increase in proinflammatory cytokines and the development of cytokine storm, which lead to lung damage and augmented COVID-19 severity. Angiotensin receptor blockers (ARBs) might increase the expression of ACE2, and TMPRSS2 assists in S protein priming, leading to enhanced binding of SARS-CoV-2 and higher proinflammatory cytokine release. The higher expression of ACE2 is reported to affect SRC and RPS3, the two crucial genes engaged in inflammatory responses and viral replication. Further, the expression of ACE2, at the same time, is enhanced by the infection. It is also evident that at the same time, SARS-CoV-2 may downregulate ACE2 expression, which further contributes to an increase in angiotensin-2-induced lung injury. The ACE2-mediated negative regulatory activity is curtailed by SARS-CoV-2 and contributes to aggravating the severity of illness.
Recently, it has been found that there was no significant difference in the expression level of the ACE2 in individuals with pre-existing comorbidities when compared to healthy populations. However, long-term smoking has been speculated as a risk factor for SARS-CoV-2 infection following the reporting of the elevated expression of ACE2 among cigarette smokers. The analytical findings of ACE2 in SARS-CoV-2-infected cells suggested the involvement of ACE2 receptors to have a regulatory role in the immune response, cytokine secretion, and replication of virus at post-infectious state, also acting as a binding receptor to facilitate viral entry. Indeed, these findings may render potential insight into the pathogenesis of COVID-19 in the purview of designing effective therapeutic strategies for battling SARS-CoV-2 infection.
It requires further investigation to determine whether SARS-CoV-2 binds to an additional target. The spike protein undergoes protease cleavage after the host protein binds to SARS-CoV-2. A model was proposed to activate spike protein of SARS-CoV and MERS-CoV comprising a two-step sequential protease cleavage, which, for priming, comprises cleavage at the S1/S2 cleavage site and, for activation, cleavage at the S1 site, which is within the S2 subunit and is a position that is adjacent to a fusion peptide [129][130][131]. S1 and S2 subunits remain non-covalently bound. The distal S1 subunit contributes to the prefusion state after cleavage at the S1/S2 cleavage site and stabilization of the membraneanchored S2 subunit [123]. Presumably, the spike for the membrane fusion is activated by the subsequent cleavage at the S 2 site through conformational and irreversible changes. This spike of the coronavirus is unusual amongst the viruses because it can be cleaved and activated through a wide range of varied proteases [132]. The existence of the furin cleavage site at the S1/S2 site, is a characteristic that is unique to SARS-CoV-2. During the biosynthesis, the S1/S2 site of the SARS-CoV-2 is completely subjected to cleavage, which is in drastic contrast to the SARS-CoV spike that has been included in the assembly without cleavage [123]. The cleavage was subjected upon the S1/D2 site through varied proteases such as cathepsin L and TMPRSS2, and the virus is made extremely pathogenic by the ubiquitous expression of furin [131,133].

Immunopathological Mechanisms of SARS-CoV-2 Infection
When considered at the whole-genome level, close relations have been found between SARS-CoV-2 and bat-SL-CoVZC45 and bat-SL-CoVZXC21, albeit its receptor binding is identical to that of SARS-CoV [134]. Though, it is pertinent to mention here that species specificity is not solely determined by receptor recognition. After binding to the receptive receptor, the innate immune response is generated to counteract the entry of the SARS-CoV-2 into host cells. The innate immune signaling evasion or inhibition is necessary for infecting its new host and enhancing viral productivity. Though, how SARS-CoV-2 has driven the pathogenesis as well as evade the immune response is still unknown. SARS-CoV-2 seemingly shares an identical pathological mechanistic to that of SARS-CoV, as SARS and COVID-19 exhibit similar clinical features [38]. While responding to the SARS-CoV-2 infections, for inhibiting the replication of the virus, expression of IFN-stimulated genes (ISGs) is stimulated by the type I interferon (IFN) system. SARS-CoV-2 encodes over eight viral antagonists that evade the ISG effector function and modulate the induction of cytokines and IFN to overcome the antiviral activity [135]. To inhibit the dissemination and replication of the virus, the host immune system responds via cellular antiviral activity and plays a critical role in subjugating inflammation. However, there is a likelihood of pathological implications coming into play due to the lytic effects of the virus on host cells and exacerbated immune response. The studies that include fever, dry cough, breathlessness, hypoxia, and severe pneumonia have identified the symptomatology in COVID-19 diseased patients [38,113]. Some patients witnessed rapid progression combined with Acute Respiratory Distress Syndrome (ARDS), Systemic Inflammatory Response Syndrome (SIRS), and Multi-Organ Dysfunction Syndrome (MODS) that lead to death in approximately 10% of the patients due to the presence of ACE2 receptor in cardiac, renal, and hepatic tissues [113,136] (Figure 3). The evolution of diffuse alveolar damage is due to the increased levels of IFN-γ-induced protein 10 (IP10), pro-inflammatory cytokines (IL-1, -2, -6, -8, -10, and -12), interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP1), and macrophage inflammatory proteins-1A (MIP1A), in patients diagnosed with SARS [137] (Figure 3). A similar picture of immunopathology has been observed in patients infected with SARS-CoV-2. Significantly higher levels of IP10, MCP1, GSCF, and TNF-α have witnessed in patients admitted to the ICU (intensive care unit) as compared to non-ICU patients, suggesting that the underlying cause of the severity of the disease can be cytokine storm [38].
Against all expectations, an uncommon phenomenon was seen during the acute phase of the viral infection wherein IL-10 and IL-4 anti-inflammatory cytokines were also increased in those patients [38]. One more interesting finding that has been explained before was that older males are more likely to be infected with SARS-CoV-2 as compared to children, with rare cases being reported in that age group [38,113]. With SARS-CoV-2, the same observation was found in primate models where the aged cynomolgus macaque was found to be more likely to be infected by the virus as compared to young adults [138]. The identification of the host genes of SARS-CoV-2 and virulence factors that facilitate virus crossing of species-specific barrier mandates further studies to recognize the cause of the lethal disease in humans.

Diagnosis of COVID-19
There are several ways to diagnose and test the impact of SARS-CoV-2 infection such as complete hematological profiling with cellular counts; functional assays of the liver and kidney with the estimate of their enzyme markers and urea levels, respectively; detection of the pathogenic DNA from quantitative PCR or the nucleic acid amplification test; inflammatory markers such as CK-MB, ESR, CRP, D-dimer, ferritin, IL-6, and procalcitonin; and radiological investigations such as chest radiographs, ultrasound, and CT. Quantitative real-time PCR is the mainstay in diagnosing COVID-19 due to its sensitivity, specificity, and feasibility as compared to viral culture. It is the gold standard diagnostic approach with high sensitivity to the viral detection. Elevated procalcitonin levels may also show a superinfection of SARS-CoV-2, as the viral infection may cause a superinfection with bacteria. Similarly, increased levels of biological markers such as IL-6, CRP, ferritin, LDH, Ddimer, and ESR may signify the critical stage of SARS-CoV-2 infection. Further, estimating the complete blood picture report including the count of RBC, WBC, and platelets is another approach to examine SARS-CoV-2 infection. Common laboratory abnormalities present amongst the patients suffering from COVID-19 include prolonged prothrombin time, lymphopenia, and elevated lactate dehydrogenase [37,111,113]. Compared to non-ICU patients, patients admitted to ICU exhibited higher laboratory abnormalities [38,111]. Some patients exhibited elevation in levels of creatine kinase, aminotransferase, creatinine, and C-reactive protein [26,37,38]. Normal serum procalcitonin levels were seen in most of the patients [37,38,111]. High levels of IFN-γ, IP10, IL1β, and MCP1 are present in COVID-19 patients. Importantly, ICU-admitted patients tend to have higher levels of MCP1A, MIP1A, granulocyte-colony stimulating factor (GCSF), IP10, and TNF-α [38]. Further, there can be variations of radiology findings in patients according to immunity status, disease progression, comorbidity, age, and initial medical intervention [139]. In a study conducted amongst the initial 41 cases of 2019-nCoV infection, all 41 patients were suffering from pneumonia, and chest computer tomography (CTscan) showed abnormal findings [38]. In another study comprising six patients, patients who showed multifocal patchy opacities with ground-glass appearances in the peripheral sections of the lungs also exhibited abnormalities on the chest CTscan [26]. As per the data derived from studies, consolidative pulmonary opacities and bilateral pulmonary parenchymal ground-glass are the typical findings of chest CTscans [37,38,[111][112][113]140]. Lung consolidation was noted predominantly among patients five or more days from the onset of disease and those who were 50 years or older compared to those at four or fewer days from onset and those who were 50 years or younger [141]. Manifested by extension and increasing density of the lung opacities, the progression of the disease was noted to be mild to moderate as the course of the disease continued [142]. Sub-segmental and bilateral multiple lobular areas of consolidation are the common findings on chest CTscan in ICU-admitted patients [38]. In a study conducted amongst 99 patients, imaging examination revealed pneumothorax in one patient [113]. The overall diagnosis methods are presented in Figure 5.

Therapeutic Strategies
Unfortunately, to date, no single medication has been reported or proposed to combat the infective viral load of SARS-CoV-2. However, previous strategies for developing proper medications to pulverize SARS-CoV can be extrapolated to COVID-19 infection effectively. Scientists, several research groups, and clinicians across the globe are working towards finding effective medications that can curtail or eliminate the viral load of SARS-CoV-2. Yet, a definitive treatment to fight COVID-19 needs to be announced across the nations. Further, identifying and implementing various natural products, antiviral drugs, anti-malarial drugs, and vaccines can aid in the treatment of the ongoing pandemic( Figure  6). Social distancing, hand washing, self-isolation, fluid management, oxygen therapy (supportive care), and antibiotics treatment for secondary bacterial infections have been recommended [143]. The deployment of primary prevention of risk factors (social distancing, hand washing, face masks,and other infection control measures) hasbeen addressed and propagated to contain the novel coronaviral agent among communities [144]. Since proper evidence-based treatment was not available for COVID-19, the WHO has framed the guidelines to manage the disease based on triage. Once sepsis in COVID-19 patients

Therapeutic Strategies
Unfortunately, to date, no single medication has been reported or proposed to combat the infective viral load of SARS-CoV-2. However, previous strategies for developing proper medications to pulverize SARS-CoV can be extrapolated to COVID-19 infection effectively. Scientists, several research groups, and clinicians across the globe are working towards finding effective medications that can curtail or eliminate the viral load of SARS-CoV-2. Yet, a definitive treatment to fight COVID-19 needs to be announced across the nations. Further, identifying and implementing various natural products, antiviral drugs, anti-malarial drugs, and vaccines can aid in the treatment of the ongoing pandemic ( Figure 6). Social distancing, hand washing, self-isolation, fluid management, oxygen therapy (supportive care), and antibiotics treatment for secondary bacterial infections have been recommended [143]. The deployment of primary prevention of risk factors (social distancing, hand washing, face masks, and other infection control measures) hasbeen addressed and propagated to contain the novel coronaviral agent among communities [144]. Since proper evidencebased treatment was not available for COVID-19, the WHO has framed the guidelines to manage the disease based on triage. Once sepsis in COVID-19 patients is suspected, the rational use of multiple antibiotics and glucocorticoids to control the cytokine storm was suggested. The routine use of glucocorticoid administration is not recommended until and unless it is definitively indicated [144]. Corticosteroid treatment is also not prescribed, as evidenced by several clinical studies [145]. A few studies suggested that the administration of intravenous immunoglobulin might help in combating SARS-CoV-2 infection in severely ill patients [146]. Immuno2021, 1, FOR PEER REVIEW 16 is suspected, the rational use of multiple antibiotics and glucocorticoids to control the cytokine storm was suggested. The routine use of glucocorticoid administration is not recommended until and unless it is definitively indicated [144]. Corticosteroid treatment is also not prescribed, as evidenced by several clinical studies [145]. A few studies suggested that the administration of intravenous immunoglobulin might help in combating SARS-CoV-2 infection in severely ill patients [146].

Antiviral Drugs
There are no proven studies available to state that anti-viral treatment can combat SARS-CoV-2 infections. Initially, anti-viral drugs, broad-spectrum antibiotics, and effective nebulization of IFN-α had been utilized to curtail this load; though, only a few drugs have displayed their potential impact against the virus [147][148][149]. Aligning to prior therapeutics investigations for treating MERS and SARS infection, drugs are being evaluated [150]. In general, there is a lack of strong evidence that antiviral drugs can considerably improve the clinical outcomes. Researchers have also used the anti-influenza drug oseltamivir in combination with empirical antibiotics as a cocktail in the management of COVID-19 patients [38]. Similarly, in the US, Remdesivir developed for the Ebola virus

Antiviral Drugs
There are no proven studies available to state that anti-viral treatment can combat SARS-CoV-2 infections. Initially, anti-viral drugs, broad-spectrum antibiotics, and effective nebulization of IFN-α had been utilized to curtail this load; though, only a few drugs have displayed their potential impact against the virus [147][148][149]. Aligning to prior therapeutics investigations for treating MERS and SARS infection, drugs are being evaluated [150]. In general, there is a lack of strong evidence that antiviral drugs can considerably improve the clinical outcomes. Researchers have also used the anti-influenza drug oseltamivir in combination with empirical antibiotics as a cocktail in the management of COVID-19 patients [38]. Similarly, in the US, Remdesivir developed for the Ebola virus has been recently utilized along with other antimicrobials to combat COVID-19 [151]. Certain potential clinical advantages were observed on a few COVID-19 patients when administered with the combination of Arbidol/ShufengJiedu Capsule (SFJDC) and Ritonavir/Lopinavir [111]. To examine the safety and effectiveness of Lopinavir/Ritonavir and interferon-2b in COVID-19 patients, a clinical study is under trial [143]. A broad-spectrum antiviral drug, Remdesivir, a nucleotide analog RNA polymerase inhibitor, has shown efficacy against SARS-CoV-2 in in-vitro and in-vivo studies and clinical trials [12,122]. It has been reported that patients suffering from coronaviruses were affirmed as clinically recovered after the administration of Remdesivir. This antiviral drug alone or in combination with chloroquine or IFN-β significantly blocks SARS-CoV-2 replication [151,152]. Intravenous Remdesivir cured the first case of COVID-19 reported from the USA, along with other supportive care [12]. However, the establishment of definitive effects of Remdesivir on SARS-CoV-2 further warrants detailed research work in animals and clinical sample-based settings. A range of other antiviral drugs is presently under evaluation against SARS-CoV-2 infection. In vitro studies on Nitazoxanide, Nafamostat, Penciclovir, Ribavirin, Arbidol, Favipiravir, Ritonavir, Baricitinib, and AAK1 showed moderate results against COVID-19 [111,[151][152][153]. Similarly, several other combinations, including combining traditional Chinese medicines with antiviral or antibiotics, have been evaluated in humans and mice against SARS-CoV-2-induced infection [152]. Additionally, the drugs from existing antiviral categories hold scope for future prospects [154,155] (Table 3).

Vaccines
Usually, vaccine preparation is a three-phase (I, II, III) trial-based procedure. The conduction of phase I trial aims at checking the generation of immune response by enrolling only multiples of 10 (approximately 30-40) individuals. A successful phase I trial proceeds further to phase II with the further enrollment of hundreds of individuals, wherein the aim is to identify the dosage concentrations, immunogenicity, and safety. Finally, the phase III trial will include the enrollment of thousands of individuals for measuring the efficacy of the vaccine in terms of its immune response against the targeted disease infection. Currently, multiple companies/universities are investigating the development of a vaccination against SARS-CoV-2 with the Coalition for Epidemic Preparedness Innovations [174]. Here we have collated the information on various vaccines that are approved (Table 4) and underdevelopment (Table 5) along with their respective details.

Other Promising Therapeutics
In China, the medical researchers collected plasma from COVID-19 recovered patients and re-infused it into clinically ill patients who showed complete recovery from COVID-19 disease with good pulmonary compliance. Recently, CR3022 (monoclonal antibody) binding with the spike RBD of SARS-CoV-2 has also been identified, and this is probably due to the antibody's epitope not overlapping with the divergent ACE2 receptor-binding motif. With the pieces of the available evidence on CR3022 (monoclonal antibody), it can be plausibly used as a therapeutic to treat COVID-19 [175]. Recently, Remdesivir, lopinavir, emetine, and homoharringtonine have also reported inhibiting SARS-CoV-2 replication in vitro [176]. Further, the sensitivity of SARS-CoV-2 to recombinant human interferons α and β (IFNα/β) has also been observed. Treatment with IFN-α or IFN-β at a concentration of 50 international units (IU) per milliliter reduces the viral titers by 3.4 log or over 4 log, respectively, in Vero cells. The noted EC50 of IFN-α and IFN-β treatment is 1.35 IU/mL and 0.76 IU/mL, respectively, in Vero cells. These results suggest the higher sensitivity of SARS-CoV-2 compared to other human pathogenic viruses, including SARS-CoV. Overall, this study shows the potential efficacy of human type-I IFN in suppressing SARS-CoV-2 infection, a finding which could inform future treatment options for COVID-19 [177]. Further, usingneutralizing antibodies against SARS-CoV-2 (anti-SARS-CoVnAbs) can be an alternative approach to prevent SARS-CoV-2 infection [178].
The utilization of nucleotide analogs can be another potential approach to treat COVID-19. These nucleotide analogs have the potential to evade the exonuclease activity of the virus. In this context, it is noteworthy that the prodrugs of five of these nucleotide analogs (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine, and Entecavir) are FDA-approved medications for treating other viral infections with well-established safety profiles. To reiterate, following the demonstration of the inhibiting potency of viral replication in cell culture, the candidature molecules are subjected to being evaluated as potential therapies for COVID-19 [179]. Further, type 1 interferons can be more helpful in treating COVID-19. The experience and knowledge inferred from the IFN-I treatment against SARS-CoV and MERS-CoV prove valuable in the selection of potential treatments against SARS-CoV-2 [180].
Interestingly, Ivermectin, a FDA-approved anti-parasitic agent, has previously showed broad-spectrum antiviral activity in vitro, as an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post-infection with SARS-CoV-2 able to effect~5000-fold reduction in viral RNA at 48 h. Therefore, this agent warrants further investigation to perquisite humans [166].
Due to the drastic improvement in the field of molecular biology and translational science, cellular therapy has emerged as a potential option for combating COVID-19 [181]. Cellular therapy deals with the usage of autologous or allogenic pooled conditioned stored cells to treat the disease and to regenerate the damaged cells, tissues, or organs. Researchers have thrown the limelight on the usage of various cells like bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and placentalderived mesenchymal stem cells (MSCs) to curb COVID-19 pneumonia. A total of 53 clinical trials have been in the recruiting status and are ongoing. The results of these trials are awaited (ClinicalTrials.Gov). Despite the advantage of regenerating the damaged pulmonary epithelium, MSCs have quite a list of challenges when administering to patients with COVID-19. The challenges in the usage of cellular therapy are isolation, harvesting, and characterization of cells, preparation protocols, route of administration, dose, and frequency of treatment, immune privilege nature, and the expected outcome of the cells that have been transfused. Outweighing the challenges, cellular therapy has a ray of hope to curb COVID-19 pneumonia.

Obstacles to Research on COVID-19 Pathogenesis
Animal models serve a critical role in unveiling pathogenicity mechanisms of the virus, from the entrance to the transmission, and targeting therapeutic strategies. Earlier, to check the replication of SARS-CoV-2, symptoms of serious infections were depicted by different animal models [201]. In small animals, MERS-CoV pathogenesis was not observed in contrast to SARS-CoV-2. As a result of the non-compatibility of the DPP4 receptor, mice are not at risk of infection with MERS-coronavirus [202]. The infectious pathogenicity of SARS-CoV-2 can be determined by the animal models as used in SARS-CoV-2 since both the viruses share 80% of the genomes and recognize ACE2 receptors. Hamsters that have been genetically modified with CRISPR or TALEN or other small animals can be used for studying the novel coronaviruses' pathogenicity. Replication of SARS-CoV-2 has been reported as a cause for severe ailments in rats (f344), wherein at spike glycoprotein, a mutation was revealed by the sequence analysis [203]. Therefore, it can emerge as a suitable alternate option for the development of spike glycoprotein targeting therapeutics against novel coronaviruses. Clinical isolates and mice models were used recently for the development of a therapeutic strategy against COVID-19 induced by SARS-CoV-2 [111,152]. Artificial Intelligence prediction has also been used in a similar study for investigating the drug's inhibitory role against SARS-CoV-2 [153]. Randomized clinical trials were also conducted on patients suffering from SARS-CoV-2 [147,151,152]. The investigation into the invivo mechanisms relevant to the pathogenesis of COVID-19 mandates the global collaboration of scientists for designing an appropriate model.

Conclusions
The sudden sprouting of COVID-19 cases across the globe has questioned the solidarity of the medical fraternity. Apart from lungs, viral infections-mediated inflammatory state and immonumodulation may have potentially adverse impacts on several other organs/organ systems, too [204,205]. The sudden outset of this viral infection has brought people into captivity across the nations. The day-to-day altering trends in symptomatology and presentation have pacified the molecular research and developmental sciences to come up with safer and effective therapeutic agents and vaccines. To date, we are comprehending and battling this pandemic with no proven therapeutics. The need of the hour is to consider a holistic approach and render supportive care, as per the presenting severity of the case. While combating a pandemic, it is of utmost importance for health care professionals to keep themselves updated with the current and emerging therapeutic trends for treating the disease with greater effectiveness [206][207][208][209][210]. A multidisciplinary team must mitigate the secondary waves of the pandemic with all the necessary precautions. At the same time, it is equally important to optimize novel ideology of cellular therapy protocols in adjunct to the development of vaccines, as these have the potential to prove as the positive shades of a rainbow amidst the storm of COVID-19 pandemic. Further research on the definitive management protocols by conducting randomized controlled trials is greatly needed for the hour because safety and efficacy parameters need to be concretely investigated.
Author Contributions: N.K.J. designed and wrote this review with input from all other authors who contributed to studies that led to the view presented in this manuscript. All authors have read and agreed to the published version of the manuscript.