SARS-CoV-2: Insights from the Immunopathogenesis and Current Clinical Diagnosis and Therapeutic Strategies

The global public health scenario is worsening gradually as the confirmed cases of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections are incessantly escalating with every passing day. The pathological condition caused by SARS-CoV-2 is termed as Coronavirus disease 2019 (COVID-19). The understanding of SARS-CoV-2 transmission dynamics, immunopathogenesis, and the need for early-stage diagnosis and the effective therapeutic regime are the few immediate challenges faced by healthcare professionals worldwide. More specifically, the role of SARS-CoV-2 in the host’s immunopathogenesis response is crucial to determine the disease severity and its clinical outcome in COVID-19 patients. In the present review, we provide insights into the SARS-CoV-2 pathology, host immune responses including innate, cellular, and humoral responses, and immunomodulatory functions of SARS-CoV-2 including cytokine storm and immune evasion. We also shed light upon the present clinical and laboratory-based applications enrolled in the SARS-CoV-2 diagnosis. Taking into consideration the pathogenesis and SARSCoV-2 immune function, in the present review, we finally provide succinct insights into the SARS-CoV-2 transmission dynamics, immunopathogenesis, with the assessment of the current diagnostic and preventive/ therapeutic strategies. SARS-CoV-2: Insights from the Immunopathogenesis and Current Clinical Diagnosis and Therapeutic Strategies Shruti Sharma1*, Ujjawal Sharma2#, Anupama Chaudhary3, Manisha Naithani4, Priyanka Naithani5, Saurabh Prashar2, Bunty Sharma6, Pramod Kumar Nagar7, Prudhvi Lal Bhukya8, Unnati Bhalerao8, Meenakshi Singh9, Manjita Srivastava8, Muneesh Kumar Barman10, Sampan Attri2, Jitender Gairolla2,11# 1Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India 2Department of Community Medicine and School of Public Health, Postgraduate Institute of Medical Education and Research, Chandigarh, India 3Orinin BioSystems, Karnal, India 4Department of Biochemistry, All India Institute of Medical Sciences, Rishikesh, India 5Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh, India 6Chitkara School of Health Sciences, Chitkara University, Punjab, India 7Hematology Oncology Unit, Advance Pediatric Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, Chandigarh, India 8National Institute of Virology, Pune, India 9Transplant Immunology and Immunogenetics Lab (HLA), ACTREC, Tata Memorial Centre 10Laboratory for HIV Research, National Centre for Cell Sciences, Pune-India 11Department of Microbiology, All India Institute of Medical Sciences, Rishikesh, India


Introduction
We are witnessing unprecedented times with the rapidly evolving global COVID-19 pandemic that has brought the world to a socio-economic halt and greatly impacted public health worldwide. The onset of the COVID-19 outbreak was marked in late December 2019, with the reporting of a group of patients suffering from a Severe Acute Respiratory Syndrome-like disease in Wuhan, China. Preliminary investigations revealed that the causative agent is a previously unknown virus and therefore it was named as 'Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), by the ICTV (International Committee on Taxonomy of Viruses) 1 . Coronaviruses belong to the Coronoviridae family that comprises of viruses which are characteristically identified by a ribonucleic acid core surrounded by a protein-spiked envelope and their structural resemblance to that of a crown (crown i.e., corona in Latin)

SARS-CoV-2, Outbreak, and Transmission Dynamics
The first few cases of SARS-CoV-2 infected cases identified infections were reported from in Wuhan City China (in the Hubei Province), with symptoms like province of China and these cases presented with an acute respiratory syndrome of idiopathic etiology 9 . An early outbreak of SARS-Cov-2 infection showed symptoms such as fever, sore throat, and significant dyspnea 10,11 . On February 11, 2020, WHO named the disease as 'COVID-19', and by the end of February, COVID-19 had spread to over 25 nations 12,13 . An early investigative study performed on the confirmed COVID-19 cases from Wuhan reported the exposure history of the patients to the seafood market 11 . Besides the live-food, the possibility of seafood contamination with bats or bat droppings was also greatly postulated. Initial phylogenetic analyses of SARS-CoV-2 suggested its resemblance to the beta-coronaviruses, however, the distinctive phylogeny of the RNA-dependent RNA polymerase of SARS-CoV-2 indicated that it was a novel strain of the genus Betacoronavirus and subgenus Sarbecovirus. The genomic analyses revealed an 88% sequence identity of SARS-CoV-2 with bat coronaviruses 11,14 , and therefore affirmed its zoonotic origin with bats serving as reservoirs for SARS-CoV-2, which further transmit the disease to humans and other animal species. Prior to the WHO's declaration of the 'COVID-19 pandemic' in March 2020, an unexpected rise in the numbers of confirmed COVID-19 positive cases were observed in China. An early report from China revealed that the basic reproduction number (R0) of COVID-19 ranges from 2.2 to 3.58 which was relatively lesser than SARS but higher than MERS 15 . Of note, the SARS-CoV-2 median incubation duration was found to be 6.4 days (Credible interval (CI)-95%, 5.6 to 7.7 days) and these were very similar to those for MERS and SARS 16,17 . The key pathology factors such as the onset of symptoms and rate of mortality were found to be dependent on age, pre-conditions (hypertension, diabetes, and cardiovascular disease) and the immune state of the infected individual 13,16,18,19 . Therefore, a higher susceptibility, severity, and mortality rate of COVID-19 was reported in individuals who were either aged (median > 59 years), or had had aforementioned comorbidities 19,20 . In the early outbreak, the mortality rate was reported to be between 1.5-3% in Wuhan, China, however, these statistics largely found to depend on the criteria of diagnosis, the scale of COVID-19 transmission, and geographical location as well 10,21 . By date, the COVID-19 outbreak has spread to over 208 countries, and the Centre for Disease Control and Prevention (CDC) and WHO are jointly engaged in closely monitoring the situation to forecast its timely impact on public health. As of March 20, 2021, a global death count of 2,694,094 with a total 121,969,223 confirmed cases have been estimated and as a result of the COVID-19 pandemic (https://covid19.who.int/). While assessing the SARS-CoV-2 transmission dynamics, the person-toperson contact established either through direct contact or droplets released in the air (by coughing or sneezing) was found to be the most common route of its transmission to a healthy person 13 . While, the indirect routes of transmission were also observed, suggesting that SARS-CoV-2 transmission can also occur by touching a surface or fomites (copper, plastic, steel, cardboard, etc.) that contains the existing virus particles 22,23 . Of note, the half-life of SARS-CoV-2 varies with environmental conditions, and it was notably high on cardboard (~ 24 hours), but found to be at a minimum in aerosols (≤ 4 hours) 23 .

SARS-CoV-2 Infection, Host Cell Entry, and Immunological Triggers
The 50-200 nanometers (nm, diameter) virion of SARS-CoV-2 is found to be composed of 4 structural proteins viz. spike (S) envelope (E), membrane (M), and nucleocapsid (N) (Fig. 1A) 24,25 . Out of these, the S protein is essential for attachment of the virus to the membrane of host cells and to gain access into the cells (Fig. 1B) 24 . Initial validations conferred that SARS-CoV-2 acquires a greater binding to ACE2 protein in humans which was later identified to be its binding receptor by which it ensures entry inside the cell 26,27 . Of note, processing of S-protein by host cell transmembrane proteases serine 2 (TMPRSS2) was found to be crucial for S-protein-ACE2 coupling, wherein the TMPRSS2 protease plays an activator function and facilitates the SARS-CoV-2 entry into the host cell (Fig. 1B) 27 . Markedly, this processing of S-protein before its binding to the ACE2 receptor includes its enzymatic modification leading to its cleavage and change in conformation by protease activity of TMPRSS2 that resides in the vicinity of ACE2 receptor i.e., a crucial step in the initiation of viral endocytosis (Fig. 1C) 28 . Insights from an earlier report analyzing SARS-CoV binding with ACE2 suggest that it enables SARS-CoV-2 internalization and trafficking into the endosomes and then the lysosomes, which subsequently helps in the viral uncoating and fusion (Fig. 1C) 29 . In this process, the S protein further undergoes a series of post translational modifications including enzymatic cleavage and modifications by both cathepsin L and B, the key lysosomal cysteine proteases, which causes the internalization of the virus into the endo-lysosomes. These steps are crucial in the fusion of viral and late endosome membranes and subsequent discharge of the viral genome (ssRNA) in the host cell cytoplasm (Fig. 1C) 30 . The latter step is highly pH-dependent, where fusion only occurs after reaching highly acidic conditions in the lysosomes, possessing the utmost cathepsin L activity (Fig. 1C) 31 . These findings are further supported by the observation that the usage of various lysosomotropic agents inhibits infection by interfering with lysosomal processing of viral membrane proteins, which is a pre-requisite for fusion 32 . Therefore, it is reasonable to speculate that inhibiting the binding of the S protein to ACE2 or intracellular blocking of its subsequent cleavage by cathepsin L could be the two key target steps in the SARS-CoV-2 infection to develop therapeutics against SARS-CoV-2 30 . Another key therapeutic target could be the Nsp15 protein, which is conserved among coronaviruses and is required for their life cycle and virulence. Nsp15 is a crucial SARS-CoV-2 protein that interferes with the host immune system and facilitates the replication of the virus genome within the host 33 .
Looking at the immunological cues of the SARS-CoV-2's viral function, frequent lymphopenia, and pneumonia have been observed in infected moderate-to-severe cases 34 . Of note, SARS-CoV-2 induced alveolar macrophage activity has been suggested to trigger the immunological response in the patient's lungs that incites cytokines/chemokines release and causes a subsequent activation of T-Cell and the Neutrophils 35 (Fig. 2). As a result of this, elevated expressions of pro-inflammatory cytokines viz. TNFα, MCP-1, MIP-1A, IL-2, IL-7, IL-10, and G-CSF were observed in a severely infected cohort of 41 hospitalized COVID-19 patients (Fig. 2) 17 . These factors suggest a crucial role of cytokine storm leading to viral sepsis and inflammationinduced lung injury. These risks may further promote pneumonitis and acute respiratory distress syndrome (ARDS) that may lead to the organ collapse, shock, and eventually death 36 . Another report by Liu and Li 37 suggests that the binding of viral proteins attacks the heme localized on the hemoglobin 1-beta chain to detach the iron and to constitute the porphyrin. The above activity of viral protein attenuates the hemoglobin function to transport oxygen and CO 2 . This also interferes with the heme anabolic pathways of humans resulting in the several debilitating manifestations of COVID-19 37 . A review of SARS-CoV-2's activity and its impact on the host's immune system is discussed in detail in subsequent sections.

SARS-CoV-2 Infection and Immune Response
Innate immunity plays a crucial role in combating the severity of any viral infection. In a recent study, the innate immune response was shown to cause an increase in the neutrophil count and cause lymphopenia 38 which indicates its critical role in disease progression and severity. The Severe Acute Respiratory Syndrome caused by contagious SARS-CoV-2 primarily targets the lungs and causes severe clinical manifestations. The frontline complement proteins, type I Interferons (IFN), and other factors of innate immune response essentially control viral replication in the host during the initial stages of the disease itself 39 . Activation of these factors on viral exposure subsequently triggers and shapes the adaptive immune response. However, an overactivation of innate immune response can result in lethality to host tissues 40 .
Previously, it has been shown that SARS-CoV attacks T cells. A minimal expression of ACE2 receptor on monocytes/ macrophages in the lungs was observed by Dandekar and Perlman 41 , which suggests a possibility of the involvement of other immune mechanisms such as antigen-antibody reactions. It is speculated that SARS-CoV-2 may modulate the host innate immune response by dampening the type I IFN response as is often the case in SARS. However, as of now, there is no evidence which indicates a similar mechanism of modulation of host innate immune responses by SARS-CoV-2 42 .
A lot has been learnt regarding the molecular pathogenesis and host immune responses of human coronaviruses from the SARS-CoV epidemic in 2003. Many studies were aimed at understanding the immune response in SARS-CoV infected patients so as to highlight upon the pathogenesis and clinical perseverance of the infection. These efforts to decipher the underlying immune mechanisms have investigated the interaction of SARS-CoV with innate immune cells e.g., dendritic cells (DC), macrophages, and also, their role in triggering other immune cells and responses including phenotype maturation, T-cell priming activity, cytokine production, and phagocytosis. Of note, the activated macrophages and DCs essentially contribute to the maintenance of innate response homeostasis within the host's lung and inside infected tissues during the course of the infection. During the early phase of the COVID-19 pandemic, the pathology of SARS-Co-V-2 was still under investigation, and its molecular pathogenesis and function in host immune response remained largely uncharacterized, and as a result, knowledge of these aspects was majorly derived from the studies that investigated SARS-CoV immunopathogenesis (Fig. 3).
SARS-CoV-2 infections are characterized with a high degree of lymphopenia, which in some cases has been reported to affect CD4+ T cells, CD8+ T cells, B cells and natural killer cells, while other studies indicate that it is more associated with CD8+ T cells. Severe cases of COVID-19 also show a marked increase in the levels of cytokines such as IL-6, IL-10 or tumor necrosis factor (TNF), which are known to have a direct effect on T cells as well as DCs and neutrophils. T cell depletion may also be a result of hyperactivation or even the excessive expression of pro-apoptotic factors such as FAS, TRAIL or caspase 3 43 .
The CD8+ T cell response in COVID-19 has been observed to vary within and among cohorts as well. Terminally differentiated or exhausted T cells have been associated with certain cases of severe COVID-19 owing either to an increased expression of the inhibitory receptors PD1, TIM3, LAG3, CTLA4, NKG2A and CD39, or as a result of hyperactivation, or in some cases, a result of decrease in the expression of inhibitory receptors has also been observed. In comparison with CD8+ T cell activation, CD4+ T cell activation is lesser. However, CD4+ responses are associated with worse clinical outcome in patients. SARS-CoV-2 S protein specific CD4+ T cells have a T H 1 cell profile, and they have the ability to produce IFNɣ, while the role of T H 2 cells in COVID-19 is less clearly understood as of yet 43 .

Attenuation of type 1 IFN response
Host interferons (IFN) are well-known to interfere with viral life cycle and limit their replication by activating several hosts antiviral effectors proteins such as Protein kinase R (PKR), RNAse L and other factors of innate response, but prior studies on SARS-CoV encoded proteins have been described to attenuate the IFN mediated antiviral signalling by inhibiting the IFN activity ( Fig. 3) 44 . In the early phase of the SARS-CoV-2 pandemic, these insights on coronaviruses in general, included the SARS-and MERS-CoVs is what provided basic conceptual as well as speculative knowledge regarding the novel virus. The SARS-CoV nsp1 protein inhibits IFN function by modulating STAT phosphorylation, and a similar control over IFN activity has been observed by nsp3 involving unknown mechanisms. The IFN response blockage by SARS-CoV membrane (M) and nucleocapsid (N) structural proteins has also been shown to be mediated by TBK1/IKK through an unknown mechanism. Of note, attenuation of IFN production and/ or function by the SARS-CoV structural and non-structural proteins activates inflammatory signalling, essentially by the progressive viral replication in host cells induced by Pathogen-Associated Molecular Patterns (PAMPs). The PAMP machinery in turn attenuates IFN function and elicits pattern recognition receptors (PRRs) promoting an inflammatory response. Deficiency of IFN response elevates counts of Ly6C low monocytes and neutrophils 18,45 (Fig. 3). These aspects of SARS CoV pathogenesis and immune evasion were speculated to apply to SARS-CoV-2 as well.
With increasing research on the novel SARS-CoV-2, a better understanding of its immune mechanisms has been obtained. Being an RNA virus, upon cellular entry, several of the SARS-CoV-2 proteins have the ability to interact with and bind to RNA, even that of host origin. Banerjee et al. shed light upon the molecular basis of SARS-CoV-2 pathogenesis describe the ability of SARS-CoV-2 proteins to interact with human RNAs. They identified a very specific interaction between the viral NSP16 protein-which localizes in the nucleus upon infection, and the U1 and U2 snRNAs which mediate post-transcriptional RNA splicing, and in doing so, NSP16 has the ability to suppress global mRNA splicing in SARS-CoV-2 infected cells so as to decrease host protein and mRNA levels by triggering nonsense-mediated decay of improperly spliced mRNAs. This mechanism may result in inhibition of IFN-mediated immune responses since most of the key genes for IFN stimulation are spliced posttranscriptionally. Additionally, the SARS CoV-2 NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the 40S subunit and disrupts translation of RNA with an ability to suppress translation of global host mRNAs and the host IFN immune response. Similarly, NSP8 and NSP9 bind to the 7SL RNA component of the SRP (signal recognition peptide) preventing its proper folding and assembly which further suppresses the integration of integral membrane proteins in the cell membrane. A very interesting finding by Banerjee et al. was that these four viral proteins are all products of the initial stages of the virus replication cycle prior to production of the viral dsRNA which is a trigger for the type I IFN response, and this disruption of cellular processes is what allows the virus to replicate with a minimization in the IFN-mediated immune response 46 .

SARS-CoV-2 and altered Macrophages and Dendritic cell immune function
Neither DCs nor MP cells were shown to acquire SARS-CoV infection, or even facilitate viral replication. However, reports have shown that exposure to the infectious SARS-CoV acquires phenotypic and functional changes in the DC and macrophages (Fig. 3). These changes alter differentiation of the DCs and modulate the expression of co-stimulatory molecules required for priming of T-cells, which serves as an important factor for stimulating the subsequent adaptive immune response. Exposure of infectious SARS-Sharma S, Sharma U, Chaudhary A, Naithani M, Naithani P, Prashar S, Sharma B, Nagar PK, Bhukya PL, Bhalerao U, Singh M, Srivastava M, Barman MK, Attri S, Gairolla J. SARS-CoV-2: Insights from the Immunopathogenesis and Current Clinical Diagnosis and Therapeutic Strategies. J Immunological Sci. (2021); 5(2): [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] CoV was earlier shown to attenuate the antigen-capture function of macrophages, wherein endocytosis uptake gets disrupted. This, in turn, substantially compromises the antigen presentation function of macrophages to T-cells. On the other hand, exposure of differentiated DCs to infectious SARS-CoV results in exponentially higher activation of the T-cells by these cells. These results offers insights into the immunomodulatory function of SARS-CoV in the innate immune cells that further determine the adaptive response in the event of SARS-CoV infections (Fig. 3) 18,48,49 .
In the case of SARS-CoV-2, alveolar macrophages are most likely the very first immune cells that are encountered by the virus during infection. A study by Dalskov et al. indicates that naïve alveolar macrophages are incapable of sensing SARS-CoV-2 and mounting an IFN response against the virus, which suggests that SARS-CoV-2 genomic RNA is not detected by alveolar macrophages and this is a postulated reason for the early asymptomatic phase of COVID-19. This is very much in contrast to the response mounted by alveolar macrophages against other respiratory viruses such as the Influenza virus and the Sendei virus. However, Dalskov et al. noted that similar to SARS and MERS, SARS-CoV-2 was incapable of causing productive infections in alveolar macrophages 50 .

Dysregulated cytokine and chemokine levels and its clinical consequences
The attenuated type 1 IFN action, as discussed earlier, compromises early viral control. Additionally, the SARS-CoV replication rate in ACE +/+ human lung epithelial cells are very rapid. Therefore, these factors potentially result in hyperactivation of dendritic cells and macrophages, aggravating the proinflammatory responses of these innate immune cells (Fig. 3). Additionally, elevated expressions of IFN-γ, IL-1, IL-6, IL-12, and TGFβ pro-inflammatory cytokines and CCL2, CXCL10, CXCL9, and IL-8 chemokine were observed in the blood serum of severely-sick SARS-CoV patients as compared to patients having moderate symptoms (Fig. 3) 51,52 . Studies have shown a significant increase in levels of pro-inflammatory cytokines (PICs) like IL-2, IL-7, CRP, IP-10, MCP-1, TNF-alpha -all further validating the presence of a modulated, hyperactive innate immune response in SARS-CoV infected patients [53][54][55] . A clinical outcome of the release of PICs in SARS-CoVinfected ACE2+ cells in patient tissues was suggested to promote acute lung injury and multi-organ failure (Fig.  3). For instance, MCP-1 functions in the induction of the migration of macrophages/monocytes and potentiate these cells in the vascular system to travel to the infested area/tissue to ensure their combative function against the viral agent(s). However, the combative function of these cells is not limited to infected cells only, thus it can also harm normal cells that are in close affinity. In severe SARS-CoV cases, it has been consistently found that there is a higher infiltration of monocyte-macrophages-neutrophils at the site of infection. This provides a framework for understanding the pathogenesis of SARS-CoV infection. Some PICs e.g., TNF-α, TGF-β1, etc. producing macrophages undergo proliferation and become activated, resulting in a higher PICs release, causing more damage of the host tissues 56 . Expression of Mac387+ and CD25+ proliferation and/or activation markers on a large number of alveolar macrophages corroborates these results. However, TGF-β1 has been implicated in alveolar epithelial cell death and this may promote Fas-mediated cell death of platelets and lymphocytes as well. These TGF-β1 function may produce acute lung injury, thrombocytopenia, and lymphopenia in SARS-CoV patients. Expression levels of apoptotic Fas and Fas Ligand (FasL) remain high in pneumocytes and epithelial cells of the lungs of SARS patients 53,56,57 .

SARS-CoV-2 infection and Antigen Presentation
Antigen presenting cells (APCs) are the host's major anti-viral defense mechanism that process and present the antigen when pathogens strike into cells. The major histocompatibility complex (MHC) or human leukocyte antigen (HLA) presents the antigenic peptides that are primarily recognized by cytotoxic T lymphocytes (CTLs) (Fig. 3). Although, the process of antigen presentation remains highly conserved and demonstrates mechanistic uniformity for diverse viral infections, lack of any evidence of these mechanisms in SARS-CoV-2 presently hindered a clear view. The gathered knowledge on SARS-CoV and MERS-CoV for their antigen presentation mechanisms offers an insight into these functions for SARS-CoV-2 (Fig. 3). The coronavirus antigen presentation is mostly governed by MHC I molecules 58 , but then MHC II similarly subsidizes to antigen presentation.

SARS-CoV-2 and Cellular and Humoral Immunity
Antigen presentation consequently activates the cellular and humoral immune response in the host, which is primarily triggered by viruses targeting B-and T-cells. The immunoreactive antibodies against SARS-CoV show a Sharma S, Sharma U, Chaudhary A, Naithani M, Naithani P, Prashar S, Sharma B, Nagar PK, Bhukya PL, Bhalerao U, Singh M, Srivastava M, Barman MK, Attri S, Gairolla J. SARS-CoV-2: Insights from the Immunopathogenesis and Current Clinical Diagnosis and Therapeutic Strategies. J Immunological Sci. (2021); 5(2): [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] characteristic IgM pattern that disappears at the end of the 12 th week, while IgG antibodies stay in the humoral system for a long time 63 . The IgG antibodies showing specificity to SARS-CoV mainly includes the S-and N-specific antigen 64 . A recent report showed the counts of CD4+ and CD8+ T lymphocytes significantly reduce in the blood along with the peripheral blood mononuclear cells (PBMCs) in COVID-19 patients, however, these cells showed a hyperactivated state as marked by high expression of HLA-DR (CD4+) and CD38 (CD8+) dual positive populations 65 . Severely infected SARS-CoV patients were also earlier shown to have a depletion of CD4+ and CD8+ T cells population. Of note, in the survived SARS-CoV patients, the CD4+ and CD8+ memory T cells population was present for about four years and has actively acquired DTH response, proliferation, and IFN-γ production 66 . A study conducted by Tang et al. 67 , showed that memory T cells retain SARS-CoV S-peptide specific memory even after six years of the viral infection that was found to be evident in 14 out of 23 patients. Similarly, a potent activity of CD8+ T cells was also validated in the mice model for the clearance of MERS-CoV viral load 68 . These evidences could provide a valuable reference to assess the host's immune response against the SARS-CoV-2 in current clinical settings (Fig. 3).

SARS-CoV-2 Immunogenicity and Cytokine Storm
Development of ARDS largely contributes to the mortality in COVID-19 patients. Huang et al. have shown that 6 out of 41 SARS-CoV-2 patients died of ARDS 18 . ARDS is a frequent immunopathological response as is evidently seen in consecutive coronavirus infections 65 . An activated systemic inflammatory response in COVID-19 patients prompts an array of pro-inflammatory chemokines and cytokines that contributes to the emergence of a robust cytokine storm in the patients. It includes the constitutive release of IFN-α, IFN-γ, TNF-a, IL-1β, IL-6, IL-12, IL-18, IL-33, and TGF-β pro-inflammatory cytokines and CXCL9, CXCL10, CCL2, CCL3, CCL5, CXCL8, chemokines (Fig. 3) 18,45,69,70 . Consistent to these findings in COVID-19, patients with severe MERS-CoV infections exhibited a higher level of pro-inflammatory cytokines and chemokines in the serum, in comparison to those who had moderate infection 71 . The clinical feature of cytokine storm involves aggressive assault of the immune system in the host body that leads to ARDS and multiple organ failures and eventually contributes to mortality in severely infected SARS-CoV2 patients, as also reported earlier in MERS and SARS-CoV pathologies (Fig. 3) 65 .

SARS-CoV-2 and Immune Evasion
MERS and SARS coronaviruses evade the host immune system to promote their replication and survival. Microbederived molecules/motifs viz. PAMPs (Pathogen-associated molecular pattern) are largely recognized by pattern recognition receptors (PRRs) or toll-like receptors (TLRs) that further affirms the response of the host's immune system. MERS and SARS coronaviruses were shown to instigate the formation of double-membrane vesicles, lacking PRR that facilitates their replication in the vesicles and escaping from the host identification of their dsRNA. Interferon-I (IFN-I) provides protection to host against viral infection, however, inhibition of its above function was observed in MERS-CoV and SARS-CoV infected mice 72,73 . The role of MERS-CoV accessory protein 4a was suggested earlier to inhibit IFN activation 74 . In a similar manner, MERS-CoV virus membrane antigens ORF4a, ORF4b, and ORF5 were seen to restrict IFN regulatory factor 3 (IRF3) nuclear localization that subsequently impairs IFN promoter activation 75 . These findings emphasized the importance of building preventive/therapeutic strategies that could target the immune evasion function of SARS-CoV-2 (Fig. 3).

COVID-19 Diagnostics: Clinical and Laboratory Approaches
In clinical diagnosis, patient history is designated as an essential primary record. Prior to an advancement in the stage of this pandemic and the diagnosis of infection, patient history, in terms of contact with suspected or confirmed COVID-19 cases, supported quicker diagnosis for COVID-19 (Fig. 4). At the physiological level, mildmoderate fever, coughing/sore throat, and shortening of breath are primary symptoms of SARS-CoV-2 infection patient (Fig. 4) 76 . Nevertheless, reports have shown that SARS-CoV-2 may persist in asymptomatic people for more than two weeks and there are chances of its recurrence after recovery as well 77 . The infected individual, at an early stage, may possess an average or reduced count of peripheral white blood cells (WBCs). A recent clinical study, comprising a 1,099 COVID-19 patient's cohort exhibited a lesser than 4×10 9 /L WBC count 78 . Also, most of the COVID-19 patients were found to have elevated enzymes, proteins like C-reactive protein (CRP), aspartate aminotransferase (AST), myoglobin, and erythrocyte sedimentation rate (Fig. 4) 74,78,79 . As a preferred clinical diagnosis method, analyses of chest X-ray of a majority of COVID-19 patients has revealed an abnormal state of the lungs, marked by shadows in small patches and interstitial changes that are excessively evident in the extrapulmonary bands 80 . Furthermore, Computed Tomography (CT) of the chest in COVID-19 patients showed several extended ground-glass opacities and infiltrations with the progression of infection's severity (Fig. 4) 81 . Often, patients with COVID-19 infection exhibit acute lung injury, unusual pneumonia, and ARDS 36 . At the advanced stage, increased inflammation, accumulation of fluid, and advanced fibrosis in the COVID-19 patient's lungs was shown to compromise the gas exchange and thus may postulate an eventual organ failure (Fig. 4) 82 . Therefore, a greater abnormality in chest radiographic scans could predict a more severity of the COVID-19 infection in the patient.
Quantification of COVID-19 nucleic acid in nasopharyngeal and oropharyngeal swab sample or secretion of the respiratory tract sample by quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) is the routine diagnostic approach enrolled presently in the laboratories worldwide ( Fig. 4) 83,84 . RT-qPCR remains the gold standard and sensitive tool for COVID-19 detection in comparison to other available symptomatic and scanning tests as it directly detects the SARS-CoV-2 genetic signature (RNA) and quantifies the viral load. Therefore, RT-qPCR offers enormous help to clinicians and epidemiologists to trace COVID-19 spread either to individual clusters or in the community, and for its clinical management as well 85 . The RT-qPCR test includes primer and probe designing at initial steps followed by the optimization of the amplification conditions, while modification in processing steps and duration can be conceived based on manufacturer's recommendation. Primers and probe for RdRP (RNAdependent RNA polymerase) gene of the ORF1ab region of open reading frame and E (Envelope -protein) gene have shown high analytical sensitivity [85][86][87] . As an early readout of RT-qPCR utility, initial reports from China revealed that its overall detection rate for SARS-CoV-2 infection was 38% in 4880 cases 88 , while other testing performed on 51 confirmed patients of SARS-CoV-2 infection showed 71% positive detection rate 89 . It has been observed that patients may show COVID-19 positive in the RT-qPCR test after 2-8 days of getting infected 84,90 . Some modified methods of RT-qPCR test known as Reverse Transcription Loop-mediated isothermal amplification (RT-LAMP) and CRISPR-Cas12-based assay for SARS-CoV-2 RNA detection were also developed and enrolled for diagnosis 91,92 . Consistent to these novel diagnostic initiatives and with evolving situation in the United States, the FDA (Food and Drug Administration, USA) has recently approved the emergency usage authorization of cobas 6800/8800 high throughput platforms for SARS-CoV-2 diagnosis which offer quality and high-volume testing capacity 93,94 . Highthroughput sequencing for SARS-CoV-2 nucleic acid and SARS-CoV-2 specific antibody-based ELISA systems have now been employed as point-of-care tests (POCT) for rapid SARS-CoV-2 diagnosis (Fig. 4) 95 .

Preventive and Therapeutic Strategies
Presently, we lack any SARS-CoV-2 -specific therapeutics that could offer a sure shot clinical outcome. Researchers and health professionals across the globe are summing up their efforts to identify and validate precise SARS-CoV-2 targeted therapeutics. Earlier, WHO had released technical guidance systematically listing the crucial measures to contain and further prevent COVID-19 spread 96 . It broadly specifies the strategic measures to prevent infection in healthcare professionals and related facilities and extensively discusses quarantine measures and requisite of rational use of masks and personal protective equipment  25,99,100 . The progress on vaccine development has already surpassed several stages of trials and vaccinologists are now assessing the immune-protection against COVID-19 at the National Institutes of Health, USA, China, and UK 96,101 . The broad-spectrum antiviral drugs like HIV protease inhibitors and nucleoside were repurposed and are also being utilized as therapeutic alternatives given their benefits against other earlier reported 2 viral infections 100,102 . As of recent data, more than 85 % percent of patients in China received various anti-viral agents including oseltamivir, ganciclovir, and lopinavir/ritonavir 15 . Another study showed that repurposing of remdesivir could help in COVID-19 treatment, and this drug has been approved for the same 103 . Additionally, a recent report discussed benefits of early-stage intravenous glucocorticoid administration (methylprednisolone, 40-80 mg, given once a day till 5 days), while its treatment course can be modified based on clinician's recommendation 104 . Furthermore, immune suppressants were recommended to all patients undergoing diagnosis of accompanying hyper-inflammation. Another insightful report citing readouts of multi-centric clinical trials in China demonstrated that chloroquine phosphate i.e., a drug used for malaria treatment, exhibits a clear efficacy and protection against COVID-19-associated pneumonia 105 . FDA's recent approval to use convalescent plasma (collected from COVID-19 exposed patients) also gave a remedial hope to critically ill COVID-19 patents 106 . Presently, a few potential therapies have been developed for treatment, one such example is the IL-6 receptor blockade. In this line, China has initiated multi-centric, randomized controlled trials to use tocilizumab for COVID-19 patients who are having pneumonia and elevated IL-6 levels 107 .   Other therapeutic options include the usage of specific cytokine blockade by tocilizumab or anakinra and through JAK inhibition (Available at https://www.clinicaltrials. gov/ct2/show/NCT04322773; accessed on 16 April 2020). Currently, unprecedented research efforts towards finding the treatment possibilities for COVID-19 have been made and key recommendations are constantly updated worldwide. Evidence-based details of recent potential therapeutic strategies for COVID-19 cure are duly enlisted in Table 1 and details of vaccines and technology used are duly listed in table 2.

Conclusion
In the present review, we have provided insights into the SARS-CoV-2 pathology by shedding light on the host's immune function comprising innate, cellular, and humoral responses. Further, we discussed the immunomodulatory functions of SARS-CoV-2 including cytokine storm and immune evasion. The review further sheds light on the clinical and laboratory strategies that are presently employed for COVID-19 diagnosis. We herein provide a detailed and up-to-date insight into the preventive and mitigating measures that are presently being considered against COVID-19 globally. The updates on the repurposing of FDA approved drugs to cure COVID-19 have been highlighted and reflect state of ongoing research and therapeutic initiatives those are being employed worldwide towards finding the treatment strategies for COVID-19. Furthermore, it conferred an update on revised diagnostic state and strategies for accelerated detection of COVID-19 infection. The ongoing clinical trials and research worldwide to develop the interventional COVID-19 treatment have been specifically envisaged. Given the fact that being a scientific community, we are now engaged in translating the health implications of ongoing COVID-19 pandemic, an extensive research further is required to address the challenges of COVID-19 pandemic with identifying the druggable targets, vaccine development, quicker diagnosis, overcoming the acute immune-response linked with SARS-CoV-2 infection.