Challenges and Opportunities to Develop Diagnostics and Therapeutic Interventions for Severe Acute Respiratory Syndrome- Corona Virus 2 (SARS-COV-2)

Severe Acute Respiratory Syndrome-Corona Virus 2 (SARS-CoV-2) or Corona Virus Disease 19 (COVID-19) is playing havoc all over the world since December 2019. Despite being a family member of coronaviridae, which has previously affected mankind twice in last one decade, the novel corona virus, as it is named left medical practitioners and scientists defenseless. The major challenge is twofold identifi cation and therapeutic intervention. Several approaches, including real-time PCR have already been taken for quick identifi cation of Covid19. Due to very fast evolving rate, accurate identifi cation is still a challenge for most of the detection methods developed in last three months. Several proposals for therapeutic intervention have also put forth by scientists, ranging from vaccine to RNA therapy. In this article, a comprehensive review is made from the scattered scientifi c literatures and is fi ne-tuned further with possible diagnostic and therapeutic interventions. ABSTRACT *Corresponding author


INTRODUCTION
The novel corona virus or SARS-CoV-2 is alleged to be originated from the city of Wuhan, China, has been currently turned into a pandemic and aff ected almost every country (213 countries) throughout the world (https://www.worldometers. info/coronavirus/countries-where-coronavirus-has-spread/). Corona virus is a unique class of virus, that exists under the large viral of coronaviridae, which is a single stranded positive RNA sense virus. The size of virus particle ranges from 70-90 nm [1]. Due to presence of spikes on the outer surface, which resembles a crown under an electron microscope, these viruses are called as corona virus, as per Latin word 'corona' which means 'crown' [2]. Further, the corona viruses are subclassifi ed into alpha, beta, gamma and delta subtypes. These viruses are endemic in non-human vertebrates, such as bats, civet cat, camel as well as Pangolin and few of them are able to infect human [3]. According to Center for Disease Control and  [5]. In the current paper, we shall mention the disease as COVID-19 and viruses as SARS-CoV-2.
SARS-CoV-2 is airborne, swiftly evolving and highly contagious in nature, which is evident by the infection numbers, which is escalating at an exponential rate in past 5 months. Till now, the mortality rate is calculated between 2%-3%, however, there are numerous factors, including age, comorbidity of the patient and health facility, that infl uence the mortality rate [6]. As a result, the reported mortality rate is found to have less than 1% in some countries whereas higher than 10% in some other countries [7].
There are various studied have been conducted on the virus incubation period in human which in turn helps the policy makers to quarantine and observe suspected individual [7].
The results suggested the incubation time may vary greatly, but 14 days of isolation and observation may be suffi cient to determine the infection [8]. There are diff erent theories of mechanism of SARS-CoV-2 infection has come forward and diff erent treatment approaches have been proposed.
Out of multiple approaches to contain the infection and treat the infected patients, one model has been adapted globally, which is early detection of the infected individual and treat them in a quarantine facility. Hence, it is evident that we need a sensitive and a rapid diagnostic system to identify the infected, but asymptomatic patients. The second part is, of course, fi nding suitable and target specifi c therapeutic interventions. Vaccine is considered to be hopeful therapeutic agent against Covid-19 to combat them and control its transmission. As of now total 115 various types of vaccine candidates have been identifi ed out of which 78 are confi rmed based on publicly available data and 5 of which has been approved for clinical trial [9]. For developing any successful and precise therapeutic and diagnostic measures, it is important to know the genetics of SARS-CoV-2 and its exclusive structure.
Among structural type of proteins, Spike protein (S) (~150 kDa) is a glycoprotein, which has a signifi cant role in the attachment of the virus to cells. It is glycosylated and it has three domains-N terminal domain consists of S1 and S2subdomain and protrude outwards, a domain of transmembrane and a cytoplasmic structure at the C terminal end. It intermingles with the Angiotensin Converting Enzyme 2 in human (hACE2), after being cleaved by Transmembrane Serine Protease 2 (TMPRSS2) at the S1 and S2 junction [12]. Membrane (M) protein is comparatively smaller (~25-30 kDa) but more abundant. It has three transmembrane domains and plays a signifi cant role in membrane dynamics like shaping the virion and membrane curvature and nucleocapsid binding [13]. The Envelope (E) protein is also a small, yet extremely Diverse Protein (~8-12 kDa). Although E protein is not fully characterized, yet it is speculated that E protein may be a transmembrane protein ion channel activity. Also, E protein enables the viral assembly and release and certain studies indicated that the E protein is essential in pathogenicity, especially viral lethality [14]. Nucleocapsid (N) protein is a heavily phosphorylated protein, which plays a variety of functions. It has an affi nity towards both host and viral RNA, but phosphorylation of the N protein changes the affi nity towards viral RNA over host RNA [15]. Also, N protein found to play role in viral genome packaging, acts as a structural subunit of replicase complex and mediates the tethering of the viral genome to replication transcription complex [16]. Also, it was found to infl uence host cell response by modulating chaperone activity and cell cycle regulation [17].
Non-Structural Proteins (Nsp) of Betacoronaviridae family members have a diverse function ranging from blocking the host immune response to RNA polymerization.
Non-structural proteins are categorized as Nsp1 to Nsp 16. Inhibition of the immune system is caused by diff erent Nsp is diff erent ways. Nsp1 inhibits interferon signaling [18], Nsp3 blocks host innate immune response and are essential in cytokine storm by promoting cytokine expression [19], Nsp5 acts like Protease (3CLpro) and cleaves pp1a [20] and inhibits interferon signaling [21]. Nsp6 restricts autophagosome expansion [22], Nsp12 acts like RNA dependent RNA polymerase by its C terminal domain [23], Nsp14 and 15 has an exo-and endo-ribonuclease activities [24] and Nsp15 helps the viral machinery to evade double stranded RNA sensors [25]. Nsp16 along with Nsp10 mediates ribose 2'-O methylation [26]. So, it is evident that beta coronaviridae family has an extremely sophisticated genome organization and understanding its function at the molecular level will help to design eff ective therapeutic measures.
The virus particle attaches the human cell via spike protein and hACE2 binding. Upon binding, spike protein is cleaved by TMPRSS2 and helps the processed S protein to fuse with host membrane [15]. In SARS-CoV-2, the cell entry is preactivated by proprotein convertase furin, hence, the dependence on host protease is reduced. Cell mediated endocytosis helps the virus to internalize into the human (host) cell [27]. After entering into the host cell, lysosomal proteases like cathepsin cleaves the viral structure and helps to release the genomic RNA inside the cell. Later, the virus develops a 'slippery' sequence (5′-UUUAAAC-3') and a pseudoknot RNA structure to translate two polypeptides by frame-shifting mechanism. The polypeptides are cleaved into Nsps and some of the Nsps take part in Replication-Transcription Complex (RTC) formation to provide an amiable environment for viral replication [15].

PATHOPHYSIOLOGY OF SARS-COV-2
The pathophysiology of any pathogen-mediated infections are required to be understood elaborately for the eff ective designing of treatment modalities and drugs to combat them [28]. SARS-CoV-2, which is a new virus pathogen that has ability to cause Severe Acute Respiratory Syndrome (SARS) and has infected 13 million people from December 2019 to May 2020 [29]. The pathophysiology of SARS-CoV-2 is in high demand to be revealed in recent times as it is a new virus (novel Coronavirus or 2019-nCoV) [4,30], no proper treatment methods until now, governments have implemented lockdown measures to reduce the crowd for mitigating the spread of this virus, which eventually declines the economy of a country [31] and understanding their pathophysiological pathways can help to combat this viral infection eff ectively in the upcoming days [32]. The SARS-CoV-2 virus was fi rst recognized in certain respiratory illness patients from December 2019, in the city of Wuhan, China [33], and received this name as its 80% of the genome sequence is similar to SARS-CoV (coronavirus family, that caused SARS), which has spread rapidly in the year 2002 [34]. These types of viruses possess Ribonucleic Acid (RNA) as their genetic materials, which provides them enhanced potential to mutate rapidly and can generate sub-species [35]. The viral genome is protected by a protein capsid [36] and a Spike (S) protein, which binds with the host cell [37]. The protein capsid in SARS-CoV-2 is made up of three unique protein groups, such as E (capsid protein), M (membrane protein) and N (nucleoprotein) [38]. Even though, other proteins in the capsid are useful in the maintenance of genome structural integrity, the spike proteins are the one, that can bind with receptors in humans to initiate its replication [39]. The SARS-CoV-2 infection and its transmission from the person to another starts via microdroplets, that are airway generated by the infected person and expelled, while sneezing, coughing or other similar activities [40] as displayed in fi gure 1. The microdroplets with the virus particles will reach another person and can enter them via epithelial conjunctiva and the upper respiratory tract cells [41]. However, the aerosol transmission of viral particles is still under investigation and it has been estimated that these particles can reside in an unventilated space for several hours [42]. Recently, several researchers suggested that the aerosol can stay longer in the air with the viral load, which may transmit the virus eff ectively in a closed or air conditioned environment [43,44].
The virus with the double domain surface glycoproteins (spike protein), that are inhaled will bind with the epithelial cell in the nasal cavity via Angiotensin Converting Enzyme Type 2 (ACE2) receptor and initiates its replication by the stages, such as entry, uncoating, replication, transcription, translation, virion assembly and release [45,46]. It has been proven in the in vitro data that the coronaviruses family possesses enhanced ability to bind with the ciliated cells in the respiratory tract as the primary target in the conducting airways [47]. The virus will replicate in the epithelial cells and spreads to other parts of the respiratory tract in the initial 1-2 days of infection, which is called as an asymptomatic state [48]. In this stage, a person infected with the virus can be identifi ed by analyzing their nose and throat swab samples, which will contain high viral load [49]. However, recent studies also proved that the existence of virus (low viral load) can be detected in saliva, sputum, tracheal and pharyngeal swabs, pleural eff usion fl uid, broncho-alveolar lavage and in certain cases, urine and semen [50]. The samples from the patients will be subjected to Real-Time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) analysis to detect the single RNA sequence of SARS-CoV-2 to label them as positive or negative cases [51].
In the next phase, the virus spread more in the respiratory tract cells and the viral load will increase along the conducting airways, which eventually activates an innate immune response to show certain mild to severe symptoms, such as uncomfortable to breathe and fever [52]. The immune response will vary with the age, gender and various other factors in covid-19 positive patients and the cytokinebased innate immune response will be triggered to protect the unaff ected cells as well as inhibit the replicating virus 222 Paul R, et al. (2020), J Biomed Res Environ Sci, DOI: https://dx.doi.org/10.37871/jbres1147 [53]. Above 80% of the SARS-CoV-2 infected patients are either asymptomatic or with mild symptoms such as fever, if their immune response is better without any comorbid conditions [54]. However, these patients can spread the virus to others through air transmission as mentioned earlier [55]. It is worthy to note that the level of CXCL 10, which is an interferon responsive gene, which is expressed in viral infected epithelial cells along with other beta and lambda interferons. This CXCL 10 gene possesses superior ratio of signal to noise in the response towards type 2 alveolar cells and are recommended to be benefi cial as a potential disease marker for SARS diagnosis [56][57][58].
The rest of 20% population aff ected by SARS-CoV-2 will progress to the next stage, which will show moderate symptoms, such as pneumonia with fever and cough without hypoxemia, severe symptoms, including pneumonia with hypoxemia, which leads to critical conditions in patients such as Acute Respiratory Distress Syndrome (ARDS), along with other conditions namely encephalopathy, shock, heart failure, acute kidney injury, myocardial injury and coagulation dysfunction [59]. Out of this 20% severely infected patients, 2% of fatality rate can be observed, mostly for patients with comorbid conditions and age [60].
In this stage, the viral load will increase in the lungs'gas exchange units and critically contaminates the sub pleural and peripheral type 2 alveolar cells [61,62]. Later, the lung cells will undergo apoptosis, due to high viral load and starts to gradually spread towards other organs [57]. The result from the pathological studies revealed that the SARS-CoV-2 possess ability to diff use damage in the alveolar region with a few giant multinucleate cells and hyaline membranes rich in fi brin [63]. Thus, vigorous response of innate and acquired immunity as well as epithelial cell regeneration are required to treat severe symptom exhibiting patients and recover them from the infection [64]. The 2% mortality rate in infected patients is mostly due to reduced immune response in old-age and comorbid populations with reduced ability to repair the damaged epithelial cells. In addition, these groups of infected patients will have reduced mucociliary clearance, which eventually facilitates the escalated virus spread in the gas exchange units of the lungs and leads to death [65].

Effect of SARS-CoV-2 in various organs
Apart from general pathophysiology, which is focused in the lungs, SARS-CoV-2 have a specifi c mechanism of action to infi ltrate damages in other organs. It has been reported that the kidney, heart and brain are the most aff ected organs, due to corona-type virus infection, especially SARS-CoV-2, other than the lungs.
Lungs: Lungs are the most aff ected organ due to any nasal pathogenic viral infections and SARS-CoV-2 also severely aff ect these organs. It can be noted that the lung serves as a replication spot for these novel coronaviruses, cause pulmonary damage and spread to other organs after severely aff ecting the lungs and nasal pathway [11]. Ziehr, et al. [66,67]. stated the respiratory pathophysiology of  Human Protein Atlas [72]. The ACE2 receptor is expressed in several organs, including kidney, and the corona virus binds and replicates in humans via ACE2 receptor to cause acute respiratory illness [73]. However, the viral RNA has also been noticed in the urine samples, which has concluded the involvement of kidney in Covid-19 infection. The study concluded that the existence of ACE2 receptor in the kidney tubules will cause injury, which eventually leads to the consequences of covid-19 infection in the kidney [74].
Further, Zhang and Liang also mentioned the latent risk of the kidney as a defenseless organ to the infection caused by novel coronavirus 2019 [75]. In the study, they have reported the prevalence of non-respiratory symptoms, such as myalgia, diarrhea and fatigue in moderate cases and acute kidney injury in severe cases, aff ected with SARS-CoV-2.
Further, pathophysiological studies exhibited that the acute kidney injury can be a multifactorial mechanism, that are triggered via 2019-nCoV direct infection, which elevates responses of infl ammation and immunity and leads to toxic reactions, due to respiratory failure [75]. Contrarily, Wang, et al. with SARS and MERS provided ample evidence that SARS-CoV-2 can enter and damage the brain. Further, Moriguchi, et al. [81] reported that the SARS-coronovirus-2 can lead to meningitis or encephalitis in severely aff ected patients. In a particular case reported by the authors, the patient had fever and fatigue in day 1 and prescribed with Laninamivir and antipyretic agents for 2-5 days. Later, the patient developed severe symptoms and became unconscious after 9th day. The nasopharyngeal swab analysis of the patient did not detect the presence of any viral load, however, the viral load was detected in cerebrospinal fl uid. Furthermore, Zanin, et al. [82] mentioned that novel coronavirus can induce spine and brain demyelinating lesions in severely infected patients.
The study suggested that the virus can cause Systemic can be milder symptoms such as fever and cough [84]. In addition, Verdoni, et al. [85] reported that the children aff ected with SARS-CoV-2 in the Italian epicenters are developing a severe Kawasaki-like disease, which is an acute self-limiting vasculitis of the medium caliber vessels.
In certain cases, the binding nature of corona virus with ACE2 has led to a potential damage in the gastrointestinal tract. Interestingly, Grassia, et al. [86] reported that the

DIAGNOSTIC APPROACHES TO IDENTIFY SARS-COV-2
Rapid diagnosis of infectious agents is a key step to fi ght against an outbreak. When a new virus infects human, diagnosis becomes a challenging task. The diagnosis is divided into 3 major parts-specimen collection, virus isolation and identifi cation. It is worthy to note that the PCR method is highly benefi cial in the detection of the virus, while it exists in a person. Another technique applied with limited success is immunodiagnostic method. Here, the detail procedure shall be discussed to shed light on diagnostic procedure.

Specimen collection
Being an airborne pathogen, in the early stage of

Covid-19 diagnosis
Diagnosis is an important part of fi ghting a pandemic. In a large population, identifying the potential candidates is a challenging task. In addition to this, diagnosis of a relatively unknown virus is always challenging. Multiple approaches have been proposed ( Table 1). Some of the key approaches are documented in the current review.

Real time PCR
It is also named as reverse transcription quantitative PCR. It is regarded as the gold standard to detect SARS-

LOOP MEDIATED ISOTHERMAL AMPLI-FICATION
It is a Nucleic Acid Amplifi cation Test (NAAT), where rapid analysis can be obtained. It is widely applied to various types of pathogen detection like virus, bacteria and protozoa (especially malaria). The simplicity of the technique enables it to be converted into mobile unit and can be used at the point of care like railway station, airport, and rural areas. Reverse transcription loop mediated isothermal amplifi cation process or RT-LAMP is a cheaper and faster process compared to qRT PCR. It depends on one step amplifi cation technique. Unlike PCR, which needs thermal cycling, RT LAMP is carried out at a temperature between 60-65C and use DNA polymerase. This technique employs a set of four primers [93] to recognize six unique sequences in target DNA sequence. There are forward as well as backward inner primers with dual sequences of primer.
The fi rst set is for initial stage priming and the second is for later stage self-priming process. Among four primers, both primers are utilized at the early stage, but later the stand displacement is done by inner primers. In the fi rst report, the reaction is carried out at 65C for 1 hour [93]. Later it was observed that the inclusion of two additional primers can shorten the reaction time by half [94]. The current LAMP method uses total six primers which are specifi c against eight unique sites. Besides inner and outer primers, which are able to identify six distinct sites, the loop of two primers elevates the speed and the effi ciency of amplifi cation [95].
Previously, the effi cacy of LAMP was shown to detect SARS [96] and RT-LAMP assay for MERS detection [97] corona virus with high sensitivity. In LAMP, reverse transcription reaction is done in the same tube prior to amplifi cation; hence it makes the identifi cation process faster. In one  it can be identifi ed in visual detection at 40 min ( [100]. In another study, researchers have targeted ORF1ab region and the reaction was conducted at 65C. The visible color change was observed within 20 min when the template RNA copy number was 1000. The detection capability was further extended by using SYBR green dye [101].

Imaging techniques
Imaging techniques are highly benefi cial in any disease diagnosis. As Covid 19 is an acute respiratory syndrome,

Serological Test
Many reports have emerged that young adults and children, who have not been exposed to any of the antibody-rich serum to help treat critically ill patients.
Another key application would be to identify people who have developed likely immunity to the virus. They might be able to treat patients safely or take on other front-line jobs during the pandemic [91,105].

Vaccine development
The were also recently recommended as a probable vaccine to produce immunity against SARS-CoV-2 in human hosts [113,114]. Furthermore, Shenzhen Geno-Immune Medical Institute of China prepared LV-SMENP-DC vaccine by using lentiviral vector with the synthetic minigene to produce antigen-specifi c cytotoxic T-lymphocytes and pathogenspecifi c artifi cial antigen-presenting cell to inhibit viral protein as an eff ective vaccine candidate against 2019-nCoV [53]. However, all these vaccines are in Phase I clinical trials and it will take a long time to reach the commercial pharmaceutical market.

Antiviral drugs
Several preexisting antiviral drugs that are used in

Rehabilitation programs
In addition to diagnostic, vaccine development, antiviral drugs and drug repurposing approaches, rehabilitation programs were also considered to be a signifi cant method for the recovery of SARS-CoV-2 infected patients. Zhu, et al.  CoV-2 infected patients, where the lung muscle is tightened and inspiratory muscle training as well as breathing exercises can be benefi cial to improve dyspnea. Moreover, physiotherapy was reported to be useful for assisting the positioning of COVID-19 patients to relax them from respiratory distress and to prevent secondary complications. Furthermore, early rehabilitation via physiotherapy can be recommended to recovered patients for limiting or preventing ICU-acquired weakness [130]. Likewise, Dehesh (2020) stated that stress may lead to negative eff ects on COVID-19 patients with back pain or musculoskeletal disorders, which can be reduced with fi rm physiotherapy guidelines [131]. Similarly, Diwate (2020) agreed to the fact that physiotherapy is useful in the physical rehabilitation and treatment of COVID-19 patients and recommended 'expert consensus and recommendation for physiotherapy management for COVID-19 in Indian set up' guidelines, which is approved by Maharashtra State Council for Occupational Therapy and Physiotherapy, Mumbai (India) for physiotherapists to decide and plan treatment for SARS-CoV-2 infected patients [132]. All these studies emphasized that physiotherapy can be a benefi cial approach for the proper rehabilitation, during and after ICU-based treatments for COVID-19 patients. which gives promising status that a vaccine will be available within next year. Even before the vaccine, rapid diagnostic tools and drugs to reduce or treat the health complications instigated by the infection of virus, will be available in markets as mentioned in this review. In the near future, more critical knowledge on the pathophysiology of SARS-CoV-2 viruses will be available, which will eventually lead to the discovery of highly effi cient and target specifi c drug or vaccine to eradicate this viral infection and save lives of several millions of people.