SARS-CoV-2/COVID-19 and Advances in Developing Potential Therapeutics and Vaccines to Counter this Emerging Pandemic Virus – A Review

length: 288 words Figures and tables: 4 (2 figures and 2 tables) Manuscript length: 12000 words Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 April 2020 doi:10.20944/preprints202004.0075.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license.


Introduction
Since December 2019, a pneumonia-like Coronavirus emerged in Wuhan, China. Within a few weeks, a novel coronavirus was given a nomenclature as 2019 novel coronavirus (2019-nCoV) by the World Health Organization (WHO) (https://www.who.int/emergencies/diseases/novel-coronavirus-2019) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses (ICTV) (Gorbalenya et al., 2020), causing the disease termed as an emerging coronavirus disease . Since then, this virus has spread to every populous continent and infected thousands around the world -through the vast majority of confirmed cases, and fatalities fall within China and spreading to Italy, Iran, South Korea, France, Spain, USA, Japan, Spain, and many others. COVID-19 has now spread to 175 countries with nearly 500,000 confirmed cases and 22,000 human deaths, and most recently (on 11 th March 2020) WHO proposed it as a pandemic situation worldwide (WHO, 2020a; Yang et al., 2020a). Unlike other coronavirus epidemics like SARS and MERS, the transmission rate of COVID-19 is much higher with spreading of the virus infection to an average of two to three individuals from every infected individual (Gates et al., 2020).
Coronaviruses, among all known RNA viruses, have the largest genomes ranging from 26 to 32 kb in length (Regenmortel et al., 2000;Schoeman and Fielding, 2019). In addition to encoding structural proteins, a significant chunk of the coronavirus genome is transcribed and translated into a polypeptide, which encodes proteins essential for viral replication and gene expression (Lai and Holmes, 2001). The ~306 aa long main protease (Mpro), which is a highly conserved sequence, is a crucial enzyme for coronavirus replication (Lai and Holmes, 2001). Mpro has been utilized for developing anti-coronaviral drugs (Xue et al., 2008;Anand et al., 2003). Nevertheless, in the past two decades, a massive amount of research has been done to understand the replication, molecular basis of the coronavirus infection and evolution, develop effective therapy in forms of both vaccines and antiviral drugs, and propose efficient measures for viral detection and prevention (Perlman and Netland, 2009

Taxonomy and Classification
ORDER: Nidovirales  Table 1 describes the broad classification of the coronavirus.

Phylogenetic analysis and Genomic organization
Recent phylogenetic analysis has revealed that the new virus spreading in China is a version of SARS-CoV. There are two significant similarities between SARS-CoV-2 (nCoV-2019) and SARS-CoV: they share nearly 80% of their genetic codes, and both are originated in bats (Malik et al., 2020;Zhu et al. 2020). The first study to analyze the viral genome analysis was conducted in the Wuhan Institute of Virology. Samples from seven patients initially reported cases of severe pneumonia were used for the analysis. It was found that the new virus genetic sequences were 79.5% similar to SARS-CoV (https://www.businessinsider.in/science).
The genome of CoVs consists, a single-stranded, positive-sense RNA of around 29.8 kb nucleotides in length with a 5′-cap structure and 3′-polyA tail. The polyprotein 1a/1ab (pp1a/pp1ab) is directly translated using genomic RNA as a template, which encodes nonstructural proteins (nsps) and forms the replication-transcription complex (RTC). Also, a nested set of subgenomic RNAs (sgRNAs) are produced by replication-transcription complex in a discontinuous manner of transcription. It has common 5′-leader sequences and 3′terminal sequences. Transcription termination and acquisition of leader RNA happens at transcription regulatory sequences, which are located between open reading frames (ORFs). These sgRNAs (minus-strand) acts as the templates for the synthesis of subgenomic mRNAs (Perlman and Netland, 2009;Chen et al., 2020b;Dhama et al., 2020a) A typical CoV's genome and subgenome contain at least six ORFs. The first ORF (ORF1a/b), contains two-third of the whole length genome and encodes 16 nsps (nsp1-16). A-1 frameshift between ORF1b and ORF1a, leads to the synthesis of two polypeptides: pp1ab and pp1a. These are processed by chymotrypsin-like protease or two papain-like protease or main protease and one into 16 nsps. Rests of the ORFs of the genome encode four major structural proteins: membrane (M), spike (S), nucleocapsid (N) and envelope (E) proteins. Other than these proteins, different CoVs encode specific accessory proteins and structural proteins, such as 3a/b protein, HE protein and 4a/b protein (Perlman and Netland, 2009;Chen et al., 2020b;Dhama et al., 2020a).
The genomic sequence alignment among different CoVs shows 58% identity on the nonstructural protein-coding region, 43% identity on the structural protein-coding region, with 54% at the whole genome level which suggests the non-structural proteins are more conserved when compared to the structural proteins and structural proteins are more diverse when in need of adaptation for new hosts. However, the CoV genome is extensive, with ~30 kb in length and is considered as the largest known RNA viruses. Such a large genome of CoVs is maintained by the unique features of the CoV RTC, which encodes for many RNA processing enzymes. The 3′-5′ exoribonuclease is one such kind of RNA processing enzyme and is unique in CoVs among different RNA viruses, also believed for providing a proofreading function to the RTC. Sequence analysis depicts that the SARS-CoV-2 has a typical genome structure same as of CoV and mainly belongs to the Betacoronaviruses cluster, which includes Bat-SL ZXC21, Bat-SARS-like (SL)-ZC45, SARS-CoV, and MERS-CoV Dhama et al., 2020a). The glycoprotein spike surface plays an essential role in receptor binding and determines host tropism (Zhu et al., 2018). These proteins of SARS-CoV and MERS-CoV bind via different receptor-binding domains (RBDs) to various host receptors. SARS-CoV utilizes angiotensin-converting enzyme 2 (ACE2) as the primary receptor and MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4, also known as CD26) as the primary receptor. Initial analysis depicted that SARS-CoV-2 has a quite close phylogenetic association with SARS-like bat coronaviruses. (Malik et al., 2020;Zhou et al., 2020) Comparison of genomes of SARS-CoV-2, SARS-CoV and MERS-CoV strains suggested that they are almost identical and possess only five nucleotide differences in the genome. The SARS-CoV-2 genome annotation shows that they possess 14 ORFs encoding for 27 different proteins. The orf1a and orf1ab genes of the genome, respectively encode the pp1a and pp1ab proteins. They together possess 15 nsps, from nsp1 to nsp10 and nsp12 to nsp16. The genome also contains eight accessory proteins and four structural proteins (S, E, M, and N). The SARS-CoV-2 is quite similar to that of SARS-CoV, at the amino acid level. Also, there are some differences. For example, the 8b protein is 84 amino acids in SARS-CoV, whereas in 2019-nCoV, 8b protein has 121 amino acids; the 8a protein is present in SARS-CoV but absent in SARS-CoV-2 Dhama et al., 2020b) An observation of the amino acid substitutions in different proteins occurs among them, and this sheds light into how SARS-CoV-2 differs from SARS-CoVs structurally and functionally. In total, 380 amino acid substitutions were there between the SARS-CoV-2 amino acid sequences and the SARS and SARS-like viruses. However, no amino acid substitutions were found in nonstructural protein 7 (nsp7), envelope, nsp13, matrix, or accessory proteins 8b and p6. Nevertheless, due to minimal knowledge about this novel virus, it is quite hard to give reasonable explanations for the amino acid substitutions between the SARS-CoV-2 and SARS or SARS-like CoVs and also whether these differences could affect the host transmission property of the SARS-CoV-2 when compared to SARS-CoV needs future investigation .

The emergence of Coronaviruses (SARS-CoV, MERS CoV and SARS-CoV-2)
The Coronaviruses were known to cause only mild respiratory disease in humans; with severe infections in the immunocompromised cases and young kids mainly acting as an asymptomatic carrier as disease symptoms are not much prominent in young ones (Shen et al., 2020). Coronaviruses have the highest known frequency of recombination of any positivestrand RNA virus (Rohde, 2020). Earlier reports suggested the emergence of SARS-CoV by recombination between bat SARS related coronaviruses (SARSr-CoVs) followed by mutations in civets before its spillover. Similarly, the MERS-CoV also circulated and attained mutations for around 30 years in camels before the MERS pandemic happened (Muller et al., 2014;Cui et al., 2019), suggesting the adaptation of the viruses to the environment and different host before their spillover to humans (Ellwanger and Chies, 2018). Coronaviruses also have an essential replication process, which involves a 2-step replication mechanism. Many RNA virus genomes contain a single open reading frame (ORF), but coronaviruses can contain up to 10 separate ORFs except for SARS-CoV-2, which has single intact ORF (Rohde, 2020). That can lead to the emergence of CoV at times.

MERS-CoV
MERS-CoV was first identified in the Kingdom of Saudi Arabia in 2012. Cases have been geographically restricted to the Arabian Peninsula. However, a smaller number of cases were also found in Europe, i.e. in people who had travelled to the Arabian Peninsula or had been in contact with people who had. Individual cases and small clusters continue to be reported in that region. MERS-CoV is thought to be transmitted from camels to humans, with the possibility that at some point bats infected camels. MERS-CoV is thought to have emerged from bats or other small animals, and have infected humans via dromedary camels acting as intermediate hosts (Ramadan and Shaib, 2019; WHO, 2020c).

SARS-CoV-2
A novel (new) coronavirus (SARS-CoV-2) that was first detected in Wuhan City, Hubei Province, China is posing a significant threat now by creating a pandemic situation of the COVID-19 (WHO, 2020d; Yang et al., 2020a). The first case was detected in December 2019, and presently nearly 500,000 confirmed cases detected (WHO, 2020a). The disease terrorized over 175 countries/territories/areas with 22,000 deaths which is probably not the last figure to be known (WHO, 2020a). In early March 2020, the pandemic started to subside in China (Flahault, 2020) but started to haunt Europe and the United States. In this context, other than China, the major blow was felt by Italy, Spain, Iran and France with 7500, 3,650, and 2077 deaths, respectively but the pain of countries like France (1,330 deaths), USA (944 deaths), UK (465 deaths), Republic of South Korea (126 deaths), Netherlands (356 deaths) and Germany (206 deaths) could not be overlooked.
However, only a few countries are left beyond the reach of this unfortunate pandemic, but their possibility of remaining untouched by this pathetic virus is meagre because within a short period the virus emerged in many new countries including India where two individual recently got victimized. Now the number of new confirmed cases and deaths are shooting day by day with no hope of sudden halt leaving the global population in a stage of physiological, psychological, and socioeconomic stress. In a study, the psychological stress of general public along with nurses involved in the treatment of COVID-19 patients was evaluated based on vicarious traumatization scores and revealed that the scores were found significantly higher for the general public than nurses .
The origin of SARS-CoV-2 was postulated to be from bats, considering them the natural reservoirs of the virus (Chan et al., 2020a). The search of probable intermediate hosts is going on keeping the main focus on animal species available for sale in Huanan seafood market. Recently, a study suggested the pangolins to serve as a natural reservoir host of SARS-CoV-2-like coronaviruses (Pangolin-CoV) based on 91.02% sequence similarities at the wholegenome level between pangolin-CoV and SARS-CoV-2. Additionally, the S1 protein of pangolin coronavirus was reported to be more closer to SARS-CoV-2 than bat coronavirus (RaTG13) . Initially, the disease was thought to be spreading via seafood consumption, but later contact transmission of SARS-CoV-2 among humans was confirmed. In nearby places like clinics and hospitals, CoV can be transmitted through the air if people remain there for a long duration of time, suggesting the risk of aerosol transmission (Cascella et al., 2020).
The emergence of SARS-CoV in 2002 established CoVs as capable of causing severe disease in humans. This ability was once again demonstrated by the emergence of a second severe CoV a decade later; the MERS-CoV in 2012. The MERS-CoV and SARS-CoV emergence are believed to be the result of spill-over of bat-adapted CoVs into an intermediate host. They are transmitted from person to person through respiratory droplets and close contact (https://www.cdc.gov/coronavirus/types.html). The virus is believed to be transmitted to patients living in or working at areas in proximity to wholesale seafood markets and places where live animals like snakes, bats, birds, marmots, and other wild or farm animals were sold (Ji et al., 2020). Rarely animal coronaviruses infect humans severely except for SARS-CoV, MERS-CoV and now SARS-CoV-2. Seldom coronaviruses cause lower respiratory infection or pneumonia but SARS-CoV-2 causes. Coronaviruses have seasonal pattern usually affecting in winter, however, can infect out of season also (Rohde, 2020).

COVID-19 Clinical Pathology
The coronavirus claimed its first life on January 10 th , 2019, and since then the death toll has climbed at an alarming and accelerating rate. The virus seems lethal, causing severe acute respiratory symptoms, including fever, dyspnea, asthenia and pneumonia, thrombocytopenia, and increased C-reactive protein and lactate dehydrogenase levels among people in Wuhan, China Lu et al., 2020b). As per reports, mild COVID-19 cases revealed higher levels of pro-inflammatory cytokines and chemokines like IFN-α, IL-1β, MCP-1 and IP-10 whereas individuals with severe COVID-19 had upregulation of IP-10, IL-8, IL-10, TNF-α, G-CSF, MCP-1 and MIP-1A (Huang et al., 2020; Qin et al., 2020) resulting into cytokine storm syndrome followed by severe pulmonary damage and death due to respiratory failure. Additionally, all the blood cells except neutrophils were reported to be decreased with fall in lymphocytes subsets like T cells, B cells and NK cells in severe COVID-19 cases (Qin et al., 2020). Chest radiographs show invasive lesions in both lungs with flaws of a variable degree in the lungs. Additionally, bilateral multilobular subsegmental consolidation, groundglass opacity with many mottling was also reported in the COVID-19 patients (Bassetti et al., 2020;Huang et al., 2020). Recently, myalgia and fatigue are found associated with rhabdomyolysis in a COVID-19 patient in Wuhan, China suggesting the need of rapid clinical diagnosis followed by favourable hydration treatment to reduce the risk of severe outcomes as a result of rhabdomyolysis (Jin and Tong, 2020).
Additionally, COVID-19 patients may also manifest neurological signs such as headache, nausea and sometimes vomiting, but even dysgeusia and anosmia. Moreover, SARS-CoV infection has been reported in nervous tissue of experimental animals and patients with heavy involvement of brainstem. In this context, acute respiratory failure in COVID-19 patients suggests the probable invasion of the brain by SARS-CoV-2 (Li et al., 2020b). Recently, a study supported the neurotropic potential of the SARS-CoV-2 virus as 36.4% of involved COVID-19 patients manifested neurological signs (Mao et al., 2020). Those who developed severe pneumonia, pulmonary oedema, hypoxemic respiratory failure, gastrointestinal infection, multiple system failure or Acute Respiratory Distress Syndrome (ARDS) succumb to the disease. The threat is still looming high on the event of this deadly virus created a pandemic situation (  A reverse real-time PCR assay (rRT-PCR) is required for useful and timely screening of COVID-19 patients, which can be carried out in clinical samples like fibre bronchoscope brush biopsies, bronchoalveolar lavage, nasal swabs, pharyngeal swabs, sputum, blood and faeces ). An interactive web-based dashboard for monitoring COVID-19 in real-time mode has also been reported (Dong et al. 2020). A fluorescence-based quantitative PCR assay based on SARS-CoV-2 N and ORF1ab regions has been developed ). Moreover, testing for COVID-19 requires travelling to a clinical setting that could lead to increased risk of disease transmission hence a rapid, cheap, user-friendly and sensitive diagnostic tool must be developed for use by ordinary people in their homes (Yang et al., 2020b). In this context, a potential RNA-based POCT diagnostic device which combines a LAMP assay technology and a paper-based POCT was described as a home-based highly accessible and sensitive COVID-19 diagnostic tool with the additional advantage of smartphone integration enabling the individuals to record and share the results with healthcare workers and subsequent clinical care (Yang et al., 2020b).

Developing Neutralizing antibodies
In general, the replication of coronavirus starts with the entry of S protein, which binds to the surface of the cells. This S protein fuses with the cell membrane and helps the syncytial formation and delivering of viral nucleocapsids into the cell for further replication ( S protein was targeted for developing a neutralizing antibody therapy to combat the novel coronavirus disease (Casadevall et al.,2015). Methods such as phage or yeast display libraries which express antibody fragments could be used efficiently to identify the candidate neutralizing antibody. Traditional methods of screening such as mice or rabbits for neutralizing antibodies would be too late during outbreaks. The only challenge is that neutralizing antibodies should be rigorously tested in animal and cell culture models to confirm that they can neutralize the SARS-CoV-2 disease infection (Shin et  The alternate strategy of generating the neutralizing antibodies against S protein is to immunize large animals like sheep, goat, cow and horse and purify the polyclonal antibodies from these animals. Monoclonal antibodies can be used as potent bio-therapeutics in the form of passive immunotherapy to neutralize the SARS-CoV-2 and to control the harmful outcomes of COVID-19 (Shanmugaraj et al., 2020). These strategies may prove to be beneficial in the condition of an outbreak since they have many advantages, such as simplifying production and manufacturing. For shorter treatment strategy, this could quickly help in SARS-CoV-2 outbreak.
For the time being, immunoglobulin G has been administered in COVID-19 critical patients as therapy . FcR has a role in pulmonary inflammation; hence blocking of FcR activation can reduce inflammatory damage in COVID-19. Thus intravenous use of immunoglobulins can prove helpful in the therapy of SARS-CoV-2 induced pulmonary inflammation (Fu et al., 2020).

Oligonucleotides targeting SARS-CoV-2 RNA genome
Apart from targeting S-protein of the nCoV-2019 virus using neutralizing antibodies, targeting of viral genomes could be other option to reduce the infectivity by degrading its genome. Recently, the RNA genome of the nCoV-2019 virus has been published (Gen Bank: MN908947.3). GS-5734, a nucleotide prodrug, showed broad-spectrum anti-coronavirus activity against bat CoV, pre-pandemic bat CoV, and existing human CoV in vitro and over primary human lung epithelial cell cultures and was found promising for treating epidemic and zoonotic coronaviruses of the near future (Sheahan et al., 2017). The siRNA or antisense oligonucleotides (ASO) can be used to combat the virus by targeting its genome (Qureshi et al., 2018).
Nevertheless, there are a few challenges associated with these methods, such as conserved RNA sequence in the genome of coronaviruses is still not known. Since the conserved sequences are essential in siRNA targeting to avoid viral escape from the oligonucleotides targeting strategy. Second, the delivery of oligonucleotides (siRNA and ASO) would be very challenging. Lipid nanoparticles technology can mediate the delivery of these oligonucleotides into the lungs

Passive antibody transfer
One of the most effective and traditional tools used in most of the infectious outbreaks is the use of serum of patients who just recovered from the active viral infection to treat patients who contract in future . Patients recovered from active viral infections develop a polyclonal immune response to different antigens of SARS-CoV-2 as they neutralize active viral infections and hence convalescent-phase plasma can be used as a therapeutic alternative (Chan et al., 2015). Passive immunotherapy in the form of convalescent serum tested in MERS-CoV infected mice either as prophylactic or in therapeutic form both showed good results and supported the use of dromedary immune serum in preventing MERS-CoV infection . Patients who have 100% recovery from the novel coronavirus infection, can simply donate their plasma to treat the infected patients Marano et al.,2016). The same strategy of convalescent serum was used during the Ebola virus outbreak in 2014-2015 (Kraft et al., 2015). Plasmaderived from the patients who recovered from the disease has also been used as therapy ). α-interferon atomization inhalation has been weakly recommended at a dose of 5 million U per time for adults in sterile injection water, twice a day . Similarly, interferon therapy has also been used; however, it aggravated pathology (Rothan and Byrareddy, 2020).

Drug repurposing using available antivirals
Drug repurposing is a promising, fast and cost-effective method that can overcome traditional de novo drug discovery and development challenges of targeting various diseases and disorders. Drug repurposing, the process of identifying new uses for the existing or candidate drugs is an effective strategy for drug discovery in multiple diseases, including infectious viral diseases. In combating the nCoV-2019 viral outbreak, use of already approved small drug molecules could inhibit the biological aspects of viral life cycle like replication, transcriptions, host protein interaction, boosting immunity, among others. Since the novel coronavirus belongs to the same category of SARS, repurposing of HIV drugs against novel coronavirus could give positive efficacy. Lopinavir may have some prospects in COVID-19 therapy as has been in the SARS, and MERS therapy; however, an extensive evaluation is required ). Efficacy of remdesivir against SARS-CoV-2 is under testing by Gilead Sciences (NASDAQ-GILD) pharmaceutical company (https://www.fool.com/investing/2020/03/04/is-gilead-sciences-the-best-buy-in-thecoronavirus.aspx). National Health Commission of the People's Republic of China has advocated the inclusion of chloroquine phosphate for the cure of COVID-19 patients, in its revised guidelines for the prevention, diagnosis, and treatment of pneumonia developed due to COVID-19 infection in the vast populous country Lin and Li, 2020). Future research could continue to screen currently clinically available small molecular antiviral drugs in tissue culture models to identify candidate drugs to combat the novel coronavirus infection.
The pangolin CoV and SARS-CoV-2 are more than 92% similar at amino acid levels. Thus, using pangolin coronavirus as a model three drugs namely, cepharanthine (CEP), selamectin, and mefloquine hydrochloride were observed to possess anti-SARS-CoV-2 activity with complete blocking of cell cytopathic effects (Fan et al., 2020).

Anti-viral proteases
Rolf Hilgenfeld, a renowned structural biologist in the University of Lubeck, Germany has developed two compounds containing viral proteases and is intended to visit Wuhan, China at the peak of SARS-CoV-2 epidemic to test the clinical efficacy of these compounds in animals so that can use them for the treatment of COVID-19 patients suffering presently and in any future coronavirus outbreak (Cyranoski, 2020).
Anti-coronavirus protease activity was exhibited by the lopinavir (LPV), and it is proposed as a treatment option for ongoing COVID-19 infection . Further, a novel vinylsulfone protease inhibitor suggested treating patients suffering from the 2019-nCoV. That would help in the development of broad-spectrum anti-coronaviral agents for future epidemics (Morse et al., 2020). Recently, a breakthrough in search of antivirals came with the elucidation of SARS-CoV-2 main protease (Mpro) structure. The same could be exploited globally to design some novel drug candidates. Lately, a Deep Docking (DD) platform was used for structure-based virtual screening of nearly 1.3 billion molecules with potential of 1,000 putative ligands for SARS-CoV-2 Mpro protein (Ton et al., 2020).

Blocking Coronavirus receptors like ACE2
It is already known that ACE2 is a crucial player in the coronavirus infection by promoting cell entry (Hoffmann et al., 2020). The metallopeptidase, ACE2, has been identified as a functional receptor for SARS-CoV (Li et al., 2003) and a potent receptor for SARS-CoV-2 . ACE2 is an important drug target for the treatment of cardiovascular and kidney diseases, and. Recently, Lei et al. (2020) demonstrated the potential of ACE2 based therapeutics against SARS-CoV-2, which could further be exploited alone or in combination, and they elucidate the molecular mechanisms of their potent and broad neutralizing activity. These ACE2 fusion proteins could be used for diagnosis and as research reagents in the development of vaccines and inhibitors. ACE2 and AT1R (angiotensin receptor one blocker) molecules such as losartan as inhibitors of the renin-angiotensin system (RAS) could be a useful therapeutic option in reducing the lung inflammation and treating pneumonia in COVID-19 patients (Gurwitz, 2020;Sun et al., 2020). The virus attachment through spike glycoprotein (S) to ACE2 receptors and subsequent priming of S protein by the host cell serine protease TMPRSS2 has been exploited as therapeutic targets. In this context, the role of TMPRSS2 protease in SARS-CoV-2 replication has been reported further supporting their probable role in the development of an active therapeutic agent (Matsuyama et al., 2020). The TMPRSS2 inhibitor proved useful in blocking the virus entry and could work as a therapeutic option (Hoffmann et al., 2020). Arbidol, an effective antiviral against SARS-CoV in combination with antibiotics (moxifloxacin or levofloxacin, nemonoxacin, linezolid, azithromycin or amoxicillin), corticosteroids and oxygen therapy has been used in COVID-19 therapy (Zhang et al. 2020c). Corticosteroids have been routinely used for the treatment of Th1 and Th2 induced lung injury reported in COVID-19 . Conventional Chinese drugs like ShuFengJieDu and Lianhuaqingwen were also used in the treatment of COVID-19, but their efficacy needs to be determined (Lu, 2020). In contrast to this, WHO has indicated that currently no effective treatment for SARS-CoV-2 is known and use of different antibiotics, antiviral drugs, traditional Chinese drugs, corticosteroids like glucocorticoid and their combinations are not recommended before clinical trials as their efficacy is not known and might be detrimental to COVID-19 patients WHO, 2020d). Besides, to manage the hypoxia in the COVID-19 patients' ventilation and salvage therapy is reported beneficial. An α-glucan-based mushroom extract, i.e. AHCC has been reported to be used as an immunostimulant for animals and humans affected by viral infections like the influenza virus, West Nile virus, hepatitis virus, herpes virus, papillomavirus and HIV. In this context, AHCC may be used in the prevention of COVID-19 after evaluation of its efficacy against SARS-CoV-2 (Di Pierro et al., 2020). For safe and successful treatment of severe respiratory illness in infants and children, probability of atelectasis due to invasive or non-invasive ventilation support and risks of oxygen toxicity must be taken into account (Marraro et al., 2020).

Combination therapy
An overview of developing COVID-19 therapeutics and drugs is illustrated in Figure 1.

COVID-19 Vaccines
Appropriate preventive measures, as advised by WHO and CDC, must be implemented in order to delay the spread of COVID-19 so that health workers, healthcare departments, research and development wing and the government will get some time to design and test the The other modes of vaccine development are the utilization of either the virus itself or its part for developing whole organism-based vaccines or subunit vaccines. These include attenuated or inactivated vaccines using cultured SARS-CoV-2 that can be attenuated by passaging or inactivated by physical and chemical methods such as UV light, formaldehyde, and βpropiolactone ). However, these may have limitations of infectivity, reversion to pathogenicity, and disease-causing potential (Roper and Rehm, 2009).  (Forbes, 2020). https://www.forbes.com/sites/alexknapp/2020/03/13/coronavirus-drug-update-the-latest-infoon-pharmaceutical-treatments-and-vaccines/.

Exploration of vaccine candidates of SARS-CoV
As per recommendations of CDC, researchers engaged in diagnosis and clinicians involved in the treatment of COVID-19 infected patients must wear all personal protective equipment including gloves, masks, goggles or face shield (Patel et al., 2020). Besides, due to short supply and unawareness of general public surgical masks are in extensive use which may only reduce the spread to some extent but not prevent the acquisition of SARS-CoV-2. Moreover, high filtration N95 masks along with PPE must be used by healthcare workers while attending patients to avoid nosocomial infection .
Though the outbreaks are spreading worldwide healthcare workers caring for COVID-19 patients and other close contacts, as well as immunocompromised individuals are under high risk (CDC, 2020; Rohde, 2020; Yang et al., 2020a). Surprisingly, pregnancy-associated immunosuppression is reported beneficial for the mother as far as the severity of SARS-CoV-2 infection is concerned possibly due to protection of lungs from cytokine storm brought by the immune system to counter the infectious agent (Who is getting sick…. statnews.com/2020/03/03/who-is-getting-sick-and-how-sick-a-breakdown-of coronavirusrisk-by-demographic-factors/). Several measures were taken and still on to curb the disease, but no acclaimed antiviral or vaccines are available till date as researchers are at large, for which purpose researchers are working day and night as discussed above, and hopefully, appropriate proven vaccines and therapeutics would be available soon (Dhama et Figure 2.

Patents on successful methodologies on various aspects of coronaviruses
Global research focuses on coronavirus infections, especially the recently happened outbreaks of SARS, MERS and the most newly COVID-19. This involves exploration of diagnostics, prophylactics and therapeutics. Accordingly, patenting of successful methodologies has gained immense importance, safeguarding the interests of scientists and institutions. Various patents are being filed, and many approved on diagnostic, prophylactic and therapeutic aspects of coronaviruses and their diseases, especially SARS-CoV caused by SARS, MERS-CoV caused by MERS and the most newly SARS-CoV-2 caused COVID-19. As per one study, around 80% of patents are related to therapeutics, 35% for vaccines and 28% for diagnostics agents or methods . Similarly, on MERS, more than 100 patents are published on therapeutics, and more than 50 on diagnostics and prophylactics ). These patents cover a range of research areas. Table 2 provides details about the various fields and research areas for which patents have been applied and granted. These areas include developing novel, rapid, and specific diagnostics which are cheap and readily available (Liu et  . This is the primary class which has attracted most of the patents ). Either a drug target is located or a direct antiviral compound, molecule or agent is established, or an indirect immunomodulator that raises the immune response against virus is identified as shown in Table 2. Vaccines which are the main backbone for the prevention strategies and are mostly lacking especially for novel coronaviruses have shown immense research prospects and are attracting a sufficient number of patents as shown in Table 2  Since there is some degree of genomic and structural identity hence therapeutics active against SARS-CoV or MERS-CoV or other broad-spectrum antivirals (e.g. remdesivir, chloroquine) are being explored for SARS-CoV-2. Identification of specific therapeutic targets and vaccines candidates will enable developing of specific and potential drugs or vaccines that can prove useful in the prevention and control of SARS-CoV-2 (Liu et al. 2020c).

Conclusion and Future Perspectives
Currently, SARS-CoV-2 outbreak has taken a disastrous turn with high toll rates alone in China itself, most likely the infection is spreading across the globe. There are no licensed vaccines or therapeutic agents (i.e., antivirals and monoclonal antibodies) indicated for this coronavirus prevention or treatment. However, researchers are working to develop countermeasures. Several vaccine candidates for both SARS-CoV-2 are in early clinical trials. This review is an accumulative hub of the latest knowledge and out comings of all novel approach to tackle this deadly disease. Validated and clinically proven therapeutic measures are in great need at this hour of crisis to ensure global safety and on stopping this pandemic before it leads to distressing global outbreaks.
Under the current COVID-19 pandemic scenario the global threats are increasing, more countries are being predisposed, and new outbreaks are being reported, increasing the number of infected cases and risks to non-infected persons-this necessitates timely intervention for appropriate management of affected ones and prevention of threats to healthy ones. Nonavailability of specific antivirals against COVID-19 is causing heavy tolls in infected persons. Utilization of conventional therapies as supportive treatment though helps in managing severity but is proving ineffective on a long term basis as the overall mortality is changing significantly daily. Non-specific antiviral therapy by oseltamivir, ganciclovir; antibacterial therapy by moxifloxacin, ceftriaxone, azithromycin; and glucocorticoid therapy is being improvised by adding broad-spectrum antivirals such as remdesivir, chloroquine, or lopinavir/ritonavir. Passive therapy by α-interferon atomization inhalation, immunoglobulin G therapy, or plasma therapy is proving beneficial.
However, there is a dire need for development and evaluation of specific antivirals against COVID-19. There have been attempts for exploring the applicability of already existing antivirals, potential antivirals or combination thereof along with supportive medicines; however, the focus should be on designing and adopting safe and effective modalities against SARS-CoV-2. These include developing neutralizing antibodies, oligonucleotides targeting SARS-CoV-2RNA genome, passive antibody transfer, drug repurposing using available antivirals, anti-viral proteases, blocking coronavirus receptors like ACE2, targeting spike proteins, and combination therapies.
Alternative measures like neutralizing antibodies, oligonucleotides, passive antibody transfer and drug repurposing can bring a revolutionary change until the core researchers are busy finding the specific target to curb the SARS-CoV-2 which can be time taking and still at large.

Author contributions
All the authors substantially contributed to the conception, design, analysis and interpretation of data, checking and approving the final version of the manuscript, and agree to be accountable for its contents. AAR, SHA, RS and SH initiated this review compilation; RT, SKP, MP, MIY, DKB, YSM and KD updated various sections. SKP and MP developed

Funding
This compilation is a review article written, analyzed and designed by its authors and required no substantial funding to be stated.    The invention relates an immunogenic composition comprising the MERS-CoVN nucleocapsid protein and/or an immunogenic fragment thereof, or a nucleic acid molecule encoding the MERS-CoV N nucleocapsid protein and/or the immunogenic fragment thereof. Furthermore, the present invention relates to a vector comprising a nucleic acid molecule encoding the MERS-CoV N nucleocapsid protein and/or an immunogenic fragment thereof, for use as a vaccine as well as a method of inducing a protective immune response against MERS-CoV. A vaccine composition for vaccinating dogs, the composition comprising: a canine respiratory coronavirus (CRCV) comprising a Spike (S) protein having the amino acid sequence listed in Figure 4, or a coronaviral S protein having at least 97% amino acid identity with the amino acid sequence of Figure 4, or an immunogenic fragment of The present disclosure provides methods and compositions for inducing an immune response that confers dual protection against infections by either or both of a rabies virus and a coronavirus, and/or which can be used therapeutically for an existing infection with rabies virus and/or a coronavirus to treat at least one symptom thereof and/or to neutralize or clear the infecting agents. In particular, the present disclosure provides a recombinant rabies virus vector comprising a nucleotide sequence encoding at least one coronavirus immunogenic glycoprotein fragment, as well as pharmaceutical compositions comprising the vaccine vectors. The invention discloses a high-efficiency ranilla luciferase gene expression recombinant human coronavirus OC43 virus and application thereof to screening of antiviral medicines. By an overlap PCR (polymerase chain reaction) method, a ranilla luciferase gene is replaced or inserted into accessory genes (ns2 and ns12.9) to be cloned into human coronavirus OC43 full-length infectious clone pBAC-OC43FL, and four ranilla luciferase gene expression recombinant viruses, including rOC43-ns2DelRluc, rOC43-ns2FusionRluc, rOC43-ns12.9StopRluc and rOC43-ns12.9FusionRluc, are obtained respectively. The virus rOC43-ns2DelRluc is efficient in ranilla luciferase gene expression and similar to a parent virus HCoV-OC43-WT in virus growth curve, the inserted reporter gene is stable in a serial passage process, and the virus rOC43-ns2DelRluc can be successfully applied to antiviral medicine screening experiments and has an extensive application prospect in high-throughput screening of anti-coronavirus medicines and host antiviral genes.

Shenliang, 2016
2016-08-10 CN105837487A Prevention /treatment Small-molecule inhibitor against MERS-CoV main protease, and preparation method and application thereof The invention provides a small-molecule inhibitor against MERS-CoV main protease. The small-molecule inhibitor is designed on the basis of the crystal structure of main protease of the novel coronavirus MERS-CoV. The invention also provides a synthetic method for the small-molecule inhibitor and application of the small-molecule inhibitor in preparation of drugs used for preventing and treating MERS-CoV infections. The small-molecule inhibitor against MERS-CoV main protease can substantially inhibit the activity of main protease of the MERS coronavirus, has good inhibitory activity to main protease of coronaviruses like SARS and MHV, and presents good application prospects in preparation of drugs used for preventing or treating coronavirus infections. The invention belongs to biomedicine field, it is related to the polypeptide for suppressing human coronary virus's infection, and in particular to polypeptide and its application of human coronary virus's infection can be suppressed wide spectrum.The S2 regions based on coronavirus S protein of the invention are more conservative and feature of with similar syncretizing mechanism provide can be to polypeptide of the infection with wide spectrum inhibitory action of two or more human coronary virus.The present invention is the results showed obtain " general character " of human coronary virus, the identical syncretizing mechanism in i.e. similar HR regions and its mediation, and provide the serial polypeptides of HCoV-EK as point of penetration, the polypeptide not only has preferable inhibition to some currently a popular human corona viruses, and equally has good inhibitory activity to the class SARS virus (RsSHC014-CoV or RsW1V1-CoV) for being possible to infect the mankind.The present invention can provide the drug candidate of prevention and treatment for the novel human coronavirus for being still possible to outburst in popular human corona virus and future at present. Priming of an immune response The present invention provides a method for expanding and improving an immune response and at the same time increasing the speed of a response against a pathogenic antigen or a cancer antigen. A two-step prime-boost method whereby the immune system is first primed with a nucleic acid construct comprising an invariant chain or a variant thereof, followed by a booster vaccine using any type of suitable vaccine Administration) to sufficiently stimulate the immune response generated by vaccine administration. The present invention is directed to compounds, methods and compositions for treating or preventing viral infections using nucleosides analogs. Specifically, the present invention provides for the design and synthesis of acyclic fleximer nucleoside analogues having increased flexibility and ability to alter their conformation structures to provide increased antiviral activity potential with the result of inhibiting several coronaviruses. The present invention provides a method of selectively recognizing base pairs in a target DNA sequence by a polypeptide, A modified polypeptide that specifically recognizes one or more base pairs within a target DNA sequence, and a DNA that is modified so that it can be specifically recognized by the polypeptide, and a specific D Use of polypeptides and DNA in NA targeting and methods of modulating expression of target genes in cells. Microfluidic device for detecting target gene, method for manufacturing same, and method for detecting using same The present invention provides a target gene capable of being differentiated by the naked eye by amplifying the target gene to selectively block the fluid path and, specifically, a microfluidic device for detecting pathogen genes, and a detection method using the same. Therefore, the present invention can conveniently detect a single target gene, such as a single pathogen, or at the same time, several target genes, such as several pathogens, without complicated mechanical devices. The present invention relates to a method for preparing a vaccine antigen comprising a membrane protein, as well as to a vaccine antigen and a vaccine, and uses thereof.