1. INTRODUCTION:

Coronaviruses are a newly recognized group of viruses which infect many animal species, causing a variety of respiratory and gastrointestinal illnesses. Among these viruses are porcine transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCV), avian infectious bronchitis virus (IBV), feline infectious peritonitis virus (FIPV), etc., which cause severe epidemiological problems in livestock and other domestic animals. Human coronaviruses are responsible for a significant number of common colds and diarrhoea, and have also been implicated in multiple sclerosis. Another member of the virus group, mouse hepatitis virus (MHV), is a frequent contaminant of laboratory mouse colonies. Coronaviruses share several common morphological and structural feature refer Fig-1

Fig-1: Structure of Coronavirus

Fig-1: Positive sense single stranded RNA, Genome ∼30 000 nucleotides long, Pleomorphic viruses 80 × 160 nm diameter, with 12–24 nm surface projections (spikes) that cause the corona (Latin: crown) appearance Major proteins: S – spike E – envelope M – membrane N – nucleocapsid

They consist of an enveloped virus particle with characteristic petal-shaped spikes, which give an appearance of a crown, or corona, thus giving the name for the virus. The viral envelope contains two glycoproteins, one of which forms the spikes and the other the matrix proteins. The spikes interact with virus receptors on the cell surface, thus determining the target-cell specificity of the virus. Inside the viral envelope is a helical nucleocapsid which is composed of a nucleocapsid protein N and the RNA genome. The genome is a single piece of RNA of more than 18,000 nucleotides. The RNA is of positive sense, meaning that the viral genome can be directly translated into proteins. The increased spread of SARS-CoV-2 causing COVID-19 infections worldwide has brought increased attention and fears surrounding the prevention and control of SAR-CoV-2 from both the scientific community and the general public. The outbreak is difficult to halting the spread of SARS-CoV-2 are being implemented, other less common transmission pathways should also be considered and addressed to reduce further spread. Environmentally mediated pathways for infection by other pathogens have been a concern in buildings for decades, most notably in hospitals. Substantial research into the presence, abundance, diversity, function, and transmission of the microorganisms taken place in recent years. This work has reports the replication of COVID-19 (coronavirus), which is aleader RNA primed transcription: an alternative mechanism of RNA-splicing, that could lend insights into potential methods to mediate the spread of SARS-2-CoV with description of therapeutic approach of some antiviral combinations which have been reported in the literature we present here the in-depth survey which could be the little contribution to the scientific community to come out with globule challenge.

Replication of virus

Fig-2: Schematic diagram showing the replication cycle of coronavirus

Coronaviruses generally infect cells of the same animal species of origin in tissue culture. Immediately after the virus enters infected cells, the viral RNA is released from the virus particles by a process termed 'un-coating'. The viral RNA is used as messenger for an RNA-dependent RNA polymerase, which transcribes the genomic RNA into a full-length negative-stranded RNA.² This negative-stranded RNA species then serves as the template for the synthesis of seven virus-specific mRNAs. Each mRNA is used for the synthesis of either a viral structural or non-structural protein. The non-structura1 proteins are probably used to regulate the replication and transcription of virus. One of these non-structural proteins is the RNA-dependent RNA polymerase which is needed to synthesize more mRNAs as well as virion genomic RNA. The structural proteins, together with the genomic RNA, are used to assemble the virus particles. These particles, unlike most other enveloped viruses, bud into the endoplasmic reticulum, instead of directly into the plasma membrane.³ The mature virus particles travel through the Golgi complex and are eventually released into the extracellular space. The virus acquires the virus-specific glycoproteins, E1 and E2, during the budding and transport process.

Coronavirus mRNASequence:

Coronavirus mRNA is comprised of a genomic-sized RNA and six subgenom- ic mRNA species. All of these RNAs are associated with polysomes, thus representing functional mRNA species

Fig-3: - mRNA structure of coronaviruses. The structure is based on that of mouse hepatitis virus. The open boxes represent the functional parts of mRNAs. The solid squares are the leader sequences and the wavy lines represent poly A tails. NS proteins represent non-structural proteins which are not packaged into virus particles. El, Matrix protein; E2, spike protein; N, nucleocapsid protein.

In infected cells. The structure of these sequences is such that they each start from the 3'-end of the genomic RNA and extend for various distances toward the S-end, depending on the size of the mRNA species (Fig. 3).⁴ Thus the sequence of each subgenomic mRNA is completely contained within the 3'- portion of the next larger mRNA, i.e. a nested-set structure. The 5'-portion of each mRNA therefore contains a unique sequence which does not overlap with the smaller mRNAs. This unique portion is the functional part of each mRNA. For instance, when mRNA 3 was added to an in vitro translation system, such as reticulocyte lysate,⁵ or was injected into frog oocytes,⁶only the E2 protein was synthesized, although this mRNA contains several downstream genes. Thus each mRNA is functionally monocistronic. This phenomenon is probably a result of the in- ability of mammalian cells to utilize internal translation initiation codons of mRNAs.

The abundance of different mRNAs varies. The small mRNAs are relatively more abundant than the larger mRNA species,⁷ but this inverse relationship between the mRNA size and abundance is not universal, Furthermore, the genetic map of different coronaviruses is not co-linear. For instance, IBV contains an additional gene inserted between the genes encoding E 1 and N. Thus genetic recombination, insertion or deletion must have occurred during the evolution of the viruses. Another unique feature of corona- virus mRNAs is that the 5'-ends of all the mRNAs, including genomic RNA, contain an identical stretch of 50-70 nucleotides^8,9 termed the leader sequence. These leader nucleotides are not repeated in the internal region of the genomic RNA. Therefore, the leader of the sub-genomic mRNAs must be de- rived from the 5'-end of the genomic RNA, analogous to the leader sequences of many eukaryotic mRNAs. The presence of the leader sequences on the coronavirus mRNAs was first suggested by an unusual oligonucleotide,¹⁰ which turns out to be the fusion site between the leader RNA and mRNAs. The identity of the leader sequences has now been confirmed by cDNA cloning and sequencing of genomic RNA and mRNAs. In the case of MHV, the leader sequence is approximately 70 nucleotides long.

The Corona-viral leader RNA is not Derived by the Conventional Pre-mRNA Splicing Mechanism:

Although the presence of a leader sequence is a common feature of eukaryotic mRNAs, the synthesis of coronavirus mRNAs does not appear to utilize the conventional RNA splicing mechanism, which involves cleavage of intervening sequences from a precursor RNA. Two pieces of data suggest that the leader sequences present at the 5'-end of coronavirus mRNAs are probably derived by a novel mechanism: (1) coronaviruses replicate exclusively in the cytoplasm of infected cells,^l1,l2 while conventional RNA splicing takes place in the nucleus; and (2) ultraviolet (UV) irradiation of coronavirus-infected cells resulted in the inhibition of individual mRNA synthesis at a rate proportional to the size of the mRNA.^I3 The UV target size of each coronavirus mRNA is approximately the saqqs its physical size, suggesting that each subgenomic mRNA is trans- cribed independently. If these mRNAs were derived from the cleavage of a precursor RNA, as in the case of conventional RNA splicing, the UV target size for inhibition of the synthesis of all the mRNAs would be expected to be equal to that of the genomic RNA. Thus a novel mechanism for the generation of these mRNAs is most likely utilized by coronaviruses. Several transcriptional models for coronavirus mRNAs have been pro- posed (Fig. 4 and 5).¹⁴

(1) The 'loop-out' model in which the RNA template forms a loop in the 'intron' region, thus bringing the leader RNA in close proximity to the initiation sites of subgenomic mRNAs.

Fig-5: Three possible models of coronavirus transcription.

The RNA polymerasecan thus jump from the leadersequence to the mRNAs in continuoustranscription. For smaller mRNAs the loops will be larger.

(2) The 'leader-primed transcription' model in which the leader RNA is transcribed and becomes dissociated from the RNA template. This 'free' leader RNA then binds to the template at the initiation sites of various mRNAs and serves as the primer for transcription.

(3) The ' post-transcriptional processing' model in which the leader RNA and the body sequences are transcribed independently and then joined together by a transsplicing mechanism post-transcriptionally. A considerable body of data¹⁴ suggests that the first and third models are not compatible with the replication mechanism of coronavirus RNA. Thus, the ' leader-primed transcription' model is considered to be the most likely mechanism utilized for the transcription of coronavirus mRNAs.

Evidence for the 'Leader-primed Transcription' Mode1:

The direct evidence in support of the 'leader-primed transcription' model is multifold.

(I) Several free leader RNA species of 50-90 nucleotides have been detected in the cytoplasm of MHV- infected cel1s.¹⁵ They are discrete RNA species, some of which are dissociated from the RNA template.

(2) A tem- perature-sensitive mutant has been isolated which synthesizes only the small leader RNA but not mRNAs at the non- permissive temperature,¹⁵ suggesting that the synthesis of the leader RNA and the synthesis of the mRNAs are discontinuous and require different viral proteins.

(3) During a mixed infection with two different MHVs, the mRNAs of each virus contain a population with the leader RNA sequences derived from the co-infecting virus.¹⁶ This result suggests that the leader RNA is a separate transcriptional unit and can be freely exchanged between the mRNAs of two co-infecting viruses. Thus the free leadercontaining RNA species detected in the cytoplasm of MHV-infected cells represent bona fide transcriptional intermediates, rather than abortive transcription products.

(4) An RNA species complementary to the leader RNA ('anti-sense' leader RNA) could be expressed in L-2 cells by a mammalian expression vector. When the cells ex- pressing this anti-sense leader RNA were infected with MHV, the RNA transcription of the superinfecting virus was inhibited, though not completely (L. Soe, unpublished observation). Thisresult suggests that the availability of the leader RNA sequences is required for MHV RNA synthesis. These data are most compatible with the interpretation that the leader RNA serves as a primer for transcription.

The Mechanism of Leader-primed Transcription:

The precise mechanism of leader RNA- primed transcription has been suggested from the sequence analysis of both the 5'-end and the mRNA initiation sites on the RNA genomes. The sequence of the 5'-end of MHV genomic RNA reveals a possible leader RNA termination signal, i.e. a hairpin loop followed by an AU-rich sequence, approximately 84 nucleotides from the 5'-end (Fig. 6).¹⁷This is consistent with the size of the free leader-containing RNA species detected in the infected ce1ls.^l5Close to the 3'-end of the putative leader RNA, there is a stretch of 9-18 nucleotides, which is homologous to the genomic sequences at the initiation points of various mRNAs^17,18. Thus the leader RNA would be able to bind to the RNA template at these sites. The homologous sequences are not located at the exact 3'-end of the free leader RNA, and there is also a mismatched nucleotide within some of the homologous regions. These mismatched nucleotides in the leader RNA have to be removed before transcription takes place. Thus the mechanism of MHV RNA transcription involves the synthesis of a free leader RNA species from the 3'-end of the negative-sense RNA template, followed by its binding to the intergenic regions via the homologous sequences. The leader RNA is then cleaved at the mismatched point, generating the primer for the initiation of mRNA transcription (Fig. 6). Therefore, the leader-body fusion sites vary with the mRNA species, depending on the location of the mismatched nucleotide.

There is a close parallel between the number of homologous nucleotides at the intergenic sites preceding each mRNA and the amount of RNA synthesized in the infected ce1ls ^l5.For instance, there are 18, 14 and 9 homologous nucleotides upstream of mRNAs 7, 6 and 4, respectively. Correspondingly, the molar ratio of these three RNAs in infected cells is roughly 100:31: 1-7.' In addition, other regulatory sequences may also be involved. The free leader RNA may be associated with RNA polymerase, which could assist in leader RNA binding.

Fig-6. The postulated model of leader-primed transcription. The solid squares represent free leader RNA, which binand to the initiation points of mRNAs on the template RNA. The expanded regions show the nucleotide sequences of the intergenic regions of mRNAs 6 and 7, and the mechanism of leader RNA binding. The solid arrows represent the postulated cleavage points of the leader RNA. Transcription starts from the 3'-end of the cleaved primer. The resulting mRNAs consist of the leader sequence fused to the gene.

Prevention:

Attempts to control transmissible gastroenteritis virus of pigs and feline coronavirus of cats through the use of vaccines have not been successful although vaccines for the avian disease infectious bronchitis virus has been modestly effective. The fact that natural infections with 229E or OC43 do not provide long-lasting immunity is instructive in this regard. Thus, so far, there is no vaccine for a HCoV that is in clinical use. The severity of SARS led to a concerted effort to develop vaccines for SARS CoV and range of vaccine strategies including inactivated whole virus vaccines, spike-subunit vaccines, DNA vaccines and vaccinia or parainfluenza virus type 3 vectored vaccines have all been tried in experimental animal models, with some providing evidence of efficacy. It has been established that antibody to the spike protein is the key correlate of protection in animal models. However, as there is perceived to be no imminent public health threat from SARS, few of these vaccines have been taken to human clinical trials. Passive immunotherapy using monoclonal antibodies that neutralize SARS CoV has also been developed and evaluated in experimental animal models of SARS.

Control:

Prophylactic strategies that target coronaviruses andrhinoviruses (the two common aetiological agents of this syndrome) would be attractive. However, there are no validated antiviral drugs or vaccines to contain coronavirus infections so far.

Treatment:

During the outbreak of SARS, given its severity and high mortality rates, a number of therapeutic options including ribavirin, interferon alpha, lopinavir/ritonavir, and nucleoside analogue protease inhibitor combination therapy were all tried. While there is evidence of activity in-vitro, these drugs were not evaluated in controlled clinical trials and their therapeutic benefit remains uncertain.

Ribavirin

Ribavirin, a nucleoside analogue, was widely prescribed for treatment of SARS-CoV infection in 2003.¹⁸, Nevertheless, ribavirin monotherapy had minimal activity against SARS-CoV with concentrations that could be achieved in the clinical setting, and it led to significant haemolysis in many patients.¹⁹

Antiviral Therapy:

The efficacy of antiviral agents including ribavirin, protease inhibitors, and INF that were used to treat patients with SARS-CoV infection in 2003 is summarized in²⁰Table-1 Because of lack of prospective randomized, placebo-controlled clinical trial data, none of these therapies have proven benefit. Good supportive care remains the mainstay of treatment of SARS-CoV infection.

Table-1: Antiviral drugs with its application and representative antiviral combinations

Agents	SARS-CoV	Refrence
Ribavirin/ Tribavirin	Used in the treatment of RSV infection, hepatitis C and some viral hemorrhagic fevers	21
Protease inhibitor	Molecules that inhibits the function of proteases used interchangeably with alpha 1-antitrypsin (A1AT, which is abbreviated PI for this reason).^[2] A1AT is indeed the protease inhibitor most often involved in disease, namely in alpha-1 antitrypsin deficiency.	22
Interferon	IFNs group of signaling proteins made and released by host cells in response to the presence of several viruses. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. This are groups of proteins that interferes the replication of virus and activate immune cells, such as natural killer cells and macrophages; they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines.	23
Tocilizumab treatment of CS	It is also known as atlizumab, is an immunosuppressive drug, mainly for the treatment of rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, a severe form of arthritis in children. It is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Interleukin 6 (IL-6) is a cytokine that plays an important role in immune response and is implicated in the pathogenesis of many diseases, such as autoimmune diseases, multiple myeloma and prostate cancer.	24
JAK inhibitors	The receptors of novel coronavirus pneumonia, might be ACE2, which is a cell-surface protein widely existed on cell in the heart, kidney, blood vessels, especially lung AT2 alveolar epithelial cells. enter cells through endocytosis	25
Anti-cytokine storm Chloroquine and hydroxychloroquine	Chloroquine (CQ) is an amine acidotropic form of quinine and hydroxychloroquine (HCQ) differs from chloroquine by the presence of a hydroxyl group at the end of the side chain: The N-ethyl substituent is β-hydroxylated. For decades, CQ and HCQ are front-line medications for the treatment and prophylaxis of malaria and are also used to treat autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).	26
COMBINED THERAPY
Clinical Approach	Combination of antiviral
Pharmacokinetic interactions approach	Oral oseltamivir + oral amantadine (NCT00416962) Oral oseltamivir + oral favipiravir (unpublished) Intravenous peramivir + oral rimantadine43 Intravenous peramivir + oral oseltamivir43 Intravenous zanamivir + oral oseltamivir44 Oral amantadine + oral ribavirin + oral oseltamivir (NCT00867139)	37, 38
Clinical eﬃcacy approach	Oral rimantadine + nebulised zanamivir4 Oral oseltamivir + inhaled zanamivir42 Oral oseltamivir + pH1N1 convalescent plasma45 Oral oseltamivir + pH1N1 hyperimmune globulin (NCT01617317) Oral oseltamivir + maxingshigan/yinqiaosan (NCT00935194) Oral oseltamivir + sirolimus + corticosteroids46 Oral amantadine + ribavirin + oseltamivir (TCAD; NCT01617317)	39,40
A randomized controlled trials of clinical eﬃcacy approach	Oral oseltamivir + convalescent plasma or hyperimmune globulin (NCT01052480) Oral amantadine + ribavirin + oseltamivir (TCAD; NCT01227967) Oseltamivir + nitazoxanide (NCT01610245)	41, 42

TCAD= Triple combination antiviral drugs

Systemic Corticosteroids theory:

Systemic corticosteroids, in the form of intravenous pulse methylprednisolone (MP) was given to some patients with SARS-CoV infection for several reasons.^27,28 Firstly, there was an assumption that clinical progression of pneumonia and respiratory failure in association with peaking of SARS-CoV viral load might be mediated by the host inflammatory response.^29,30. Also, in many patients there were HRCT31 and histologic features of COP, which was a steroid-responsive condition. Systemic corticosteroids significantly reduced IL-8, MCP-1, and IP-10 concentrations from 5 to 8 days after treatment in 20 adults with SARS-CoV infection.

Inaddition, in patients with fatal SARS-CoV infection, there was evidence of hemophagocytosis in the lungs attributed to cytokine dysregulation. Intervention with systemic corticosteroids was thus given to modulate these immune responses. Although there was clinical improvement in some patients with resolution of fever and lung consolidation following treatment with intravenously pulsed MP, a retrospective cohort analysis in Hong Kong showed that the use of pulsed MP was actually associated with an increased risk of 30-day mortality (adjusted odds ratio [OR] 26.0; 95% CI, 4.4–154.8).^32,33,34 In addition, prolonged use of systemic corticosteroid therapy

Convalescent Plasma/Passive Immunotherapy:

In the absence of well-proven and effective antiviral therapy, convalescent plasma and human monoclonal antibody are worth further study for treatment of SARS-CoV if it returns.

CONCLUSION:

There is no proven treatment for coronavirus infections and no vaccine. Control measures that were effective in stopping the spread of SARS included isolation of patients, quarantine of those who had been exposed, and travel restrictions, as well as the use of gloves, gowns, goggles, and respirators by health care workers.

In conclusion, COVID-19 is a viral infectious disease mainly manifested as fever and pneumonia, anti-viral and respiratory supportive therapies are the mainstream of treatments for severe cases. As CS occurs in critical ill patients, which leads to ARDS and multiple organ damage, and even death, anti-inflammation treatment may be applied. However, given the viral nature of the COVID-19 CS, and considering a substantial impairness of host immune system in severe cases, it is critical to balance the risk and benefit ratio before starting anti-inflammation therapy. In addition, a timely anti-inflammation treatment initiated at the right window time is of pivotal importance and should be tailored in individual patient to achieve the most favorable effects.

More scientific data could swing the balance of evidence to favor one hypothesis over another. Obtaining related viral sequences from animal sources would be the most definitive way of revealing viral origins. For example, a future observation of an intermediate or fully formed polybasic cleavage site in a SARS-CoV-2-like virus from animals would lend even further support to the natural-selection hypotheses. It would also be helpful to obtain more genetic and functional data about SARSCoV-2, including animal studies. The identification of a potential intermediate host of SARS-CoV-2, as well as sequencing of the virus from very early cases, would similarly be highly informative. Irrespective of the exact mechanisms by which SARSCoV-2 originated via natural selection, the ongoing surveillance of pneumonia in humans and other animals is clearly of utmost importance.

SARS- CoV-2 is thought to infect host cells through ACE2 to cause COVID-19, while also causing damage to the myocardium, although the specific mechanisms are uncertain. Patients with underlying CVD and SARS- CoV-2 infection have an adverse prognosis. Therefore, particular attention should be given to cardiovascular protection during treatment for COVID-19.

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Received on 15.04.2020 Modified on 11.05.2020

Asian J. Research Chem. 2020; 13(4):291-298.

DOI: 10.5958/0974-4150.2020.00057.7

Anti-cytokine storm

Clinical Approach

Pharmacokinetic interactions approach

Clinical eﬃcacy approach

A randomized controlled trials of clinical eﬃcacy approach