Journal of Microbial & Biochemical Technology

Pandemic COVID-19 Emerging Global Concern: An Overview to Understand the Potential of Ca+2 in Pathogenesis and Advance Therapeutics Strategy on its Outbreak

Prem Raj Meena¹, Anjali Vashishth², Priyanka¹ and Arvind P. Singh^{1* 1}Department of Microbiology, School of Life Sciences, Central University of Rajasthan, Ajmer, India
²Department of Sport Biosciences, School of Sport Science, Central University of Rajasthan, Ajmer, India
^*Correspondence: Arvind P. Singh, Department of Microbiology, School of Life Sciences, Central University of Rajasthan, Ajmer, India, Email:

Received: 26-Feb-2020 Published: 17-Jul-2020, DOI: 10.35248/1948-5948.20.12.433

Introduction

New public health crisis is threatening the world with the emerging and spread of COVID-19. In December 2019, acluster of viral pneumonia cases was first observed in Wuhan, China (South China Seafood city food market, China), in January 2020 and it was suggested that the causative agent of pneumonia is a novel coronavirus (initially named "2019-nCoV" but changed to "SARS-CoV-2" by International Committee on Taxonomy of Viruses (ICTV)), which belonged to coronavirus family and subfamily orthocoronavirinae with the same lineage but with slight genetically distinct from the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). These were zoonotic in origin and linked with the respiratory, enteric, hepatic and neurological disease which associated to fatal illness in human [1- 4]. The various outbreak of SARS-CoV in 2002 and the MERSCoV in 2012, firstly demonstrated the transmission from animal to human and human to human. Recently the mystery of ongoing coronavirus health threats has emerged in China. Nevertheless, it is spreading rapidly in other countries that have attracted tremendous attention worldwide, and the World Health Organization (WHO) has already declared a health emergency and pandemic disease. On 12 January 2020, it was formally named Coronavirus Disease 2019 (COVID-2019) by the WHO [5,6]. A total of more than one crore (29 June 2020) cases of pneumonia have been registered worldwide, according to WHO. Approximately five lakh people died as a result of this pandemic disease by the end of March 2020, the USA (1.2 lakh deaths) and Brazil (56109 deaths) was the nation most affected by this pandemic and the death occurred in Italy (more than 34000), Spain (27,000), France (approx. 28000).

Putative One-Health insights for the origin and evolution of "SARS-CoV-2"

Mutation and adaptation have driven co-evolution in coronaviruses and their host, including humans. Two human coronaviruses (HCoV) SARS and MERS were identified as a causative agent in humans in 2003, and these HCoVs were detected primarily from the bat as zoonotic sources. Based on different protein sequence coronaviruses (CoV) are divided into four genera α-/, β-/, δ-/, and γ-CoV. α-/, β-CoV can cause infection in mammalian while δ-/, γ-CoV tend to infect in birds. Based on the genome sequence of "SARS-CoV-2" is 96.2% identical to a bat CoV- RaTG13, whereas it shares 76% identity to SARS-CoV, that is why bat might be suspected as a natural host of nCoV-19 and could be transmitted from bat to human [7,8]. Based on phylogenetic analysis and specific protein residues, some studies also confirm that SARSCoV- 2 mainly linked to bat-SL-CoV-ZC45 and bat-SL-CoV-ZX21 [9], currently research devoted to the potentiation alternative host for SARS-CoV2 maybe such as snake, turtles, and pangolin [10]. Such findings have drawn attention, along with sequence identification, to the probable zoonotic roots of coronavirus. Infection with SARS-CoV-2 is potentially another vital example of the One-Health principle following SARS-CoV, MERS-CoV, and Ebola virus which overlaps human, animal, and environmental health [11]. Identifying the SARS-CoV-2 reservoir could be one of the milestones for monitoring, contaminating, and mitigating the animal outbreak.

Genome and critical virulence factors of SARS-CoV-2

The "SARS-CoV2" genome is a non-segmented single-strand positive-sense (+ ss-RNA) with a 5'-cap structure and a 3'-poly-A tail with a genome size of at least 6-11 Open Reading Frames (ORF) [9,12,13]. There are 16 non-structural proteins (NSPs 1-16, except for γ--coronavirus due to lack of 1-NSP) that encode first ORFs (ORF 1a/b), which is 2/3 of the viral genome while remaining to encode structural proteins. For NSPs, genomic RNA directly translates polyprotein 1a/1ab (pp1a/pp1ab) and after translation, this protein forms Replication- Transcription Complex (RTC) in a double vesicular membrane (DMVs) [14]. The central four structural proteins Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N), and other accessory proteins that interfere with the immune response of the host, coded near 3'- N-terminus by some ORFs [2] (Figure 1). Among them, spike (S) protein plays the most crucial role in viral attachment, fusion, and invasion of the virus.Individually, each protein plays a minor role in the structure of virus particles, but also involved other aspects of the replication cycle. SgRNAs of CoVs [15] translates all accessory and structural proteins. Recent, study devoted that the mutation in NSP2 and NSP3 plays a role in the infection capability of different mechanisms of infection [16]. The Chinese scientist Thang et al. [17] performed a population genetic study of 103 SARS-CoV-2 genomes, classifying SARS-CoV-2 into two types L (70%) and S (30%), S-type L strains, and is more aggressive and pathogenic in nature.

Figure 1: Schematic Model representation of SARS-CoV-2 with four main structural proteins (S-protein, M-protein, E-protein and N-protein) with their specific functions. Structure of S-protein shows the AEC-2 binding side and RGD-motif which bind to host cell receptor.

Due to the existence of the RGD (Arg-Gly-Asp) motif, the S (spike) protein of SARS-CoV-2 differs from the normal coronavirus [18]. The primary function of this type's motif is to improve the binding capacity of SARS-CoV-2 to host lung cells, and this is perhaps the primary reason why novel coronavirus is more contagious within the lung cells than other respiratory syndrome viruses. Ca⁺², and Mg+2, and other ions play a vital role in binding in and pathogenesis mechanism of SARS-CoV-2.

Function of structural and non- structural proteins

Most of the function of 16-NSPs and 4-structural proteins have been reported already. Many of 16-NSPs play a vital role in CoVs replication, while structural protein plays a key role in pathogenesis [19]. The function of structural and non- structural proteins is summarized in Figure 1 and Table 1.

Table 1: Summarize of 16 Non-structural Proteins (NSP) in coronavirus and their function

Mechanism of Transmission of COVID-19

A large number of persons have been identified in Wuhan, Chinarelated to ( South China, Seafood city food market, China) its suggested that its likely a zoonotic origin of COVID-19, and many research ongoing to the proof reservoir of COVID-19, but there are no consistent evidence of coronavirus reservoirs other than mammals and birds [35,36]. Based on the phylogenetic analysis and similar protein residues, some study also supports that SARSCoV- 2 mostly related to bat-SL-CoV-ZC45 and bat-SL-CoV-ZX21 [19]. Recent research also supports the possibility of alternative hosts for SARS-CoV-2.Apart from different types of hosts, some factors even facilities SARS-CoV-2 to break cross species barriers, like a high mutation rate in RNA replication and adaptation of novel host. The mutation rate of SARS-CoV-2 is very high. It may be suggested that direct contact within termed the host animals and the consumption of wild animals was suspected to be the main route of SARS-CoV-2 transmission [19]. However, the transmission route and source remain a puzzle for the researcher.

Recent research shows the human to human transmission route of SARS-CoV-2, these studies data shows a person who has been visited Wuhan city market their family members also find infected by this virus [37,38]. According to WHO guideline person to person transmission occurred via direct contact or droplet spreading by the infected individual via coughing and sneezing. Likewise [39], has suggested the presence of this dangerous virus in fecal swab and blood that indicates the multiple transmission routes of infection. It's has been already shown that the transmission of SARS-CoV and MERS-CoV occurred through nosocomial transmission and considered airborne pathogens.

Host-pathogen interaction and Role of Ca⁺² in Invasion and survival mechanism

A very important stage after transmission is the binding of coronavirus to host cell receptors. It is noteworthy that SARSCoV- 2 share the same cellular receptor with SARS-CoV genera. The spike protein of coronavirus from all four families, guides to coronavirus entry into the host cell [40]. Corona viruses entre into the host cell by a two-step process: first host cell receptor recognized for viral attachment and fuse viral and host cell membrane. The spike protein is present in two very different forms pre-fused (before fusion to host) and post-fused (after fusion to host cell). The pre-fused spike protein displays a homo-trimer structure with three receptor binding S1 receptor binding side and resting at the top of trimeric S2 [41-43]. The post-fusion structure is a coiled-coil structure with contained only S2 [44,45]. The virus invasion may have two pathways (a) the ACE-2 receptor (b) using the integrin receptor.Angiotensin-Converting Enzyme-2 (ACE-2) receptor presents the cell membrane of the cells of the lungs, heart, and kidney. ACE-2 is expressed by type I and type II alveolar epithelial cells. Among them, type II is shown more than 80% ACE-2 receptor. Men had a higher level of ACE-2 receptor rather than women. This enzyme considers as the main entry point for coronavirus [45,46]. SARS-CoV-2 can also fuse directly to the cell surface in the Detection Beta-CoV receptor reveals that human cells expressing ACE-2 receptor have a crucial role to play in binding SARS-CoV-2, Spike (S) glycoprotein, and ACE-2 host receptor [47].

A 30 % difference in the S1 unit of S protein sequence between SARS-CoV and SARS-CoV-2. The RGD-motif of S protein, which is different in sequence from SARS-CoV and MERS-CoV, shows tightly binding to lung cells. It has long been known that SARSCoV is primarily a respiratory disease, so it also needed protease from the respiratory tract such as trans-membrane protein serine-2 (TMPRSS-2) and HAT [48-51] (Figure 2). The TMPRSS-2 and HAT both activate the binding affinity of the S-protein cleavage trimer. Some studies support that the S-protein and ACE-2 increase the affinity 10 to 20-fold for SARS-CoV-2 [52,53]. After binding SARSCoV- 2 to host cell receptor, it required a serial activation of kinase and protease like activities for the internalization of the virus. The phagocytosis mechanism is complex, where the interconnected, and cross-activation of proteins take participates inside the cells. TMPRSS-2 and HAT cleaves pattern for S-fragments differ from each other; HAT cleaves S protein mainly at R667, where TMPRSS-2 cleaves at multiple sites, both cleavages enhance the cell-virus fusion [50]. The infection of the target cell by SARSCoV- 2 occurred due to S-pseudotypedvirions, which is less sensitive to cathepsin inhibitor when the target cell expresses TMPRSS-2 [50,51]. Pseudo virions are still producing by SARS-CoV-2; still, TMPRSS-2, rely on endosomal cathepsin for the entry. Meanwhile, other accessory proteins may be involved in viral binding and invasion, such as cathepsin [54] and clathrin (Figure 2), while potential molecules facilitated an uncertain membrane invasion of SARS-CoV-2 [53-55]. SARS-CoV-2 Cell Entry Depends on ACE-2 and TMPRSS2, and it is Blocked by a Clinically Proven Protease Inhibitor.

Role of Ca⁺² ion in invasion and host-pathogen interaction the SARS-CoV-2 spike protein (S-Protein) protein has RGD-motif which show highly binding and invasion capacity of SARAS-CoV-2. RGD-Motif bind to Integrin receptor α-subunit and β-subunit serve up a docking site to numerous kinases, as Focal adhesion kinase (FAK) for β-subunit and talin adaptor proteins. Phosphorylated FAK promote to phosphorylation of phospholipase C- γ (PLC-γ) that direct participate into generation and catalysis of inositol phosphate-3 (IP-3) via intermediated coronin after this IP3 diffused toward endoplasmic reticulum and bind at IP-3 receptor and present on E.R. Thus, this binding opens the Ca⁺²ion channel and the Ca⁺²ions influx inside cytosol and the increased concentration of Ca⁺² ions inside cell which led to binding and invasion capacity of virus.

The lack of a complete understanding of the phagocytosis mechanism that is critical for SARS-CoV-2 to the host's which pathway involvement. A recent study shows that Ca⁺² ions increase infectivity and entry into MERS-CoV and Rubella virus cells [56,57] because of the presence of negatively charged peptide on fusion protein (S-protein). Studies show that the spike protein of coronavirus has evolutionary changes and obtain some features for its adaptations in human host cells [18,58].

In our speculation, the RGD motif of the spike protein of SARSCoV- 2 may be bound to integrin receptor [59] of host cells that participate in host-pathogen interaction and leads to internalization. In some research article [56,57] it has been showing the involvement of Ca⁺² ions play a significant role in which is several receptorbased events and initiates internalization of pathogenicity of the virus by altering the actin filaments and cytoskeleton arrangements through affecting the actions of several proteins [60]. When a virus binds an integrin receptor association (α5β1), a serial activation of kinase activates that contributes to the internalization is needed. The binding of virus or virus particle induce Ca⁺² response inside the host cell that lead cellular response.The integrin α5-subunit and β1-subunit provide a docking site for various kinases, such as β-subunit Focal Adhesion Kinase (FAK) [61] and α-subunit Talin Adapter Proteins [62]. The tyrosine kinase FAK plays a vital role as a key mediator of the integrin signaling event controller. RGD motif of spike protein (S-Protein) interaction to integrin stimulates FAK tyrosine phosphorylation result's in FAK signaling activate, meanwhile at time stimulated FAK promotes phospholipase C-γ (PLC-γ) activities that directly participate in the generation and catalyzed of inositol triphosphate 3 (IP3) and Diacylglycerol (DAG). Thus, the PLC-γ activates coronin, found as an actin-binding protein within cytosol and thus further stimulation of PLC-γ these proteins proceed IP3 and this diffuse towards Endoplasmic reticulum (E.R.) and binds to inositol triphosphate-3 receptor (IP3R), present at E.R. this results in immobilization of Ca⁺² into the cytosol [61,63]. Phagocytosis is a complex mechanism by interconnected and crossactivation of intracellular proteins.Moreover, in this complex mechanism, cytosolic proteins of the host also play a role in virus engulf, one of the best proteins talin, which a ubiquitous cytosolic protein that docks the α5-subunit of integrin and acts as a substrate for the Ca⁺² activated protease, called calpain. Thus, an increase in Ca⁺²+ concentration in host cells leads to re-armament or deform the actin filaments by binding on α-actin that provide an intact binding between actin filaments and help into the invasion of the virus into lung cells (Figure 2). So, this fact can be possible; the concentration of Ca⁺² into lung cells also increases the binding and entry of SARS-CoV-2 inside the cell, and that plays a vital role in the pathogenicity of the virus. Nonetheless, if the concentration of Ca⁺² ions in lung cells reduces, this may be a step towards reducing the degree of coronavirus infection.

Figure 2: Schematic representation of the Transmission route of SARS-CoV-2 and Association of spike binding protein (S-protein) encodes by SARSCoV- 2 during host-pathogen interactions and Ca⁺² role in pathogenesis mechanism: Possible origin from the bat of corona virus and transmission into a human. The bindingSARS-CoV-2 expressed spike (S-protein) protein that binds to the ACE-2 receptor that acts as a link to stimulate the TMPRSS-2 receptor to serve up a docking site to numerous kinases and protease, as AP-2 kinase. There is some other protease like HAT, cathepsin L and Elastase which also play a vital role in viral fusion and invasion. The ligand and receptor interaction stimulate AP-2 complex, TMPRSS-2 cleavage the S-protein which triggers the SARS-CoV-2 binding to the host cell.

After SARS-CoV-2 's invasion (Figure 3a), their formation of an early phagosome into the host cell, but it still remains unclear how SARS-CoV escape from the host immune system, it appears that the virus secretes Rab5, like protein that displays well on the phagosome, thus playing phagosome-lysosome fusion that helps to escape the immune system. One interesting phenomenon occurred, still, SARS-CoV secretes the pseudotypesvirion, which is less sensitive, and this led to the neutralization of the host antibody. This may be the main reason for the delay in the appearance of the symptoms of SARS-CoV-2, which are 4 to 14 days the incubation period of the virus. This effect was attributed to the release of the spike (S) fragments inside the body that lures antibodies and may be of great importance for the spread of the virus [50]. After immune compromise, the SARS-CoV-2 virus goes to replication, the viral RNA (+ve) released and uncoating, and translated into viral replicase polyproteins pp1a and 1b, which are cleaves into the small product by using viral proteinase. Negative strand synthesized from some discourteous transcription and plus-strand used a templet RNA for mRNA synthesis. Viral Nucleocapsid are assembled same time N protein in the cytoplasm is arranged onto virus RNA into ERGIC (Endoplasmic Reticulum-Golgi Intermediate Compartment). Then the virion is released from the cell through exocytosis mechanism (Figure 3b) [64].

Figure 3a: Schematic representations of the clathrin mediated invagination, life cycle of SARS-CoV-2 and membrane vesicle organization Immune Response against SARS-CoV-2. SARS-CoV-2 membrane surface adhesions virulence factors like S-Protein and clathrin mediated, where S-protein is cleaved by the endosomal acid proteases (cathepsin L)105 to activate its fusion activity after invasion of the virus after invagination of SARS-CoV-2 some virulence protein expression in the early phagosome and use of origin of membrane vesicles (M.V.s) which are budding off from the early phagosome and present their peptides to Antigen Presenting Cell (APC) surface through MHC-II class protein pathway.

Figure 3b: After immune compromise the SARS-CoV-2 bacteria goes to replication, the viral RNS(+ve) released and uncoating, and translated into viral replicase polyproteins pp1a and 1b, which are cleaves into small product by using viral proteinase. Negative strand synthesized from some discourteous transcription and plus strand used a templet RNA for mRNA synthesis. Viral Nucleocapsid are assembled same time N protein in the cytoplasm is arranged onto virus RNA into ERGIC (Endoplasmic Reticulum-Golgi Intermediate Compartment). Then the virion is released from the cell through exocytosis mechanism. Virion are then released from cell through exocytosis mechanism. (where S-spike, N-Nucleocapsid, M-Membrane and E-Envelop proteins).

Clinical Characteristic and Diagnosis

The incubation period of COVID-19 is 4 to 14 days, and the most common symptoms in the patient are mild flu-like fever, cough, headache, shortness of breath, some gastrointestinal infection like dihedra, vomiting, and malaise [65]. SARS-CoV-2, firstly Diagnosis by the viral research institute in china, primary identification was made by classical Koch's postulates, and the morphology was observed by electron microscopy [66]. Real-time PCR (RT-PCR) used to detection of COVID-19 and further confirmation by Next- Generation sequencing. Moreover, Computerized tomography scans can also be used to detect infections with COVID-19; images display an abnormality in the chest [66,67]. Yet the chest image often shows a regular scan image of positive corona patients; for clarification, the presence of viral RNA in the patient body needs to be further tested. Recently, it has been seen every country used to rapid test kit, which is based on the Antibodies detection method.

Therapeutics strategy and prevention of COVID-19

At the present time, there are no specific drugs and definitive treatment against COVID-19 for potential therapy in human infection. All patients accept oxygen therapy and, the WHO recommends extracorporeal membrane oxygenation (ECMO) with refractory hypoxemia [58]. For the critical condition, convalescent plasma therapy and immunoglobin G also used according to conditions being detected [68]; in 2014, WHO has recommended this convalescent plasma therapy against the Ebola virus.Based on previous experience combating SARS-CoV and MERS-CoV, some therapeutic strategy against COVID-19 can be used [19]. Antiviral drugs and corticosteroids (such as oseltamivir, peramivir, zanamivir, ganciclovir, acyclovir, and ribavirin) that were previously used for influenza virus in the clinical setting are invalid for COVID-19 [69,70]. Present time only broad-spectrum antiviral drugs (such as sofosbuvir, lopinavir/Ritonavir) and some antiviral like nucleoside (Remdesivir) or its analogs, and HIV-protease inhibitor is used for the treatment of COVID-19 [71-73].

Recently, chloroquine and hydroxychloroquine which has been used for several years to treat malaria and can prevent the replication of many viruses often used to treat COVID-19 [74- 76]. In vitro, study reveal, a combination of Remdesivir and chloroquine effectively inhibits the SARS-CoV-2 that has recently emerged [18]. In addition, various vaccine strategies such as live attenuated, inactivated viruses, and vector-based, DNA vaccine have been developed but only tested in an animal model [77,78]. Nevertheless, the efficacy of these drugs still needs to be verified by clinical trials. One more sign that the demand for chloroquine, which is used to treat malaria for many years, increased now's days that indicate the possible link between SARS-CoV-2 and Plasmodium falciform protein sequence and pathogenesis. Data are not available, but it may be possible those patients who have already suffered from these known bacterial diseases can develop probably resistance against COVID-19, and this may be led to the developed vaccine against the corona virus. The RGD motif of spike protein and TMPSRSS-2 which plays an important role in binding to the host cell and the concentration of Ca⁺² ions plays a major role in binding and invading the virus into the host cell if the receptor binding block is somehow reduced by any protease inhibitor and the concentration of Ca⁺² ions in the lung cells by some inhalation that may be a potential pre-therapy strategy for reducing the level of corona infection.ACE2 receptor binding to S-Protein can be blocked by monoclonal antibodies (such as Tocilizumab) [72,73]. Currently, there are numerous companies and research institute applied for clinical trial for the repurposing of existing drugs, for this the repurposing, randomized controlled treatment (RCT) being carried out to identify disease specific drugs [72]. Moreover, Indian traditional medicinal particles such as Ayurveda, Yoga and Unani, Siddha, Naturopathy & Homoeopathy (AYUSH) are considered as one of the oldest treatments in human history for successfully treatment of various diseases [72,79,80]. Indian herb also used for the preventive strategy against corona virus disease, for this The Ministry of AYUSH (Govt. of India) give focus on the preventive through modification in daily lifestyle for improving immunity [81]. According to Vellingiri et. al. there are various medicine plant recommended by AYUSH for fighting COVID-19 [72]. But it should be remembered still there is no efficient drug again SARS-CoV-2. Based on China's and other research data, the WHO released a guideline on how to prevent the pandemic COVID-19. The best majors now to control the source of infection early Diagnosis and effective treatment; isolation of patients also led to recovery. Recently, studies show, initial death COVID-19 outbreak occurred in older people, possibly due to a weak immune system that allows the rapid growth of viruses [82,83]. People should be aware of COVID-19 and avoid physical contact with a positive corona user, and avoid touching the wet substance such as fecal, urine, and cough droplet that will help regulate the spread of the infection. All countries release advisory to stop the spread of the pandemic disease, the passenger who has travel history from an infected country has been routinely checked. WHO gives some instruction to reduce the spread of COVID-19 which includes (i) wash your hand properly with the surfactant at least 20 sec, (ii) Avoid touching your face, (iii) Practice good respiratory hygiene, (iv) Maintain social distancing and avoiding crowded place and (v) Seek medical care early if you show symptoms [84].

Conclusion

SARS-CoV-2's intermittent emergency and epidemic reminds us that the CoV's of zoonosis are many global health threats. An effective vaccine against nCoV-19 urgently needs to be developed. The research should focus on strict preventive and control measures to reduce the potential risk of transmission of diseases. The study will rely on enforcing strict preventive and control measures that minimize the possible risk of disease transmission. Several preclinical data support that S-protein is considered a critical viral antigen for virulence, and this will lead to the development of new drugs against SARS-CoV-2 in the future. Moreover, in addition, this review will be supports understanding the role of the Ca⁺² ions in SARS-CoV-2 pathogenesis and survival mechanism. In this study we revealed the potential role of Ca⁺² and yet unknown, and these ions may play a role in raising the efficiency of SARS-CoV-2 binding or invasion into pulmonary cells.

Alternatively, plasma therapy can help to reduce the degree of COVID-19 infection. If we lower the concentration of these ions inside the cell, this will help to lower the level of infection. This latest virus epidemic has threatened the worldwide economic, medical, and public health infrastructure. This study provided context to understanding the pathogenesis and survival mechanism of SARS-CoV-2 viral infection, diagnosis, possible treatment, and CoV prevention. Along with this, one-health's role in controlling such zoonotic pathogens is highlighted. We hope this research will be useful in combating infection with CoVs.

Acknowledgements

The author is thankful to other authors, and gratefully acknowledges to the Central University of Rajasthan, Ajmer India, we are also thankful to the Department of Science and Technology, Science and Engineering Research Board No. SB/YS/LS-98/2014 and EEQ/2017/000496), Government of India.

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