An approach to the understanding of the clinical-etiopathological aspect of COVID-19 (SARS-CoV-2)

Meena Kumari1, Monika Agrawal2, Rakesh Kumar Singh3, Parameswarappa S Byadgi*4 1Department of Vikriti Vigyan, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India 2Government Medical Of icer, Rajakiya Ayurvedic Chikitsalaya, Sandeela, Pratapagarh district, Uttar Pradesh, India 3Department of Shalakya Tantra, Vijay Ayurvedic Medical College Hospital and Research Centre, Bhandaha Kala, Kaithi Varanasi– 221116, Uttar Pradesh, India 4Department of Vikriti Vigyan, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India


INTRODUCTION
The ongoing outbreak of novel coronavirus  was identi ied irst in Wuhan, China on 1 December 2019, (Kai and Kai, 2020;Wu et al., 2020) and subsequently spread to other areas of the world in early April 2020 to 190 countries (Tang et al., 2020).
On 16 July 2020, data from WHO has shown that outbreak and sporadic human infections of COVID-19 have reported 13,378,853 con irmed cases with 580,045 fatalities across the world.
SARS-CoV-2, related to β-corona viridae family have an identical genomic sequence (almost 96%) with its natural host bat β-coronavirus (Fiesco-Sepúlveda and Serrano-Bermúdez, 2020). This virus is most likely link to humans and attacks the upper and lower respiratory tract in humans. Patients with pre-existing underlying heart and respiratory diseases and aged people (>70 years) who are more susceptible to infection and prone to a severe outcome (Mona et al., 2020).
Coronavirus is enveloped, non-segmented positive -sense, and single-stranded RNA viruses and highly contagious, belongs an extensive family of viruses that can cause disease in both animals and humans (Tang et al., 2020;He et al., 2020;Ortiz-Prado et al., 2020). There no speci ic treatment and any vaccine yet. So, the best solution will be selfisolation (Fiesco-Sepúlveda and Serrano-Bermúdez, 2020), the adaptation of preventive measures and sensitive diagnostic approaches (Nitulescu et al., 2020;Lot i et al., 2020)and implementing proper management for controlling the recent ongoing SARS-CoV infection. This pandemic infection in micro and macro environment causing more damage to the society by all means (Lot i et al., 2020) & currently available antiviral drugs are not capable of containing this deadly infection.
The outbreak of COVID-19 will have a signi icant global concern on the social health, cultural, and economic infrastructures at various levels because the virus is contagious, to be long-lasting and responsible for lethal respiratory infection in the human being which takes many years to recover. The genetic characterization of the viral strains will have implications related to pathogenicity, transmissibility, and response to preventive therapy of the viral makeup for global populations . So it's tough to the understanding of this mechanism for preventive drug discovery and formation of effective vaccines.

Aetiology
This new coronavirus origin is not known precisely. Still, at the beginning of this outbreak, many patients were diagnosed primarily with pneumonia of an unknown aetiology in the hospital in a particular area which linked epidemiologically to a Hunanan south China seafood and wet animal wholesale market in Wuhan, Hubei Province, China. These are indicative of the zoonotic origin of COVID -19 based on the wide-ranging of the infected population there and early infections animal to human transmission (Rothan and Byrareddy, 2020).
According to WHO, severe acute respiratory syndrome coronavirus -2 (SARS-CoV-2) was the primary etiological source of new developed COVID pandemic that generally attack the host respiratory system. Severe acute respiratory syndrome coronavirus 2 (SARS Co-2) is one of the most acute atypical respiratory pathogens that initially infects the human respiratory tract after which other organs involved and represents the causative factor of probably brutal death in close to 10% of cases (Rothan and Byrareddy, 2020) due to progressive respiratory failure and rapidly spreading across the world to affect thousands of populations per day .
Coronavirus disease (COVID-19) is an acute pulmonary syndrome characterized by atypical pneumonia and represents as an etiological agent of SARS and mostly produced from bat origin. coronaviruses and affect the human populations recently (Rothan and Byrareddy, 2020). These viruses affect the animal, and some viruses attack humans in a few cases.

Pathogenesis
In the past two decades, the single-strand RNA (ssRNA) beta-coronavirus was divided into three epidemic disease-severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus and SARS-CoV-2 (Li et al., 2020b). Severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) are the most causative factor for producing severe viral pneumonia Guo et al., 2020). SARS-CoV-2 correlates with SARS CoV as 79 % identical genomic sequences (Tay et al., 2020).

In lammatory mechanism
In this process initially, SARS-CoV-2 attacks respiratory mucosal epithelial cells causing stimulated response in macrophage and lymphocyte, SARS-CoV-2 antigen presentation on the host cell surface and activation of B cells along with this phenomena involvement of T-helper and T-cytotoxic cells also. Immunogenic structural proteins of SARS-CoV-2 virus attack the peripheral white blood cells (WBC) and immune cells of the host, particularly T lymphocytes. So in COVID -19 patients are indicated Cellular immune de iciency and lymphopenia against the pathogen (Dhama et al., 2020).
Disturbance the ratio of ACE/ACE2 (↑ ACE2) by SARS CoV-2, which is responsible for the aggravation of the in lammatory process, pulmonary vasoconstriction and alveolar tissue damage, ultimately causing acute lung injury.
SARS-CoV-2 infection with aggressive up-regulation of pro-in lammatory cytokines including, interferon (IFN-γ), TNF-α, interleukin IL-6, IL-8, IL-10 responses (Henry et al., 2020). In serum strongly implicated in the resulting severe destruction of lung cells and in lammation (Li et al., 2020a). Excessive production of proteases enzymes and oxygen species by the in lammatory cell in iltration directly damage the lungs cells resulting from the virus (Tay et al., 2020).Because Cytokine Strom syndrome correlated with progressive in lammation is playing an essential role in the immune mechanism (Li et al., 2020a). Dysregulated local host immune response(adaptive T and B cell immune response) causes severity in COVID 19 infected patients (Dhama et al., 2020).
Activation of macrophages and monocytes that respond to the infection, release in lammatory cytokines Tay et al. (2020) and is also correlated with the epidemiologically of SARS-CoV and MERS-CoV. SARS-CoV-2 against T and B cells response shown in the host blood after one week of symptoms onset of COVID-19 (Tay et al., 2020). Hyper in lammatory response activated by SARS-CoV-2 is a major etiological factor for disease severity and death in COVID -19 patients (Merad and Martin, 2020).
At the cellular levels, SARS Co-2 infection promotes the activation of CD-I cells and production of GM-CSF, which results of activation of CD14, CD16 with interleukin 6(IL6) and other in lammatory cytokines (Li et al., 2020a) .CD 8 T cells responsible for direct attach and killing the viruses (Tay et al., 2020).
T cell lymphopenia and in lammatory cytokines con-centrations are indicative of poor prognosis along this interleukin 6(IL6), IL10 and D-dimer continuously elevation (Helmy et al., 2020) within 7-14 days after onset the symptoms marking of condition toward the severe stage of the disease (Nitulescu et al., 2020).
Lymphopenia( in iltration of lymphocyte into the respiratory tract ) in 70% cases (Mungroo et al., 2020) and an increased ratio of the neutrophillymphocyte present in 80% patients of COVID-19. Lymphopenia is a standard clinical feature and associated with severity and mortality rate of COVID -19 patients (Merad and Martin, 2020). Corona virus-speci ic T cells reduce and control the disease development by eliminating the viruses and should be help of production of several vaccine formulations (Tay et al., 2020).

Role of ACE 2 receptor
Dipeptidyl peptidase 4(DPP4) and angiotensinconverting enzyme 2 (ACE2) in the lower respiratory tract are two different sites for viral infection. The DPP4 are the fundamental human receptors for the surface spike (S) glycoprotein of MERS-CoV and SARS-CoV additionally recognizes its ACE2 receptor (Li et al., 2020b) SARS-CoV-2 is more linked to SARS-CoV(due to similar genetic sequences ) instead of MERS-CoV (Tang et al., 2020).
ACE2 (mono-carboxyl peptidase) (Dariya and Nagaraju, 2020) against af inity -puri ied antibody is responsible for blocking of syncytial epithelium between infected host cells. So SARS-CoV-2 S protein receptor binding and membrane integration is dependent on the expression of ACE2 receptor type2 alveolar epithelial cells (Tang et al., 2020) and is essential for regulating of host tropism and transmission capacity (Vallamkondu et al., 2020).
SARS -CoV-2 enter into the host cell and attach to the ACE 2 (crucial cell receptor for SARS-CoV-2 ) (Guo et al., 2020) is the irst step of viral infection (Tay et al., 2020). S protein of virus interacts with surface ACE2 enzyme and transmembrane protease, serine 2 (TMPRSS2) for activation of S protein which leads to the internalization of the viral substance (Ortiz-Prado et al., 2020).
It is responsible for the permission of the virus to entering into the host cells and binds the binding domain of the ACE2 receptor resulting in spreading of SARS CoV-2 rapidly within the human body .
During viral infection, the interaction between ACE2 and S protein which is essential for the assembly and infection of coronavirus, the structural protein S is functionally divided into S1 and S2 subunits (Tang et al., 2020). The S1 subunit determines the virus-host range and containing the receptorbinding domain ( RBD) is bind to the cellular receptor tropism and released during the transition to the postfusion conformation (Tang et al., 2020) which is responsible for targeting to the neutralizing of antibodies (Li et al., 2020a).
The membrane-anchored S2 subunit facilitates the cellular membrane integration through the two tandem HR1 (Heptad repeats) and HR2, and entrance process (Al-Qahtani, 2020).
The attachment of SARS-CoV S glycoprotein to ACE 2, can contribute to the severity of alveolar damage lung pathologies due to loss of apical airway epithelial cilia, squamous metaplasia and macrophages number elevation in the respiratory tract (Mona et al., 2020).
S glycoprotein of SARS CoV bound to the ACE2 receptor on the host cell surface through the S1 subunit. It triggered due to type II transmembrane protease serine 2 (TMPRSS2) (Nitulescu et al., 2020;Guo et al., 2020) and cathepsins B and L to encourage the viral and host membrane fusion process through the S2 subunit within the endosome microenvironment. (Mona et al., 2020). After membrane fusion, initially, the virus interacts with speci ic human cells that exhibit distinct receptors for the viral spike protein (Nitulescu et al., 2020). Then using the host cellular machinery, it starts viral translation process after the viral genomic RNA is released into the cytoplasm and activates pathogenic responses and replicating itself, and expressing speci ic sequences in which translation of 2 polyproteins such as pp1a and pp1ab by uncoated RNA (Dariya and Nagaraju, 2020).
The polyproteins cleavage into 15 mature replicase proteins ( processed through internal viral proteases ) into the replication transcription complex (RTC) which continuously replicates and responsi-ble for synthesis of a set a subgenomic RNAs that encode structural proteins, (Dariya and Nagaraju, 2020) endoplasmic reticulum and Golgi which facilitating the adaptation of this virus to its host .
After this process, new multiple subgenomic negative-strand RNAs and structural protein genes are produced and eventually released into the host cytoplasm through exocytosis (Tang et al., 2020).This virion-containing vesicles fuse in the cytoplasm and release a million copies of the new viruses (Guo et al., 2020) and newly formed virus attack the other healthy host cells (Vallamkondu et al., 2020).
Knowledge of this virus pathogenesis may be helpful for the improvement of new therapeutics to invent new antiviral strategies against SARS-CoV-2 infections by the breakdown of the SARS-CoV-2 life cycle through the blockage of the host target ACE2 receptor (Tay et al., 2020).

At a molecular level
The pathogenesis of COVID-19 virus and SARS CoV are differentiated at the molecular basis due to ACE expression on different levels which help to understand the disease and its preventive measures.
COVID -19 patient's respiratory and serum specimen contains SARS -CoV RNA which easily detected by RT-PCR mechanism (major con irmatory test)  and speci ic antibodies react with SARS antigen (Medhi et al., 2020) . This process contributes to in lammatory responses and cell-mediated responses which are responsible for disease severity, as well as alveolar tissue destruction. The adaptive protective immunity formed antigen-speci ic antibody, which binds to the S spike protein of SARS-CoV-2 and decreases the ability of the virus to attach the respiratory tissues.
Genetic alteration changes of coronavirus are lesser than to other RNA viruses, but homologous recombination may be rapidly occurred and primarily acting on the antiviral drug activity (Nitulescu et al., 2020) due to the proofreading activity of RNA dependent RNA polymerase (RdRp) enzyme of these viruses (Mona et al., 2020).
In the intra, alveolar spaces contain large nuclei amphophilic granular cytoplasm with atypical enlarged pneumocytes and Multinucleated syncytial cells, prominent nucleoli (showing viral cytopathic like changes) (He et al., 2020).
Mostly laboratory testing focused on Virus-speci ic nucleotide -positive and viral protein seroconversion of known respiratory pathogens of these viruses, which speci ically attacks the lower respi-ratory tract through the progression of disease and correlation between disease and viruses.
Human tissues PCR analysis has observed ef icient expression of ACE2 on 293 T cells by an anti-ACE2 antibody and messenger RNA in the pulmonary parenchyma as well as in the tissues of heart, kidney and gastrointestinal tract. The primary sites of expression of murine ACE2 are lung and kidney cells, which is consistent with the pathology of acute viral infection of SARS (Vallamkondu et al., 2020).
In SARS-CoV infection Lungs, histopathology images show the destruction of bronchial epithelium, loss of alveolar cilia and giant cell in iltrate with a marked high levels macrophages in the alveoli and lung interstitium (Merad and Martin, 2020).
Recently some evidence has revealed that SARS CoV is a newly emerged virus from a zoonotic source, (Mona et al., 2020) because the amino acid and genomic sequences of the receptor-binding domain (RBD) of human SARS-CoV are homologous to animal genetic phenomena, that's indicative of identical receptor for those viruses . It is originated from animals as per earlier outbreaks in humans.
In SARS CoV-2 infection, an adaptive immune response required to control of disease progression by the production of antigen-antibody complex and destroy the virus. In the early stage, if the host has strong endogenous immune, better personal health and speci ic genetic environment which has antiviral activity, then immunity booster strategies help protect from the virus individuals. The innate immunity of the host is the irst line of defence mechanism against the viral infection (Naqvi et al., 2020;Shi et al., 2020).

Clinical features
SARS-CoV and MERS-CoV infection features are approximately similar experienced but progression rate to respiratory failure is high due to MERS-CoV infection.
COVID-19 infection represents sign and symptoms after almost 5.2 days. Incubation period starts from the onset of COVID-19 symptoms to ranging between 2 days to 14 days with a median of 7 days. The incubation period and the severity and outcome of the disease depend upon the patient age ( >55 years old) and immune system status (Rothan and Byrareddy, 2020). Therefore COVID-19 spreading rate reduced by self -isolation and social distancing, especially in more susceptible people as adults or patients with comorbidities.
COVID-19 clinical features are classi ied into a stage-I( Asymptomatic or non-speci ic symptoms ), stage -II( symptoms with a virus ) with severe dyspnoea and associated pneumonia, Stage III( severe respiratory symptoms with excessive virus presence) (Shi et al., 2020) with in lammatory responses related to multiple organ damage pathology (Nitulescu et al., 2020) and critically based on clinically aggravated symptoms and their imaging reports and some severe cases leading to death (Shi et al., 2020). SARS-CoV-2 patients may respond initially with loss of smell (anosmia), loss of taste (ageusia) (Mungroo et al., 2020) or bitter taste, loss of appetite diarrhoea, nausea and mild raised body temperature but fever is not a premier symptom of this infection (Zu et al., 2020). 97.5% of COVID -19 patients show signs within 11.5 days (Tay et al., 2020). SARS-CoV-2 patients prominent clinical features, namely fever associated with a dry cough, coarse breathing sounds (shortness of breath), headache, pharyngula, myalgia, acute severe respiratory misery, aspiratory pneumonia. The symptoms are aggravated if the patient isn't properly diagnosed and treated during this stage. RT-PCR (real-time polymerase chain reaction) of the patient's sputum reports are showed the con irmation of the COVID-19 infection (Rothan and Byrareddy, 2020).
Most severe forms of COVID -19 infection and high risk of death were mainly observed in elderly (adults ≥ 55 years )and low immune patients with an underlying pre-existing disease like diabetes mellitus, hypertension, chronic kidney diseases, and coronary artery disease. So the recovery rate is higher in people who have healthy immune responses. Death due to progressive respiratory failure and massive alveolar damage in critically ill patients of COVID 19 is associated with hyper in lammation in alveolar tissues and aggravates in lethal pneumonia (Dhama et al., 2020).
COVID-19 symptoms ranges including from asymptomatic upper respiratory tract infections to severe form of pneumonia which correlated with acute respiratory distress syndrome and fatality. The COVID-19 patients have the numerous marginal groundglass opacities experiential in subpleural regions of both lungs (Mungroo et al., 2020) that probably induced both local systemic and generalized immune response that led to accelerated in lammation. Signs and symptoms between COVID-19 and prior beta coronavirus are comparable likes fever with a dry cough, breathlessness and both sided consolidated lungs and ground-glass opacities on chest radiographs (CT scans) (Dhama et al., 2020).
Severe forms of COVID-19 patients with dyspnoea or hypoxemia after one week (McGonagle et al., 2020) and after which these symptoms rapidly progres-sion and converts to septic shock, ARDS, metabolic acidosis (Medhi et al., 2020) and coagulation dysfunction (Guo et al., 2020) that develop injury to the kidney, heart, and other organs, and even multiple organ failures (Zu et al., 2020). ARD(severe COVID-19 symptoms aggravated to ARDS around 8-10 days after onset of symptoms) is a cause of death in COVID-19 (Mehta et al., 2020) in 70% cases of fatal disease due to respiratory failure (Tay et al., 2020).
Generally, animal coronaviruses do not spread between humans, but SARS and MERS affect the human populations mainly through direct contact or respiratory droplets (Adhikari et al., 2020).SARS CoV-2 might be transmitted to humans from bats (natural hosts of SARS-CoV-2) through an unde ined intermediate host. The SARS CoV-2 virus particles transmitted from one person to another person primarily through direct contact or aerosol droplets by coughing and sneezing of symptomatic patient individuals without covering of mouth and nose with mask into the air (Mungroo et al., 2020) but symptoms may develop in later who infected by asymptomatic patients. But the faecal-oral route, (recent FDA guidelines) and intermediate fomites may be other sources of viral transmission from both symptomatic and asymptomatic patients during the incubation phase of infection. SARS -CoV-2 virus infects the respiratory tract of individuals because this virus particles survival rate in the air around 14-16 hours but depending on the environmental temperature and travels a distance of 3 -4 feet (Vallamkondu et al., 2020).
Currently, many countries have decided social distancing and lockdown methods for reducing mitigation and cross-transmission of this emerging disease (Nitulescu et al., 2020) and the rapidly increasing cases number with high mortality rate.
WHO recommended that necessary precautions like hand washing, use PPE and face masks, selfisolation, avoid the travelling in high-risk areas of COVID -19 and contact of symptomatic patients are essential (Sohrabi et al., 2020) .

CONCLUSIONS
The pandemic by COVID-19 is a live issue distressing people globally. Estimation of the clinical and epidemiological distinctiveness of SARS-CoV-2 cases suggests that its diverse nature in producing infections. Symptomatic patients are usually considered as infectious. Research and therapeutic strategies for COVID-19 have focused on the viral genomic proteins and the development of better approaches. Understanding of clinical-etiopathogenesis is necessary to invent new vaccines and therapeutics for its control and management.