COVID-19: Review on its etiology, pathogenesis, and existence in humans

The world is facing a new healthcare crisis with the rise and spread of novel coronavirus since December 2019 Also known as Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2), the disease associated with SARS-CoV-2 is even called COVID-19 COVID-19 is having a pulverizing impact on the scientific community As of August 13, 2020, the number of confirmed cases had reached up to 20,439,814 and the death toll to 744,385, affecting more than 188 territories across the globe In these difficult times, the world is looking towards research and clinical work from different scientific communities to lead the way to a solution to the issue In this review, we are focusing on COVID-19 emergence, pathogenicity, and existence in humans, as well as the SARS-CoV-2 infection mechanism and its similarities to previous coronavirus strains


Introduction
The first case of mysterious pneumonia emerged in December 2019 in Wuhan, Hubei region of China (Wang et al., 2020a). A new coronavirus was identified by  from the samples taken from the Wuhan seafood market, and later it was named Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) by the international committee on taxonomy of viruses (ICTV) (Yang and Wang, 2020a). As of June 12 , 2020, the number of confirmed cases had reached up to 20,439,814 and the death toll to 744,385, affecting approximately 188 territories across the globe (https://www.who.int/docs/defaultsource/coronaviruse/situationreports/20200512-covid-19-sitrep115.pdf?sfvrsn=feac3b6d_2). The number of COVID-19 cases are increasing rapidly, and the situation is getting worsen day by day. Fig. 1 shows the global increase in COVID-19 confirmed cases and the number of deaths with time. Tab. 1 shows the total number of COVID-19 cases and confirmed death in six regions, and Tab. 2 shows a comparative analysis between some countries that are now (on August 13, 2020) the epicenter of COVID-19 outbreak (https://www.who.int/docs/ default-source/coronaviruse/situationreports).
Coronaviruses have an outer spike-like projection that gives them a crown-like appearance, henceforth the name coronavirus (Wormser and Aitken, 2010). Before SARS-CoV-2, humans had two previous encounters with coronaviruses: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) (Drosten et al., 2003;Ksiazek et al., 2003) and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (Zaki et al., 2012). In 2002, the SARS-CoV outbreak was reported in the Guangdong region of China and spread to five mainlands, infecting 8,098 individuals and causing 774 deaths (mortality rate approximately 9.6%) (http://www.who.int/csr/sars/ country/table2004_04_21/en/index.html). A decade later, in 2012, another coronavirus MERS-CoV emerged in Arabian Peninsula, where it stays a significant well-being concern and later spread to 27 countries infecting 2,494 people and claiming 858 lives (fatality rate 34%) (Walls et al., 2020). Both SARS-CoV and MERS-CoV originated from bats and then crossed the species barrier to humans trough intermediate host civet (Wang et al., 2005) and camels, respectively (Briese et al., 2014;Hemida et al., 2013).
Morphology and structure of SARS-CoV-2 Coronaviruses belong to the Coronaviridae family. Members of this family are enveloped, single-stranded, and positive sensed RNA viruses. SARS-CoV-2 has a similar genome structure as like other coronaviruses having at least ten open reading frames (ORFs) (Chan et al., 2020). They contain 5'-terminal non-coding region, an open reading box 1a/b region coding a non-structural polyprotein that is further cleaved by viral proteinase 3C-like serine protease (3CLpro) and papain-like cysteine protease (PLpro) to form RNAdependent RNA polymerase and helicase (Zumla et al., 2016). At 3' end, genes encoding structural proteins, i.e., Spike (S), Membrane (M), Envelop (E), and Nucleocapsid (N) are present. E and M protein together form a viral envelope. N protein, along with the 3' terminal non-coding region, is responsible for the assembly of virus , and S protein forms a spike-like glycoprotein that helps in the process of virus attachment with the host surface. Fig. 2a shows the genetic organization of SARS-CoV-2 . On the basis of genomic structure and phylogenetic examinations, coronaviruses can be divided into four genera α, β, γ, and δ, where α and β genera infect humans and mammals, whereas the other two genera infect the birds. SARS-CoV-2 belongs to the β family, having a round or oval shape with a diameter of 60-140 nm .

Origin and host range of SARS-CoV-2
After the novel coronavirus is discovered, it is important to understand where it originates from. It helps to decide what a suitable approach needs to be taken to control its transmission. It also helps to develop the therapeutics and vaccine against the virus (https://apps.who.int/iris/handle/ 10665/332197). Initially, the novel coronavirus outbreak has been referred to as 2019-nCov but, in February 2020, theinternational committee on taxonomy of viruses (ICTV) announced that it should be referred to as SARS-CoV-2 and the disease associated with it referred to as COVID-19 (Gorbalenya et al., 2020). Coronavirus study group (CSG), a working group of ICTV, taxonomically proved that SARS-CoV-2 had more than 70% nucleotide identity with SARS-CoV and 90% nucleotide identity with SARSr-CoV RaTG13 (Gorbalenya et al., 2020;Lu et al., 2020).
In recent years, bats have been an essential reservoir for different types of coronaviruses (Cui et al., 2019). In February 2017, during an outbreak of severe watery diarrhea of piglets in Guangdong Province of China, two novel swine enteric alpha coronaviruses (SeACoV) were detected by two different groups, which were very similar to bat coronaviruses (Gong et al., 2017;Pan et al., 2017). The confinement of these two SeACoV from sick piglets extends our insight into the host range of coronaviruses.
SARS-CoV and MERS-CoV infections started from bats, both viruses bouncing species to infect humans through a different intermediate host (Gong et al., 2017). Civet sold in the live animal market was the intermediate host of SARS-CoV, and the dromedary camel was the intermediate host of MERS-CoV. Fig. 3 explains the possible interspecies transmission routes of coronaviruses (Gong et al., 2017;Su et al., 2016). Hence, it is clear that the previous two coronaviruses required an intermediate host before passing on to humans (Cui et al., 2019), and this fact indicates that SARS-CoV-2 may have passed to humans through an intermediate host. SARS-CoV-2 shares 96% whole-genome identity with BAT CoVRaTG .
Although there is no recognizable proof available for an intermediate host of novel coronaviruses, early investigation suggests that it may have jumped from bat to humans (Wu

Pathogenesis
Although the pathogenesis of SARS-CoV-2 is poorly understood, the clinical symptoms are similar to that of  SARS-CoV and MERS-CoV like fever, headache, sore throat, nonproductive cough, respiratory distress, myalgia pneumonia. The incubation period of disease ranges from 4 to 15 days, with an initial fatality rate of around 1.4% Peiris et al., 2004), but as of 13th August 2020, the fatality rate has increased up to 3.6% (Tab. 1) (https://www.who.int/ docs/default-source/coronaviruse/situationreports/20200512covid-19-sitrep115.pdf?sfvrsn=feac3b6d_2). SARS-CoV-2 infects both the upper as well as the lower respiratory tract. Studies have shown that the higher viral load is detected in the nasal cavity than the throat (Zou et al., 2020). All the age groups among humans are susceptible, but the infection and fatality rate are higher in people above 60 years of age. Many people having COVID-19 disease show mild symptoms, while some may be asymptomatic. But people with compromised immunity and other co-morbidities may progress to acute respiratory distress syndrome and multi-organ dysfunction .

Similarities between SARS-CoV-2 and other CoVs
Recent studies conducted by Lu et al. Lu et al. (2020) suggest that SARS-CoV-2 is more related to bat coronaviruses than the SARS-CoV. SARS-CoV-2 and SARS-CoV share around 79% of genetic similarities. They also have shown structural similarities between the receptor-binding domain (RBD) of SARS-CoV-2 and SARS-CoV. Both viruses utilize similar host cell receptors and viral proteins for entry into the host cell . Furthermore, on comparing the specific domain of the viral proteins that bind to the cell surface receptor, it was found that SARS-CoV-2 binds more efficiently to the host cell than the SARS-CoV (Walls et al., 2020).

Entry of SARS-CoV-2
Viral infection begins with the binding of viral particles to the host cell surface receptors; hence it becomes very essential to identify the cell receptors involved in this process. Except for HCoV-OC43 and HKU1, the other four coronaviruses (HCoV-229E, SARS-CoV, HCoV-NL63, MERS-CoV) recognize proteinaceous peptidase as a receptor Lu et al., 2013;Raj et al., 2013). The former two viruses are shown to engage with sugar receptors for the cell attachment . After the recent episode of COVID-19, Chinese scientists quickly verified that SARS-CoV-2 likewise uses human angiotensin-converting enzyme-2 (hACE-2) for attachment to the cell (Fig. 4)  .
Novel coronaviruses use its spike-like glycoprotein S for fusion and entry into the cell (Lu et al., 2015). Further, S protein is cleaved into S1 and S2 subunit by the host cell protease, Furin. These two subunits are responsible for receptor recognition and fusion, respectively (Dijkman and Van Der Hoek, 2009). SARS-CoV and MERS-CoV utilize the C-terminal domain (CTD) of the S1 subunit for receptor recognition called the receptor-binding domain Lu et al., 2020). S1 CTD is the critical region in SARS-CoV-2 that interacts with the human ACE2 (Walls et al., 2020). Ou et al. (2020) showed that human lung cancer cell, Calu 3, is highly susceptible to the SARS-CoV-2v (Ou et al., 2020). The binding of S protein to hACE2 requires conformational changes and protease activation. A furin site is present between S1 and S2 (AA 682-685 RRAR) in SARS-CoV-2 S protein (Fig. 2b)  . Regardless of whether the proximity of this furin site has any impact on the viral pathogenesis and spreading among people, it stays to be resolved.

Immune system activation and invasion
Whenever our body is under attack by a viral pathogen, our immune system gets activated and antigens are presented to antigen-presenting cells (APC) by major histocompatibility complex (MHC) or human leukocyte antigen (HLA) where it is recognized by the cytotoxic T lymphocyte (CTL). Till now, there are no reports available regarding the mechanism of SARS-CoV-2 infection, but we can get some ideas from the previous research studies available on SARS-CoV and MERS-CoV. MHC-1 molecules are mainly responsible for the antigen presentation in SARS-CoV . Previous studies have confirmed that polymorphism in the HLA gene is associated with the susceptibility to SARS-CoV and MERS-CoV (Keicho et al., 2009;Chen et al., 2006;Tu et al., 2015).
Some initial reports during the early stage of infection have shown that out of 41 patients, six died due to the Acute Respiratory Distress Syndrome (ARDS) . ARDS is considered as a main pathological event in SARS-CoV and MERS-CoV . ARDS mainly occurs due to the cytokine storm, in which a large amount of pro-inflammatory cytokines is released due to uncontrolled inflammatory responses. In severe SARS-CoV-2 infections, a large amount of pro-inflammatory cytokines (IL1B, IFNγ, IL15, and IL17) are released . This cytokine storm results in an attack on the body by the immune system causing ARDS, multiple organ failure, and finally death in severe cases, just like in SARS-CoV and MERS-CoV .
Immune invasion of SARS-CoV-2 is not yet fully understood; however, its previous versions, SARS-CoV and MERS-CoV2, were able to evade the immune system. They can incite the creation of double-layered vesicles lacking the Pattern Recognition Receptor, and then they avoid the host detection system of their RNA by rapidly dividing in these vesicles (Snijder et al., 2006). In mice infected with SARS-CoV and MERS-CoV, the IFN1 pathway was found to be inhibited, which has a proactive effect on these viruses (Niemeyer et al., 2013).

Detection and diagnosis of COVID-19
According to the National Health Commission, detection of genetic material, neutralizing antibody, culture, and isolation of viral particles are the key laboratory diagnosis that can be performed for the rapid detection of SARS-CoV-2. The reverse transcriptase-polymerase chain reaction is also used to detect SARS-CoV-2 nucleic acid. Nasal swab, throat swab, and blood samples are used as a diagnostic standard for detecting the SARS-CoV-2 infection (http://en.nhc.gov. cn/index.html). US Centre for Disease Control (CDC) developed the specific primers and probes for the universal detection of SARS-CoV-2 by real-time RT-PCR. Recently, a novel method is developed by (Wang et al., 2020b) for the rapid detection of COVID-19, known as CRISPR/Cas 12a based assay with a naked eye readout, CRISPR/Cas12a-NER. This assay is very sensitive and can detect as few as 10 copies of the viral gene within 45 min.

Prevention and control of COVID-19
Since, as of now, there are no affirmed medicines and treatments for the SARS-CoV-2 infection, avoidance is the best remedy. The best ways to control the infection are to take out the source of infection, cutting off the route of transmission, and isolation of the infected or suspected individuals. People can contract SARS-CoV-2 infections from other people already having the virus. This disease spreads principally from individual to individual through the small droplets from the nose or mouth, which are expelled when the individual with SARS-CoV-2 virus coughs, sneezes, or speaks. Since these droplets are relatively heavy, they cannot travel too far. That is why it is essential to maintain a distance of at least one meter from an infected person (https://www.who.int/emergencies/diseases/ novel-coronavirus-2019/question-and-answers-hub/q-a-detail/ q-a-coronaviruses). No vaccine is available for the prevention of SARS-CoV-2; although, many research institutes and private companies are trying to develop the vaccine. For instance, Moderna, a biotech organization based in Cambridge, Massachusetts, is working with the National Institutes of Health on a potential mRNA vaccine candidate against SARS-CoV-2. AbMax Biotechnology Co., Ltd., reported that it had effectively developed a SARS-CoV-2's N protein-specific monoclonal antibody (Yang and Wang, 2020b).

Conclusion
The COVID-19 outbreak was declared a pandemic on 11 th March 2020 by the World Health Organization. After this, SARS-CoV-2 infection has tested the financial, clinical, and general well-being framework of the whole world. Until now, very little understanding is available regarding how SARS-CoV-2 crossed the species barrier to humans. More scientific data is required for understanding the mechanism of SARS-CoV-2 survival in humans and how it becomes so contagious (more than SARS-CoV and MERS-CoV). It is also essential to pay attention to COVID-19 vaccine development. Today, all the efforts are being made towards controlling this flareup, but endeavors ought to be made to devise accurate measures to forestall future episodes of a zoonotic outbreak.
Author Contributions: VK wrote the paper and finalized it. VA edited the manuscript.
Availability of Data and Materials: All data generated or analyzed during this study are included in the manuscript.  . SARS-CoV-2 entry into host cell and immune system activation. SARS-CoV-2 attaches to cell through hACE-2 receptor (indicated in red) and then fusion to the cell membrane occurs. After entry into the host cell it activates host immune system such as T-cell, B-cell activation and proinflammatory cytokine production in uncontrolled way that may result in Acute Respiratory Distress Syndrome (ARDS).
Ethical Statement: This article does not contain any studies with animals performed by any of the authors.