key: cord-292751-tk1oggi9
authors: Hosseini, Elahe Seyed; Kashani, Narjes Riahi; Nikzad, Hossein; Azadbakht, Javid; Bafrani, Hassan Hassani; Kashani, Hamed Haddad
title: The novel coronavirus Disease-2019 (COVID-19): Mechanism of action, detection and recent therapeutic strategies
date: 2020-09-24
journal: Virology
DOI: 10.1016/j.virol.2020.08.011
sha: 
doc_id: 292751
cord_uid: tk1oggi9

Novel coronavirus SARS-CoV-2, designated as COVID-19 by the World Health Organization (WHO) on the February 11, 2020, is one of the highly pathogenic β‐coronaviruses which infects human. Early diagnosis of COVID-19 is the most critical step to treat infection. The diagnostic tools are generally molecular methods, serology and viral culture. Recently CRISPR-based method has been investigated to diagnose and treat coronavirus infection. The emergence of 2019-nCoV during the influenza season, has led to the extensive use of antibiotics and neuraminidase enzyme inhibitors, taken orally and intravenously. Currently, antiviral inhibitors of SARS and MERS spike proteins, neuraminidase inhibitors, anti-inflammatory drugs and EK1 peptide are the available therapeutic options for SARS-CoV-2 infected individuals. In addition, Chloroquine, which was previously used for malarial and autoimmune disease, has shown efficacy in the 2019-nCoV infection treatment. In severe hypoxaemia, a combination of antibiotics, α-interferon, lopinavir and mechanical ventilation can effectively mitigate the symptoms. Comprehensive knowledge on the innate and adaptive immune responses, will make it possible to propose potent antiviral drugs with their effective therapeutic measures for the prevention of viral infection. This therapeutic strategy will help patients worldwide to protect themselves against severe and fatal viral infections, that potentially can evolve and develop drug resistance, and to reduce mortality rates.

Recent pharmaceutical strategies to treat coronavirus 12-15

Improve immune system to fight with COVID- 19 15-18

Abbreviations [18] [19] Introduction Over the last two decades, three coronaviruses have periodically crossed animal species such as bats, transmitted to human populations, and caused an ever-increasing outbreak of a large-scale pandemic [1, 2] . The previously reported viral zoonotic pathogens include SARS-CoV (severe acute respiratory syndrome coronavirus) and MERS (Middle East respiratory syndrome coronavirus) [3, 4] , that can cause severe respiratory disease in human [5, 6] . SARS-CoV-2, a novel coronavirus (which causes COVID- 19) , has fast spread like a pandemic since its outbreak in Wuhan, China, in December 2019 [7] . It causes an acute and deadly disease with a 2% J o u r n a l P r e -p r o o f mortality rate. However, this novel coronavirus is usually associated with a mild to severe respiratory disease in humans [1, 8, 9] . This virus has the ability of jumping between species, and causing a variety of diseases as a strange and complex pathogen [10] . Due to the frequent interaction between humans and animals, a virus is a common source of zoonotic infection.

COVID-19, due to its human-to-human transmission, has become a health emergency of global concern [7, 11] . Currently, we have no sufficient evidence to propose that a specific wildlife animal is the virus origin. A proper study of the viral source, evolution, mode of zoonotic transmission and infectivity, would help to prevent further infections. [3, 4] .

Coronaviruses (CoVs) belong to the Nidovirales order, Coronaviridae family, which comprises of two subfamilies, namely Orthocoronavirinae and Letovirinae (International Committee on Taxonomy of Viruses) [12, 13] . CoVs are genotypically classified into four genera: Alpha coronaviruses (a), Beta coronaviruses (b), Gamma coronaviruses (g), and Delta coronaviruses (d), according to their phylogenetic and genomic data. Further, β-coronavirus is subdivided into four viral lineages of A to D [14, 15] . Coronavirus is an enveloped and non-segmented virus, which has a large positive-sense single-stranded RNA virus genome (27-32 kb) , capped and polyadenylated [16] . Coronavirus also has crown-shape spikes projecting from its surface (80- 160 nM in size), from which its name derived [17] . The CoV Spike (S) glycoprotein attaches to cellular receptors on the host cell and mediates viral entry resulting in interspecies transmission and pathogenesis [15, 18] Figure 2 ). It seems that the high sequence identity in SARS-CoV-2 and Pangolin-CoV may be due to coincidental convergent evolution [32] . Phylogenetic analysis of RBD region of the spike protein, has also led to a further presumption that the identity of SARS-CoV-2 RBD to pangolins-CoV RBD might be result of accidental mutations followed by natural selection, and/or recombination events in pangolins [32] (Figure 2 ). 

Most common clinical symptoms of COVID-19 disease are dry cough, fever and shortness of breath in the majority of patients. Some patients also experience other signs such as sore throat, headache, myalgia, fatigue and diarrhea [33, 34] . In the initial phase of the disease, patients can J o u r n a l P r e -p r o o f be afebrile, only presenting with chills and respiratory symptoms. Although most cases appear to be mild, all patients have new pulmonary signs as ground-glass lung opacity on chest X-ray [17, 35] . The symptoms in patients with mild pneumonia are fever, cough, sore throat, tiredness, headache or myalgia [36] . They do not obviously show any of the serious symptoms or complications. Some patients were reported to have upper respiratory infection (URI), bilateral patchy opacity in lung [7] , decreased white blood cell or lymphocyte number [37] and increased ALT, AST, LDH, CK-MB, CRP and ESR in these stages of infection [38] . Patients with severe pneumonia, suffer from acute respiratory distress syndrome (ARDS) and refractory hypoxemia.

nCoV-2019 can cause severe pulmonary infection, respiratory failure, along with organ damage and dysfunction. In case of extra-pulmonary system dysfunctions, such as derangements in hematologic and digestive system, the risk of sepsis and septic shock will be serious, resulting in considerable increase in fatality rate. The findings showed that the disease is mild in the majority of patients (81%) and only a few of them develop severe pneumonia, pulmonary edema, ARDS, or different organ damages with case morality rate of 2.3%. In children, infection generally presents with much milder clinical symptoms or even asymptomatic, compared with adult.

According to previous studies, pregnant women do not seem to have a severe disease, while older patients are at a high risk of developing critical illness [33, 36] . The Case Fatality Rate (CFR) increased in 50 % of patients older than 80 with a history of chronic diseases, such as high blood pressure, diabetes, heart diseases, respiratory diseases, cerebrovascular diseases, endocrine system disorders, digestive system disorders and cancers . In most of cases, the cause of death is respiratory failure, septic shock or several organ failure [33] . In fact, increased C-reactive protein (CRP) is an important factor of impaired immunity, characterized by lymphopenia. So, SARS-CoV-2 is more probably to affect older people with chronic disease due to their poorer immune function [39] . Covid-19 has also been found to infect more males (average age of 55.5 years) than females [39] . The less susceptibility of females to viral infections is likely associated with the protective role of X chromosome and sex hormones, which result in stronger immune response to virus [40] . CT imaging findings of patients with COVID-19 revealed that most of cases had ground-glass opacities, which may manifest as crazy paving pattern, organizing pneumonia and architectural distortion. On X-rays or chest CT imaging of the examined patients, J o u r n a l P r e -p r o o f

Early diagnosis is the most important step to manage and treat COVID-19. The diagnostic tools are generally molecular methods, serology and viral culture. Initial laboratory investigations of hospitalized patients consist of a complete blood count, coagulation testing and serum biochemical test such as creatine kinase (CK), lactate dehydrogenase, procalcitonin, and electrolytes [29, 41] . Based on laboratory tests, most patients showed a significant decrease in total number of lymphocytes, suggesting that lymphocytes (particularly T lymphocytes) are likely target of SARS-CoV-2. In the COVID infection, virus particles begin to spread through the respiratory tract and infect the surrounding uninfected cells. This leads to initiate a cytokine storm and consequently trigger a series of sever immune responses. This process results in some changes in immune cells, particularly lymphocytes, and then leads to immune system dysfunction [29] . Hence, the decreased number of the circulating lymphocytes could be considered as a diagnostic marker for SARS-CoV-2 infection and its severity [33] . Previous studies reported that there is a correlation between elevated level of pro-inflammatory cytokines like IL1B, IL6, IL12, IFNγ, IP10, and MCP1, and cytokines such as IFNγ, TNFα, IL15, and IL17, in SARS-CoV and MERS-CoV infection respectively, with pulmonary inflammation and lung injury. Notably, the high value of cytokines like IL1B, IFNγ, IP10, and MCP1, may activate T helper 1 cells (Th1) response. This cytokine storm is probably associated with disease severity.

However, in SARS-CoV2 infection, enhanced secretion of T helper 2 (Th2) and cytokines like IL4 and IL10, reduce peripheral white blood cells and immune cells such as lymphocytes,

probably leading to suppression of the inflammatory response and immune system function followed by serious lung damage, which differs from SARS-CoV infection [41] . These findings suggest that sever and uncontrolled inflammatory response have a more damaging effect on COVID-19 induced lung injury than viral pathogenicity. Therefore, in SARS-CoV-2 pneumonia, it is vital to control cytokines or chemokines to detect the impact of 2019 coronavirus on their production in the critical phase of the disease [29, 41] . RT-PCR (reverse-transcription polymerase chain reaction) or Real-Time PCR and genome sequencing for respiratory or blood specimens are the next methods to confirm COVID-19 infection (Table 1) . together with its time-consuming process and the problems associated to performance of RT-PCR kit are the main obstacles to control this epidemic [49] . Chest CT, Compared to RT-PCR, is a fast, sensitive, easy to perform and more accurate and reliable tool for screening and diagnosis of COVID-19 [50] . Chest CT can also show pulmonary abnormalities in COVID-19 patients with early negative RT-PCR results [51, 52] . In the primary stage of pneumonia itself, CT images can demonstrate several small ground-glass opacity as well as some interstitial changes [9] , remarkable in the lung periphery [7] [53] . 

CRISPR-Cas13-based SHERLOCK system consists of two RNA guides, which are combined with a Cas13 protein, and form a SHERLOCK system to recognize the presence of COVID-19 viral RNA. At first the team used synthetic fragments of SARS-CoV-2 RNA as a pattern for designing two RNA guides, which are able to bind to their complementary sequences in COVID-19 RNA. In order to visual readout, they used a paper strip (as a paper strip in pregnancy test) for dipping into a prepared sample. Then, appearance of a line on the paper strip indicates the existence of virus in the sample [54, 55] .

Also, another research group has recently proposed a RNA-targeting CRISPR system to target RNA genome of SARS-CoV-2 in the laboratory, to limit its ability to reproduce [56] . This CRISPR 

CoV-2 patients, particularly to target their lung, which is main infected organ [80] . Before therapeutic application of CRISPR/Cas13d system to patients, it is necessary to determine the safety and efficacy of this system in clearance of 2019-nCov and other viruses in animals. If researchers find this therapeutic strategy secure and beneficial, then it would be applied to kill the viruses that have the potential to evolve and also develop drug resistance [58] .

Since the emergence of 2019-nCoV, due to its rapid spread and being a serious threat to human health, researchers have made great efforts to understand the pathogenetic characteristics of this virus to develop effective drugs. Due to appearance of the 2019-nCoV during the influenza season, orally and intravenously antibiotics and neuraminidase inhibitors such as oseltamivir having been widely used as an experimental treatment for 2019-nCoV in China [59, 60] .

However, there is no reliable evidence that shows oseltamivir is an effective treatment in 2019-nCoV [60] . At present, there is no antiviral drug assumed to provide protection against COVID-19 infection; also it will take long time to develop and a vaccine and gets approval for it. [9] .

Nowadays, Griffithsin, as an inhibitor of SARS and MERS spike, Remdesivir, favipiravir and ribavirin (nucleoside analogues), lopinavir/ritonavir (protease enzyme inhibitors) [61] , oseltamivir (neuraminidase inhibitors), anti-inflammatory drugs and EK1 peptide [62] , the clinical potential to be applied against the 2019-nCoV infection [67, 68] . Chloroquine shows its inhibitory effects against 2019-nCoV through increasing endosomal pH required for virus cell fusion, also affecting the glycosylation of cellular receptors of SARS-CoV [69] . Chloroquine is a cheap and safe anti-viral medicine, which is orally administrated and widely distributes all over body especially in the lungs [70] . Also, remdesivir, which has a structure chemically similar to HIV reverse-transcriptase inhibitors, is currently in the phase of clinical trials for 2019-nCov [58] . The spike (S) protein is a promising target for the development of antivirals drugs, due to its major role in the virus-receptor interaction. Griffithsin, a medicine targeting the oligosaccharides on the surface of different spike proteins [71] , has also been tested in phase I trials for the treatment of HIV and SARS-CoV [72] . However, the efficacy of spike inhibitors for the treatment of 2019-nCoV should be re-evaluated [71] . There are multiple mechanisms of action for nucleoside analogues (adenine or guanine derivative), including lethal mutagenesis, inhibition of RNA biosynthesis, or RNA chain premature termination [72] . They inhibit synthesis of viral RNA in human coronaviruses by targeting the RNA-dependent RNA polymerase [73] . Favipiravir (T-705), a guanine analogue, is an antiviral drug targeting the viral RNA-dependent RNA polymerase of RNA viruses such as Ebola, influenza, yellow fever, J o u r n a l P r e -p r o o f chikungunya, norovirus and enterovirus. It has been recently proposed to be effective also against coronaviruses, in vitro [73] . Using fabiravir and ribavirin combined with oseltamivir, for the treatment of coronaviruses diseases such as in severe influenza, is better than oseltamivir alone [74] . At present, lopinavir/ritonavir and IFN-alpha are in the initial phase of clinical trials in patients infected with 2019-nCoV [74, 75] . According to the guidelines [74] , lopinavir (400 mg/100 mg bid po) used to treat HIV/AIDS infection and ritonavir as a booster, as well as IFNalpha (5 million U bid inh) usually utilized for treatment of HBV, are recommended as antiviral therapy [61, 66] . A scholar's report from Hong Kong illustrated that use of the combination of lopinavir/ritonavir (LPV/RTV) (Anti-HIV drugs) with ribavirin, reduces the risk of acute respiratory distress syndrome (ARDS) or death in treated patients [61] . Further researches are needed to understand whether these inhibitors can effectively block the 3-chymotrypsin-like and papain-like proteases of 2019-nCoV [9] . In addition, remdesivir, earlier used against the Ebola virus, has been presently determined as antiviral drug for treating MERS/SARS and related zoonotic bat CoVs in the cell cultures and animal models [76] [77] [78] . Based on animal experiments, remdesivir effectively decreases the virus titer of mice infected with MERS-CoV and improves the lung tissue damage, in comparison to other cases treated with a compound of lopinavir/ritonavir and interferon-β [60] . Remdesivir has also been recently suggested to inhibit 2019-nCoV [35] by terminating the viral RNA transcription in early stage [79, 80] . 

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The financial support for the current research was provided by Research Deputy of Kashan University of Medical Sciences, Kashan, Iran.

Not applicable

ESH, HHK and NRK provided direction and guidance throughout the preparation of this manuscript. HHB, HN and HHK conducted the literature and drafted the manuscript. Other authors reviewed the manuscript and made significant revisions on the drafts. All authors read and approved the final version.

The authors declared that they have no competing interests.

Not applicable.