Emergence of a Novel Coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2: Biology and Therapeutic Options.

The new decade of the 21st century (2020) started with the emergence of a novel coronavirus known as SARS-CoV-2 that caused an epidemic of coronavirus disease (COVID-19) in Wuhan, China. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans, and mechanisms associated with the pathogenicity of SARS-CoV-2 are not yet clear, however, its resemblance to SARS-CoV and several other bat coronaviruses was recently confirmed through genome sequencing-related studies. The development of therapeutic strategies is necessary in order to prevent further epidemics and cure infections. In this review, we summarize current information about the emergence, origin, diversity, and epidemiology of three pathogenic coronaviruses with a specific focus on the current outbreak in Wuhan, China. Furthermore, we discuss the clinical features and potential therapeutic options that may be effective against SARS-CoV-2.

INTRODUCTION

Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with a nucleocapsid of helical symmetry (1). Coronaviruses have been widely identified as causing respiratory and intestinal infections in humans after the outbreak of severe acute respiratory syndrome (SARS) in Guangdong, China in 2002 and 2003 (2, 3). SARS was determined to be caused by SARS-CoV and emerged in a market where civets were sold (2, 3). Only a decade later, the world witnessed another outbreak in the form of Middle East respiratory syndrome (MERS) caused by MERS-CoV in the Middle East (4, 5). While the researchers were still investigating the underlying mechanisms of pathogenicity and developing effective therapeutic strategies against MERS, the world witnessed the deadliest outbreak in the form of COVID-19 (6). The causative coronavirus of this outbreak was named SARS-CoV-2 due to its resemblance to SARS-CoV (7–9). The SARS-CoV infects ciliated bronchial epithelial cells and type-II pneumocytes through angiotensin-converting enzyme 2 (ACE2) as a receptor (2, 10). MERS infects unciliated bronchial epithelial cells and type-II pneumocytes by using dipeptidyl peptidase 4 (DPP4), also known as CD26, as a receptor (2, 11). The mechanisms associated with the infectiousness of SARS-CoV-2 are not clear, however, structural analysis suggests it is likely entering human cells through the ACE2 receptor (12). This newly emerged virus has much greater similarity to SARS-CoV than to MERS-CoV, thus both SARS-CoV and SARS-CoV-2 may cause pathogenesis through similar mechanisms. The transmission of SARS-CoV to humans was reported from market civets, while that of MERS-CoV was from dromedary camels (13, 14). Similarly, the newly emerged SARS-CoV-2 also appears to be transmitted to humans from markets where wild animals are sold (8). However, the zoonotic source of its transmission is not yet clear. According to previous reports, the aforementioned three coronaviruses are thought to have originated in bats (2, 11, 15, 16).

Since the first epidemic of SARS, the pathogenic coronaviruses have harmed thousands of people worldwide (1, 17). Considering the adverse outcomes of the current COVID-19 epidemic, developing effective therapeutic strategies is necessary to cope with the lack of effective drugs, high mortality rate, and the potential of the virus to cause further epidemics. In this review, we focus on the origin, evolution, and pathogenicity of SARS-CoV, MERS-CoV, and SARS-CoV-2. We also discuss the therapeutic options for SARS-CoV-2 given its importance in the current scenario of COVID-19 outbreak in Wuhan, China. This review will be useful in preparing against future outbreaks and continuing pathogenic infections by this class of novel coronaviruses virulent to humans.

DIVERSITY AND ORIGIN OF HIGHLY PATHOGENIC CORONAVIRUSES

Coronaviruses are members of the subfamily Coronavirinae (family Coronaviridae; order Nidovirales), which contains four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (2). Gamma- and deltacoronaviruses generally infect birds, although some of them can cause infection in mammals, whereas, alpha- and betacoronaviruses are known to harm humans and animals. The viruses SARS-CoV (betacoronavirus), 229E (alphacoronavirus), HKU1 (betacoronavirus), NL63 (alphacoronavirus), OC43 (betacoronavirus), and MERS-CoV (betacoronavirus) can all cause infections in humans (2). However, betacoronaviruses are the most important group because they comprise the most highly pathogenic viruses against humans, including SARS-CoV-2, MERS-CoV, and SARS-CoV (2, 18, 19). The highly pathogenic MERS and SARS coronaviruses originated in bats (2, 18, 19), however, the origin of the newly emerged SARS-CoV-2 remains debatable. Investigations have revealed that the SARS-CoV strains detected in market civets (20, 21) were transmitted from horseshoe bats (22). These viruses were found to be phylogenetically related to SARS-CoV in bats from China, Europe, Southeast Asia, and Africa (2, 22, 23). In addition, the genome sequences of SARS-CoV strains isolated from humans were highly similar to those in bats (21). However, some variations were found among the S gene and ORF3 and ORF8 gene sequences, which encode a binding and fusion protein and dispensable proteins for replication, respectively (2, 23). Nevertheless, clade2 of the S genetic region (22, 24), ORF8 (23), and ORF3b in SARS-CoV from bats contain major variations compared to SARS-CoV from humans (23).

Different strains of MERS-CoV obtained from camels were found to be similar to those isolated from humans (14, 25, 26) except for variations among the S, ORF4b, and ORF3 genomic regions (26). Furthermore, genome sequencing-based studies have revealed that MERS-CoV strains from humans are phylogenetically related to those from bats. The strains have identical genomic and protein structures except for the S proteins (27). In addition, recombination analysis of genes encoding orf1ab and S revealed that MERS-CoV originated from the exchange of genetic elements between coronaviruses in camels and bats (26, 28).

Although the zoonotic source of SARS-CoV-2 is not confirmed, its genome sequence exhibits close relatedness (88% identity) with two bat-derived SARS-like coronaviruses (bat-SL-CoVZC45 and bat-SL-CoVZXC21). Phylogenetic analysis reveals that SARS-CoV-2 is genetically distinct from SARS-CoV and MERS-CoV. However, homology modeling reveals that both SARS-CoV and SARS-CoV-2 have similar receptor-binding domain structures, despite amino acid variation at some key residues, including the absence of the 8a protein and the fluctuation in the number of amino acids in the 8b and 3c proteins in SARS-CoV2 (29). In contrast, the primary protease is highly conserved between SARS-CoV-2 and SARS-CoV, with a 96% overall identity (30). These observations suggest that bats are the source of origin, while an animal sold at the Wuhan seafood market might represent an intermediate host facilitating the emergence of the virus in humans (12, 31).

EPIDEMIOLOGY AND CLINICAL FEATURES OF HUMAN CORONAVIRUSES

After the emergence of SARS-CoV in the Guangdong province of China, it rapidly spread around the globe (2, 3). During November 2002, an epidemic of pneumonia with a high rate of transmission to other people occurred in Guangdong, China (32), followed by subsequent outbreaks in Hong Kong. In Hong Kong, a total of 138 people contracted the infection within 2 weeks after the exposure to an infected patient in the general ward of a hospital (1, 32). Overall, SARS-CoV infected 8,098 people and caused 774 fatalities in 29 different countries by the end of the epidemic (1). Later, during June 2012, a patient infected by MERS-CoV developed severe pneumonia and died in Jeddah, Saudi Arabia (1, 33). Analysis of cluster of nosocomial cases in Jordan during April 2012 confirmed that MERS-CoV caused the outbreak (34). The spread of MERS-CoV continued beyond the Middle East, causing further reports of infected individuals (1, 4). Until 2020, 2,468 cases and 851 fatalities had been reported globally (35, 36). During December 2019, clusters of patients with atypical pneumonia were reported by local health facilities in Wuhan, China. On December 31, 2019, a rapid response team was dispatched by the Chinese Center for Disease Control and Prevention (China CDC) to conduct an epidemiologic and etiologic investigation (37). The patients were found epidemiologically linked to the wet animal wholesale seafood market in Wuhan, China. Later, the infectious agent responsible for this atypical pneumonia was confirmed and reported as coronavirus SARS-CoV-2, which caused the first fatality in early January 2020 (15). During the first 6 weeks of the outbreak, several cases were reported in more than 37 countries, including the USA, Japan, Iran, and South Korea (38). The infection rapidly spread across the globe from Wuhan, China. Therefore, the Chinese authorities implemented several strategies, including massive lockdown in Wuhan and suspension of transport to and from Wuhan to control the spread (17). According to Situation Report 35, published on the WHO website, SARS-CoV-2 caused 79,331 confirmed cases and 2,618 deaths around the globe. However, COVID-19 caused 77,262 confirmed cases and 2,595 deaths inside mainland China alone (38). Through February 24, WHO had reported 8 deaths in Iran. It is now the second country after China, bearing the highest fatalities due to SARS-CoV-2 infection. (38). The spread of SARS-CoV-2 in Iran poses a higher risk of pandemics in the Middle East and South Asian countries. Between December 10 and January 4, analysis of the growth rate of the epidemic gave a basic reproductive number (R0) of 2.2, meaning each patient has been spreading the infection to 2.2 other individuals (39). The estimated R0 value for SARS was around 3, however, SARS was successfully controlled by isolation of patients (39). Moreover, the R0 for MERS ranged from 0.45 in Saudi Arabia to 8.1 in South Korea (36). Considering the lower R0 value, the rapid increase in suspected as well confirmed cases of COVID-19 may be inferred via viral transmission through the fecal-oral route and aerosol formation. Moreover, asymptomatic persons are thought to be potential sources of SARS-CoV-2 infection (40), which may have caused the rapid spread of SARS-CoV-2. This asymptomatic spread may be one reason that the control strategy based on the isolation of patients has not been fully successful. To overcome these problems, a complete quarantine is necessary, allowing all of the infected individuals to develop symptoms without spreading the virus randomly. Thus, the direct and indirect contacts of infected individuals can be easily identified and isolated.

Clinical features associated with patients infected with SARS-CoV, MERS-CoV, and SARS-CoV-2 range from mild respiratory illness to severe acute respiratory disease (1, 17). Both MERS and SARS patients in later stages develop respiratory distress and renal failure (1, 17). Pneumonia appears to be the most frequent manifestation of SARS-CoV-2 infection, characterized primarily by fever, cough, dyspnea, and bilateral infiltrates on chest imaging (17). The period from infection to appearance of symptoms varies. Generally, it is thought to be 14 days, however, a research group at Guangzhou Medical University reported the incubation period to be 24 days. In a family cluster of infections, the onset of fever and respiratory symptoms occurred approximately 3 to 6 days after presumptive exposure (41).

DIAGNOSTIC TESTING

Diagnostic testing for the SARS-CoV-2 is primarily done in public health laboratories. Delays in testing result from the need for administrative oversight of testing at the national or regional level, as well as the time needed to transport specimens and the high volume of testing needed in some regions. More rapid testing should be widely available in the event of epidemics. High-level testing facilities at regional hospitals and commercial laboratories are needed, in addition to the commercially available tests that have undergone regulatory approval. Several tests have been validated by public health authorities, including those in China, Germany, Thailand, Japan, and the United States (WHO, COVID-19, technical guidance, Feb 12, 2020). These tests are reverse transcriptase PCR (RT-PCR) tests that use primers and probes designed to detect a variety of targets in the SARS-CoV-2 genome. Although these have been designed and validated, there is currently very limited information available related to the performance of these tests. The sensitivity and specificity of the tests are not widely known, and some of them might detect other related coronaviruses, such as SARS-CoV. In addition, the utility of different specimen types for detection of the viruses is not known. As a result, testing of multiple specimen types is recommended by some agencies, including the CDC (42). The availability of serological tests is unclear and, presumably, such tests are in development. Moreover, the collection and submission of sera from potentially infected patients is recommended by some public health laboratories.

The CDC and WHO have both issued recommendations for laboratory safety when testing specimens from patients suspected of being infected with SARS-CoV-2 (42, 43). Both guidelines recommend that manipulation of potentially infectious specimens should be done in a biosafety cabinet if there is potential for splashes or generation of droplets or aerosols. Viral isolation (culture) should be done only in BL-3 laboratories. Testing in chemistry and hematology laboratories can be done following routine laboratory precautions recommended for such work.

THERAPEUTIC OPTIONS FOR HUMAN CORONAVIRUSES

Currently there are no promising antiviral treatments available, however, numerous compounds have been proven effective against SARS-CoV and MERS-CoV but have not been tested widely for the newly emerged SARS-CoV-2. Remdesivir and chloroquine were found highly effective in vitro for the control of 2019-nCoV infection (44). Treatment with remdesivir alone or in combination with chloroquine or interferon beta was found effective against COVID-19 infection. This strategy has not caused any obvious side effects yet (35, 44, 45). However, more investigations are necessary to confirm the impacts of remdesivir. As coronaviruses share key genomic elements, common therapeutic targets are likely to be of greater importance. Therapeutic agents targeting nucleosides, nucleotides, viral nucleic acids, and enzymes/proteins involved in the replication and transcription of coronaviruses can be promising strategies to treat coronavirus diseases (Table 1) (1). The surface spike glycoprotein (S) is an important potential target for antiviral agents, due to its vital role in the interaction between the virus and the cell receptor. S consists of two subunits: S1, the amino-terminal receptor binding subunit, and S2, the carboxy-terminal membrane fusion subunit (46). In addition, activation of membrane fusion and virus entry requires the cleavage at the junction of S1-S2 (46). Hence, S1 subunit-targeting monoclonal antibodies and S2 subunit-targeting fusion inhibitors may be effective therapeutic agents for coronaviruses (1). Furin (a serine endoprotease) cleaves off S1/S2 (47) could thus be a suitable antiviral agent. Further, the helical nucleocapsid interacts with S protein, envelope proteins, and membrane proteins to form the assembled virion (1). Therefore, targeting the structural genes using small interfering RNAs could be an effective therapeutic strategy against coronaviruses (1). The host receptors are also associated with the viral entry into host cells, thus agents targeting these receptors also inhibit coronaviruses (46). Inhibitors of endosomal cysteine protease and transmembrane protease serine 2 can partially block viral entry into the cell (48). K22 targets membrane-bound RNA synthesis in coronaviruses and inhibits double-membrane vesicle formation (49), and thus could be effective against SARS-CoV-2.

TABLE 1

Therapeutic options for COVID-19

Therapeutic name	Activity	Effectiveness	Reference
K22	Targets membrane-bound replication complexes of virus in host cell to inhibit RNA synthesis	Effective against SARS and MERS, thus could be effective against SARS-CoV-2.	(58)
DRACO	Targets viral dsRNA to induce apoptosis in cells containing virus	Effective against a large group of viruses, therefore may have potential to target SARS-CoV-2.	(51, 59)
Mycophenolic acid	Targets nucleosides and/or nucleotides to inhibit synthesis of guanine monophosphate	Effective against a wide range of viruses, however combinatorial therapy with interferon beta1b may be useful for SARS-CoV-2.	(56)
Lopinavir	Targets 3CLpro enzyme to inhibit its activity	Effective against a wide range of viruses, including SARS-CoV and MERS-CoV, thus could be a suitable choice for treatment of SARS-CoV-2 infection.	(60)
Remdesivir	Terminates transcription of viral RNA transcription at premature level	Effective against a broad spectrum of viruses including MERS-CoV and SARS-CoV. The efficacy is very promising when combined with IFNb, hence could be a suitable therapeutic strategy for SARS-CoV-2.	(35)
Ribavirin	Targets RdRp (RNA‑dependent RNA polymerase) enzyme to inhibit synthesis of viral RNA synthesis and capping of mRNA	Effective against a wide range of viruses, including MERS-CoV and SARS-CoV, but the high doses required may have severe side effects. Could be reevaluated for SARS-CoV-2 and recommended if low doses are found effective.	(35, 60)
Bcx4430	Targets RdRp (RNA‑dependent RNA polymerase) enzyme to inhibit synthesis of viral RNA synthesis and capping of mRNA	Broad spectrum and effective against SARS-CoV and MERS-CoV, thus may be effective against SARS-CoV-2, however, evaluation using animal models is required.	(61)
Bananins	Target helicase to inhibit unwinding and activity of ATPase	Have effects against broad-spectrum viruses and can be evaluated for SARS-CoV-2.	(1)
Aryl diketoacids (Adks)	Target helicase to inhibit its unwinding	Effective for a broad range viruses, including SARS/MERS-CoV, and may be a suitable choice for SARS-CoV-2.	(1)
Griffithsin	Targets oligosaccharides on S to block viral binding with host cell	Has effects against SARS/MERS-CoV and other high pathogenic viruses, thus might be used against SARS-CoV-2.	(62)
Hexamethylene amiloride	Targets viral envelope to inhibit ion channel activity	Has effects against different coronaviruses, thus one of the most suitable treatment options for SARS-CoV-2.	(63)
Jl103	Targets lipid membrane and causes modification of phospholipids	Has effects against different viruses and may be promising as an anti-SARS-CoV-2 agent.	(1, 60)
Recombinant interferons	Induce the innate interferon responses against viral pathogens	Induce immune responses through recombinant interferons, found effective against a wide range of viruses and can be the most suitable option for SARS-CoV-2.	(1, 60)
Nitazoxanide	Induces the innate interferon responses against viral pathogens	Induces immune responses through recombinant interferons, has been found effective against a wide range of viruses and may be promising to use against SARS-CoV-2.	(64)
Cyclosporine, alisporivir	Inhibit cyclophilin to affect calcineurin–NFAT pathway	Inhibit broad-spectrum viruses, specifically coronaviruses, and thus could be suitable option to treat SARS-CoV-2.	(1)
Rapamycin	Inhibits kinase signaling associated pathways to block viral entry	Effective against SARS/MERS-CoV and might possibly be effective against SARS-CoV-2.	(1, 65)
Imatinib	Inhibits kinase signaling associated pathways to block viral entry	Effective against SARS/MERS-CoV and might possibly be effective against SARS-CoV-2.	(1)
Dasatinib	Inhibits kinase signaling associated pathways to block viral entry	Effective against SARS/MERS-CoV and might possibly be effective against SARS-CoV-2.	(1)

Broad-spectrum antivirals, such as dsRNA-activated caspase oligomerizer (DRACO), which selectively induces apoptosis in virus-containing host cells, provide another class to evaluate for effectiveness against SARS-CoV-2 (50). However, this class does not provide a promising strategy alone, as they cannot block virus entry or disrupt viral nucleic acid. In contrast, thiopurine compounds, naphthalene inhibitors, protease inhibitors, zinc, and mercury conjugates target 3CLpro (3C-like protease) and PLpro (papain-like protease) enzymes in coronaviruses and have been shown to block their pathogenicity (51, 52). Therefore, combinational therapy of these antiviral agents with DRACO may enhance the overall impact on the recovery of patients. Despite the higher rate of infectiousness, coronaviruses are thought to have the ability to suppress counteracting responses from host innate interferons. This response can be augmented by the utilization of interferon inducers or recombinant interferons (1). Recombinant interferons such as interferon alpha and interferon beta that have been tested against SARS-CoV (1) might be utilized either alone or in combination with other potential antiviral drugs, including remdesivir. Both interferon alpha and beta inhibit viral replication (1). The use of interferon inducers in combination with effective antiviral agents is worth evaluating for possible synergistic effects against SARS-CoV-2. In addition, calcineurin inhibitors such as cyclosporine (53), in combination with antibiotics and traditional Chinese medicines, could also be evaluated for activity against SARS-CoV-2.

VACCINES

Effective vaccines are important to prevent and control sporadic viral attacks and emerging virus-mediated epidemics, such as the recent outbreak caused by SARS-CoV-2. Although SARS-CoV was fully controlled during 2003, and MERS-CoV has been prevented from causing high mortality rates, the newly emerged SARS-CoV-2 is spreading efficiently with a significant increase in the number of cases and fatalities during each passing day. Vaccines are required to prevent SARS-CoV-2 from causing COVID-19. Live-attenuated vaccines, such as has been designed for SARS (1), may be evaluated for SARS-CoV-2 infected patients. In addition rhesus θ-defensin 1 and protein cage nanoparticles are innate immunomodulators with high anti-SARS-CoV efficiency (54, 55). Based on the high similarity and phylogenetic relatedness between SARS-CoV and SARS-CoV-2, protein cage nanoparticles designed for SARS-CoV can be evaluated for SARS-CoV-2. Meanwhile, following similar strategies utilized for SARS-CoV, novel protein cage nanoparticles specified for novel coronavirus can be designed on an emergency basis. Given the urgency in the current scenario of COVID-19 outbreak, vaccination strategies based on viral vectors, recombinant protein, and viral-like particles, which have been developed or are being developed for SARS and/or MERS, could be modified for utilization against SARS-CoV-2 (56). Despite the current progress, further work is needed to develop safe and effective vaccines, available for individuals at high risk of SARS-CoV-2 endemics, to control the ongoing and risk of future epidemics. Further, an interesting feature of plasma from recovered patients is the presence of active antibodies. The transfer of plasma from people who have recovered from COVID-19 into infected individuals might enhance immunity against SARS-CoV-2. Monoclonal antibodies that could inhibit virus-cell receptor binding and interrupt virus-cell fusion have been developed (1). Combining two or more monoclonal antibodies may be suitable for the quicker recovery of patients. Lastly, antiviral peptides that target different regions of S, such as HP2P-M2 peptide (effective against viral infections) (1), should also be considered against COVID-19.

Although some strategies against SARS-CoV are being developed, including receptor-binding domain (RBD)-based vaccines, they require further evaluation (2). Given the importance of the current outbreak in Wuhan, further studies are needed to deepen understanding of replication, pathogenesis, and biological properties using reverse genetics and related molecular techniques. These investigations will help the control and prevention of SARS-CoV-2-mediated pneumonia disease and novel emerging diseases in the future.

FUTURE PERSPECTIVES

SARS-CoV and MERS-CoV were reported to originate from recombination within bat viruses before their introduction into Guangdong province through civets and the Middle East through camels, respectively. Some of the SARS-CoV strains became epidemic after several independent spillovers to humans during the outbreak of 2002 in Guangdong, China (3). Similarly, MERS-CoV became epidemic after a series of human infections in 2012 in Middle Eastern countries (33). Both viruses transferred to several countries beyond the countries of origin. However, unlike the continuous propagation of the MERS-CoV epidemic, the SARS-CoV outbreak was successfully controlled in 2003. Based on the origin of other coronaviruses, SARS-CoV-2 is likely to have originated in bats and have been introduced to Wuhan, China through an unknown intermediate. Until now, no effective clinical treatments or prevention strategies are available to be used against human coronaviruses.

Testing drugs against coronaviruses requires suitable animal models prior to their use in humans. The currently established models are not very promising for the studies of pathogenesis and treatment of highly pathogenic coronaviruses. For instance, nonhuman primates were unable to reproduce the characteristics of the severe human disease and mortality was not observed (57). However, some small animals have developed clinical symptoms of disease (57), such as transgenic mice expressing human ACE2, and mouse-adapted SARS-CoV strains are one of the most suitable models (1). Additionally, transgenic mice expressing human DPP4 are a suitable small animal model for MERS (1). Like the animal models for SARS-CoV, transgenic animal models may also be standardized for SARS-CoV-2. The development of clinical drugs for coronaviruses is challenging because of the repeated emergence of novel coronaviruses with diverse features, thus requiring specific drugs for each newly emerged virus. Moreover, only a limited number of animal models are available and most of them can only be used in highly demanding biosafety level 3 labs (1). From the perspective of the current outbreak, designing effective therapeutics for SARS-CoV-2 is yet another challenge for scientists. Although a large number of antiviral treatment options for SARS and MERS have been reported with potent in vitro activities, a very limited number of these may have potential in the clinical setting.

Moving forward there are treatment options available that could be utilized clinically during the ongoing SARS-CoV-2 epidemic. Some of the broad-spectrum antiviral drugs may be effective for SARS-CoV-2, and there is opportunity to test them in the current scenario of pneumonia in Wuhan, China. Broad-range combinational therapies, including lopinavir and interferon antiviral peptides, can also be evaluated and examined, as these agents have shown significant effects against MERS in nonhuman primates (1). The design and development of novel broad-spectrum antiviral drugs that can potentially target all coronaviruses may prove to be the best treatment option against reemerging and circulating coronaviruses.