From SARS to MERS, Thrusting Coronaviruses into the Spotlight

1. Introduction

Before the first outbreak of severe acute respiratory syndrome (SARS), a limited number of coronaviruses were known to be circulating in humans, causing only mild illnesses, such as the common cold [1]. Following the 2003 SARS pandemic [2,3], it became apparent that coronaviruses could cross the species barrier and cause life-threatening infections in humans. Therefore, further attention needs to be paid to these new coronaviruses.

The 21st century has seen the worldwide spread of two previously unrecognized coronaviruses, the severe acute respiratory syndrome coronavirus (SARS-CoV) [4] and Middle East respiratory syndrome coronavirus (MERS-CoV), both of which are highly pathogenic. Starting from November 2002 in China [5], there have been unprecedented nosocomial transmissions from person to person of SARS-CoV, accompanied by high fatality rates. A united global effort led to the rapid identification of the SARS coronavirus and remarkable scientific advancements in epidemic prevention. Additionally, the zoonotic transmission of SARS from December 2003 to January 2004 [6] provided insight for researchers into the origin of this novel coronavirus. Notably, the SARS pandemic was declared to be over in 2004 when no more infections in patients were being detected. Subsequently, certain SARS-CoV-like viruses found in bats demonstrated the ability to infect human cells without prior adaptation [7,8] which indicates the possibility of the re-emergence of SARS-CoV or SARS-CoV-like viruses.

A decade later in June 2012, another highly pathogenic and novel coronavirus, MERS-CoV, was isolated from the sputum of a male patient who died from acute pneumonia and renal failure in Saudi Arabia [9]. Nosocomial infections were reported, and international travel led to the transmission of MERS-CoV to countries outside of the Arabian Peninsula, causing it to become a global pathophoresis. In May 2015, an outbreak of MERS occurred in South Korea due to an individual returning from the Middle East [10]. Based on the lessons learned from managing SARS-CoV prevalence over the last decade, tremendous progress toward unraveling the biological characteristics of MERS-CoV has been achieved at an unprecedented speed. Scientific advancements have allowed for rapid and systemic progress in our understanding of the epidemiology and pathogenesis of MERS-CoV.

SARS-CoV and MERS-CoV share several important common features that contribute to nosocomial transmission, preferential viral replication in the lower respiratory tract, and viral immunopathology. This review highlights the epidemiology and pathogenesis of these viruses, including our current understanding of their biological characteristics, their transmission, and their replication in the host. The spike proteins (S proteins) of CoVs play pivotal roles in viral infection and pathogenesis. As critical surface-located trimeric glycoproteins of CoVs, they guide entry into host cells. In this review, we summarize the structure and function of the S proteins and therapeutics designed to target them. Moreover, we will explore how CoV–host interactions cause pathogenic outcomes and discuss potential treatment options, as well as describe recent mammalian models that closely recapitulate the pathogenic process and have contributed to the development of prevention and treatment strategies for SARS-CoV and MERS-CoV. Although several potential therapies have been identified with SARS and MERS in animal and in vitro models, human clinical trials remain lacking, hindering the advancement of these potential countermeasures.

2. Epidemiology of SARS-CoV and MERS-CoV

Prior to the outbreaks of SARS and MERS [2,9], the clinical importance and epidemic possibility of CoVs had been recognized by researchers, (Table 1). In 2002, a SARS epidemic that originated in Guangdong Province in China resulted in 916 deaths among more than 8098 patients in 29 countries [11], identifying SARS as the first new infectious disease of the 21st century. Ten years later, the World Health Organization (WHO) published 2254 laboratory-confirmed cases of MERS-CoV that occurred from 2012 to 16 September 2018, with at least 800 deaths in 27 countries. Remarkably, more than 80% of recent research into the virology and genetics of this infection indicated that bats could be the possible natural reservoirs of both SARS and MERS-CoV. Palm civets [12] and dromedary camels [13] are also possible intermediary hosts of SARS and MERS, respectively, before dissemination to humans [14].

The transmission mechanism of SARS-CoV and MERS-CoV has yet to be fully understood. For transmission from animals to humans, direct contact with the intermediary host might be one route. Recent reports demonstrated that camel workers in Saudi Arabia with high prevalence of MERS-CoV infection may contribute to the transmission of MERS [15]. Some customs and habits may also be conducive to transmission, such as the consumption of milk, urine, or uncooked meat. In this way, MERS-CoV was transmitted from dromedary camels directly to humans, principally in the Arabian Peninsula, and this is considered to be the main route of transmission from animals to humans, causing significant morbidity and mortality [16,17]. Human-to-human spread has also been detected, especially through nosocomial transmission. Delays in diagnosis in hospitals might lead to secondary cases among healthcare workers, family members, or other patients sharing rooms [18,19,20,21,22]. Among the reported cases of SARS, 22% were healthcare workers in China and more than 40% were healthcare workers in Canada [23]. Nosocomial transmission for MERS has similarly been seen in the Middle East [16] and in the Republic of Korea [22]. Outbreaks in other countries all resulted from the reported cases in the Middle East or North Africa, and transmission was the result of international travel. Both SARS and MERS caused large outbreaks with significant public health and economic consequences.

3. Pathogenesis of SARS-CoV and MERS-CoV

Although our current understanding of the pathogenesis of the SARS-CoV and MERS-CoV infection remains unclear, we summarize what is presently known (Table 2).

Coronaviruses are the largest kind of positive-strand RNA viruses (26–32 kb) as they are about 125 nm in diameter [24], and comprise four genera (alpha-, beta-, gamma-, and delta-coronavirus) [25]. Currently, six human CoVs (HCoVs) have been confirmed: HCoV-NL63 and HCoV-229E, which belong to the alpha-coronavirus genus; and HCoV-OC43, HCoV-HKU1, SARS-CoV, and MERS-CoV, which belong to the beta-coronavirus genus. SARS-CoV and MERS-CoV are the two major causes of severe pneumonia in humans and share some common coronavirus structural characteristics. Similarly, their genomic organization is typical of coronaviruses, having an enveloped, single, positive-stranded RNA genome that encodes four major viral structural proteins, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins 3–5, that follow the characteristic gene order [5′-replicase (rep gene), spike (S), envelope (E), membrane (M), nucleocapsid (N)-3′] with short untranslated regions at both termini (Figure 1). The viral membrane contains S, E, and M proteins, and the spike protein plays a vital functional role in viral entry. The rep gene encodes the non-structural protein and constitutes approximately two-thirds of the genome at the 5′ end. In detail, the S protein is in charge of receptor-binding and subsequent viral entry into host cells, and is therefore a major therapeutic target [26,27]. The M and E proteins play important roles in viral assembly, and the N protein is necessary for RNA synthesis.

The SARS-CoV genome has 29,727 nucleotides in length, including 11 open reading frames (ORFs). The SARS-CoV rep gene, containing about two-thirds of the genome, encodes at least two polyproteins (encoded by ORF1a and ORF1b) that undergo the process of cotranslational proteolysis. Between ORF1b and S of group 2 and some group 3 coronaviruses, there is a gene that encodes hemagglutinin-esterase [4], while this was not detected in SARS-CoV. This virus is significantly different from previously reported coronaviruses for many reasons, such as the short anchor of the S protein, the specific number and location of small ORFs, and the presence of only one copy of PLP^pro.

The MERS-CoV genome is larger than that of SARS-CoV at 30,119 nucleotides in length, and comprises a 5′ terminal cap structure, along with a poly (A) tail at the 3′ end, as well as the rep gene containing 16 non-structural proteins (nsp1–16) at the 5′ end of the genome. Four structural proteins (S, E, M, and N) and five accessory proteins (ORF3, ORF4a, ORF4b, ORF5, and ORF8) constitute about 10 kb at the 3′ end of the genome. Unlike some other beta-coronaviruses, the MERS-CoV genome does not encode a hemagglutinin-esterase (HE) protein [1]. Genomic analysis of MERS-CoV implies the potential for genetic recombination during a MERS-CoV outbreak [9]. MERS-CoV and SARS-CoV possess five and eight accessory proteins, respectively, which might help the virus evade the immune system by being harmful to the innate immune response. These differences might lead to greater sensitivity to the effects of induction and signaling of type 1 interferons (IFNs) in MERS-CoV than SARS-CoV.

4. Comparative Pathology and Life Cycles of SARS-CoV and MERS-CoV

Both SARS and MERS cause severe pneumonia resulting from these novel coronaviruses, sharing some similarities in their pathogenesis (Figure 2) [28].

SARS is an emerging infectious viral disease characterized by severe clinical manifestations of the lower respiratory tract, resulting in diffuse alveolar damage. SARS-CoV spreads through respiratory secretions, such as droplets, via direct person-to-person contact. Upon exposure of the host to the virus, the virus binds to cells expressing the virus receptors, of which the angiotensin-converting enzyme 2 (ACE2) is one of the main receptors, and CD209L is an alternative receptor with a much lower affinity [29]. In the respiratory tract, ACE2 is widely expressed on the epithelial cells of alveoli, trachea, bronchi, bronchial serous glands [30], and alveolar monocytes and macrophages [31]. The virus enters and replicates in these target cells. The mature virions are then released from primary cells and infect new target cells [32]. Furthermore, as a surface molecule, ACE2 is also diffusely localized on the endothelial cells of arteries and veins, the mucosal cells of the intestines, tubular epithelial cells of the kidneys, epithelial cells of the renal tubules, and cerebral neurons and immune cells, providing a variety of susceptible cells to SARS-CoV [33,34]. Respiratory secretions, urine, stools, and sweat from patients with SARS contain infective viral particles, which may be excreted into and contaminate the environment. Atypical pneumonia with rapid respiratory deterioration and failure can be induced by SARS-CoV infection because of increased levels of activated proinflammatory chemokines and cytokines [35].

For MERS-CoV infection of humans, the primary receptor is a multifunctional cell surface protein, dipeptidyl peptidase 4 (DPP4, also known as CD26) [36], which is widely expressed on epithelial cells in the kidney, alveoli, small intestine, liver, and prostate, and on activated leukocytes [37]. Consistent with this, MERS-CoV can infect several human cell lines, including lower respiratory, kidney, intestinal, and liver cells, as well as histiocytes, as shown by a cell-line susceptibility study [38], indicating that the range of MERS-CoV tissue tropism in vitro was broader than that of any other CoV. MERS-CoV causes acute, highly lethal pneumonia and renal dysfunction with various clinical symptoms, including—but not restricted to—fever, cough, sore throat, myalgia, chest pain, diarrhea, vomiting, and abdominal pain [39,40]. Lung infection in the MERS animal model demonstrated infiltration of neutrophils and macrophages and alveolar edema [41]. The entry receptor (DPP4) for MERS-CoV is also highly expressed in the kidney, causing renal dysfunctions by either hypoxic damage or direct infection of the epithelia [42]. Remarkably, unlike SARS-CoV, MERS-CoV has the ability to infect human dendritic cells [43] and macrophages [44] in vitro, thus helping the virus to disrupt the immune system. T cells are another target for MERS-CoV because of their high amounts of CD26 [45]. This virus might deregulate antiviral T-cell responses due to the stimulation of T-cell apoptosis [45,46]. MERS-CoV might also lead to immune dysregulation [47] by stimulating attenuated innate immune responses, with delayed proinflammatory cytokine induction in vitro and in vivo [44,48,49].

5. SARS and MERS-CoV Spike Protein: A Key Target for Antivirals

6. The Animal Models of SARS and MERS-CoV

International coordination and cooperation led to the rapid identification of SARS-CoV and MERS-CoV. Emergency control measures and laboratory detection systems which were put in place in response to SARS-CoV and MERS-CoV outbreaks were both exemplary. To establish optimal prevention and control strategies for SARS and MERS, numerous efforts to develop animal models were undertaken in several laboratories, despite the fact that some conflicting results have been reported. It is therefore necessary to compare and document the features and disadvantages of different animal models to better understand viral replication, transmission, pathogenesis, prevention, and treatment. Notably, several animal species were suggested as suitable disease models of SARS-CoV, but most laboratory animals are refractory or only semi-permissive to MERS-CoV infection.

7. Role of Host Receptors in Animal Models of SARS-CoV and MERS-CoV

The reasons for host restriction, none or limited clinical symptoms observed in varies animal models are complexity. The interaction between the host receptor and functional proteins of SARS and MERS, respectively, plays an important and predominant role. In the context of animal models of SARS-CoV infection, researchers have compared the ACE2 amino acids that interact with the S protein RBD from several species. In agreement with the permissive nature of these species, the ACE2 residues of marmoset and hamster are similar to those of hACE2 [53]. By comparison, many residues of mouse ACE2 are different from those of hACE2, and this meets with decreased replication of SARS-CoV in mouse cells [207] and the lungs of young mice [149]. The changes at positions 353 (histidine) and 82 (asparagine) of rat ACE2 relative to hACE2 partially disrupt the S protein-DPP4 interaction and contribute to abrogation of binding. Interestingly, ferrets are permissive to SARS-CoV infection, but most of their ACE2 interaction residues are different from those of hACE2 [53], while many of the ACE2 residues between civet and ferret are the same, which may result in similar affinity [208]. For MERS-CoV, 14 residues of the S protein RBD have direct contact with 15 residues of hDPP4 [57]. Comparisons of human DPP4 binding affinity to that of other species indicated that human DPP4 had the highest affinity to the S protein of MERS-CoV, where the decreasing order of affinity is as follows: human > horses > camels > goats > bats [209]. Further evidence demonstrates that the host restriction of MERS-CoV remarkably depends on the sequence of DPP4, such as the characterization of amino acid residues at the connector of DPP4 with the RBD of S proteins in mice [187,210], hamsters, and cotton rats [210]. However, the multiplicity in severity of disease between rhesus macaques and common marmosets indicate that other host factors can perhaps affect the infection and replication of the virus, such as the presence of S-cleaving proteases [187]. In general, although the structural analysis of receptors-S protein interactions cannot fully explain all the observations for host restriction, they agree with the improved replication in several animal models and that it should be the premier and remarkable focus of small-animal model development. These special residues for host affinity are important to build up transgenic animal models enhancing the permissiveness and infection of SARS and MERS.

8. Outlook and Summary

Unlike SARS-CoV, which resolved without more reported cases, continued outbreaks of MERS-CoV present an ongoing threat to public health. It should be noted that no specific treatment is currently available for HCoVs, and further research into the pathogenesis of HCoV infection is therefore imperative to identify appropriate therapeutic targets. Accordingly, at present, the prevention of viral transmission is of utmost importance to limit the spread of MERS. The enormous ratio of nosocomial infections indicates that preventive measures in hospitals have not been sufficiently implemented. Additionally, as an emerging zoonotic virus, prevention of transmission from dromedary camels is another possibility to reduce the quantity of MERS cases. Regarding clinical therapies, a combination of treatment administered as early as possible and aimed at synchronously disrupting viral replication, inhibiting viral dissemination, and restraining the host response is likely to be most suitable, due to the acute clinical features of MERS with diffuse lung damage and the important role of immunopathology.

Potential treatments must undergo in vitro and in vivo studies to select the most promising options. The development of stable and reproducible animal models of MERS, especially in NHPs, is therefore a decisive step forward. The next step in the development of standardized and controllable therapies against SARS and MERS will be clinical trials in humans, validating a standard protocol for dosage and timing, and accruing data in real time during future outbreaks to monitor specific adverse effects and help inform treatment.

The comprehensive lessons and experiences that have resulted from the outbreaks of SARS and MERS provide valuable insight and advancements in how to react to future emerging and re-emerging infectious agents. Rapid identification of the pathogen via effective diagnostic assays is the first step, followed by the implementation of preventive measures, including raising awareness of the new agent, reporting and recording (suspected) cases, and infection control management in medical facilities. Studies are currently needed that focus on the epidemiology of these organisms, especially in terms of pathogen transmission and potential reservoirs and/or intermediate hosts. Animal models and prophylactic and therapeutic approaches should be promoted, followed by fast-tracked clinical trials.

Our increasing understanding of novel emerging coronaviruses will be accompanied by increasing opportunities for the reasonable design of therapeutics. Importantly, understanding this basic information will not only aid our public health preparedness against SARS-CoV and MERS-CoV, but also help prepare for novel coronaviruses that may emerge.

Funding

This research was funded by CAMS Innovation Fund for Medical Science (CIFMS), grant number 2016-12M-2-006.

Conflicts of Interest

The authors declare no conflict of interest.