1 Introduction
The world witnessed the devastating outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, resulting in up to 8000 cases and 700 deaths worldwide [
1]. SARS-CoV is a novel zoonotic pathogen, causing typical fever and respiratory symptoms. No specific antiviral drugs and vaccines were available for SARS-CoV. Fortunately, SARS-CoV remained in the population for only 8 months and vanished without a trace. In 2012, a new interspecies coronavirus outbreak occurred in Saudi Arabia, and this infection has spread to 26 countries across the globe, infecting 1698 patients, and resulting in 609 deaths as of March 23, 2016 [
2]. The virus was initially designated HCoV-EMC [
3]. All individuals diagnosed with Middle East respiratory syndrome (MERS) have been linked directly or indirectly to one of four countries in the Middle East. Therefore, the virus was renamed Middle East respiratory syndrome coronavirus (MERS-CoV). Compared with SARS, the novel coronavirus emerged with a more vaguely defined epidemiology, more severe symptoms, higher case fatality rate, absence of prophylactic or therapeutic measures, and most importantly, circulation in humans with mixed features of both epidemic and sporadic nature [
4]. The largest outbreak of MERS-CoV outside of Saudi Arabia occurred in South Korea with 185 confirmed cases and 36 deaths [
5], including the fourth-generation descendants of MERS cases, which emerged sporadically but later as an epidemic. In this review, we summarize the recent findings in the field of MERS-CoV, especially its molecular virology, interspecies mechanism, clinical features, and antiviral therapies, as well as our speculations.
2 Virology
MERS-CoV belongs to lineage C of the genus
Betacoronavirus in the family
Coronaviridae under the order
Nidovirales. SARS-CoV belongs to
Betacoronavirus lineage B. Similar to SARS-CoV, MERS-CoV is a positive-sense, enveloped, single-stranded RNA virus, which is spherical with a diameter of approximately 125 nm; MERS-CoV also represents one of the largest identified RNA genomes, with up to 30 kilobases (kb) [
6]. MERS-CoV is the sixth CoV known to cause human infection and is closely related to bat coronaviruses HKU4 and HKU5, compared with SARS [
7]. The genome of MERS-CoV contains a 5′ cap structure along with a 3′ poly (A) tail and comprises four main structural proteins, namely, spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins; all these proteins are encoded within the 3′ end of the viral genome. The viral genome is organized in the following order: 5′UTR-ORF1a/1b-S-E-M-N-3′UTR-poly (A) tail. The 5′ cap structure and 3′ poly (A) tail play a vital role in the replication and transcription of the viral genome. The ORF1a/1b occupies 2/3 of the genome and encodes a series of non-structural proteins. Most of these proteins are involved in the formation of the viral replication/transcription complex. The S glycoprotein mediates receptor recognition and membrane fusion and is also the focus of most immunization strategies against MERS-CoV. The S glycoprotein is cleaved into two subunits: S1 and S2. The S1 subunit contains the receptor-binding domain (RBD), which mediates viral attachment to its host receptor. When S1 binds to its receptor, the S2 subunit changes its conformation and partially inserts into the target cell, drawing viral and host cell surfaces closer to facilitate plasma membrane fusion. This conformational change requires the formation of a fusion core by two heptad repeats (HR1 and HR2) of the S2 subunit. Interference with either HR1 or HR2 blocks the viral entry. The three other structural proteins relate to virion assembly and release. MERS-CoV contains five accessory proteins, namely, 3, 4a, 4b, 5, and 8b (Fig. 1) [
8], which present no homology with other coronaviruses. The accessory protein 4a exhibits a potent antagonistic activity against interferon response through both cytoplasmic and nuclear targets. The functions of the other accessory proteins have yet to be clarified.
3 Animal origin
Accumulating evidence suggests that the dromedary camel is the animal reservoir of MERS-CoV. Serologic studies have shown the presence of cross-reactive antibodies to MERS-CoV in dromedary camels in Oman, Qatar, and Mongolia [
9–
11]. Live MERS-CoV was isolated directly from infected camels and confirmed in camel-to-human transmission. However, the exact mode of transmission remains largely under investigation. The effects of raw meat consumption, intake of camel milk, or exposure to other infections remain unclear. To date, studies have not found any regularity in MERS-CoV transmission from the existing cases. A comparative analysis indicated that the virus isolated from camels shows more genetic variation, with few genomic variants not infectious to humans [
12]; this finding partially explained the lower infection rate in humans than in camels. Therefore, camels serve as an important reservoir for the maintenance and diversification of MERS-CoVs.
The existence of an intermediate host between camels and humans has yet to be established. During the SARS epidemic, virus transmission from bats to humans was intermediate via civet (
Paguma larvata) and spread by raccoon. We speculated whether MERS-CoV was transmitted similarly, given the evidence that MERS-CoV replicated in cell lines of other animals, including goat, pig, rabbit, horse, and civet [
13–
15]. However, no evidence of other intermediate hosts was found mediating MERS transmission from camels to humans.
The origin of MERS-CoV infection in camel remains unclear. The transmission from bats is supported by the following evidence: (1) MERS-CoV is phylogenetically and closely related to
Tylonycteris bat CoV HKU4 (Ty-BatCoV-HKU4) and
Pipistrellus bat CoV HKU5 (Pi- BatCoV-HKU5), which were discovered in
Tylonycteris pachypus and
Pipistrellus abramus, respectively, in Hong Kong in 2006, revealing the MERS-CoV genomic origins in bats [
16]; (2) the MERS-CoV receptor is also the receptor for HKU4 but not HKU5, hence showing functional identity [
17]; and (3) viral gene fragments identical or quite similar to those of MERS-CoV have also been recovered in bats. However, an infectious virus was not isolated directly from bats [
18]. If MERS-CoV originated directly from bats, the spread from the bats to humans cannot be explained. The exact mode of transmission, geographical distribution, and origins cannot also be explained.
4 Cross-species transmission mechanism
The initial entry of the virus into human cells is mediated by specific receptors. Scientists have identified dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for MERS-CoV, whereas SARS enters the target cells via angiotensin converting enzyme 2 (ACE2). DPP4 is a membrane-bound peptidase with a type II topology and forms homodimers on the cell surface. DPP4 contributes critically to our understanding of the pathogenesis and epidemiology of this newly emerging human coronavirus and may facilitate the development of antiviral therapies and vaccines [
19]. Patients with chronic obstructive pulmonary disease and cystic fibrosis demonstrate increased DPP4 immunostaining in alveolar epithelia and alveolar macrophages. This finding suggests that a preexisting pulmonary disease increases MERS-CoV receptor abundance and predisposes individuals to MERS morbidity and mortality, which is consistent with current clinical observations [
20].
When MERS-CoV enters the body, it will impair the innate immune response of cells, including the RIG-I-like receptor signaling and MDA-5, which are associated with the expression of interferon α. Additionally, type I and II major histocompatibility (MHC) genes are affected, thereby decreasing the expression levels of the major cytokines involved in the activation of lymphocytes [
21–
23].
The host-viral interaction suggests that the interspecies transmission of MERS-CoV might be associated with the following two elements for efficient infection and replication: (1) viral adsorption capacity on the surface of human cells, particularly respiratory epithelial cells; and (2) inhibition of innate immunity of subsequent viral entry into the human cells and attenuation of the activation of adaptive immune response to take over the host metabolic apparatus and replicate efficiently.
5 Clinical presentation
The severity and outcome of MERS are related to gender, older age, and underlying diseases. Majority of the MERS patients are elderly males with median age of 56 years. A study involving 47 patients showed an increase in case-fatality rates with age, from 39% (seven of 18) in those younger than 50 years, to 48% (13 of 27) in the group aged under 60 years, and 75% (15 of 20) in patients aged 60 years or older [
24]. A study involving 70 consecutive patients found the age of 65 years associated with increased mortality (OR 4.39, 95% CI 2.13‒9.05;
P<0.001) [
25]. Most patients present underlying co-morbid medical disorders, such as diabetes and hypertension [
26].
Patients with MERS present with symptoms of influenza-like illness (ILI). Clinically, MERS and SARS show few similar features, including fever, cough (predominantly dry), and even renal failure. The majority of MERS patients are reported to suffer from multiple co-morbid conditions. However, few MERS patients show no fever or cough at all and present with walking pneumonia at early stages, which can progress rapidly. A quarter to a third of all the patients shows digestive tract symptoms. More than half of the patients develop acute renal impairment at a median time of around 11 days after symptom onset, with most cases requiring renal replacement therapy [
24,
27,
28–
31]. However, about 6.6% of patients with SARS show acute renal failure occurring at 20 days after the onset of symptoms, and only 5% need replacement therapy [
24]. The possible factors contributing to the common presentation of acute renal failure in MERS patients include (1) increased number of chronic renal diseases in MERS patients, with progressive respiratory failure; and (2) direct renal injury; DPP4 is present in the renal cells, and MERS-CoV was detected in urine.
Without a travel history, linking the patient to the Arabian Peninsula, or a known MERS case, few clinical and radiological features can reliably differentiate MERS from acute pneumonia caused by other microbial agents. Asymptomatic cases have been reported among female healthcare workers and children [
28]. MERS-CoV incidence in children is less frequent and seems to be associated with less mortality in patients without underlying comorbidities [
32]. Low mortality in children with MERS-CoV was also reported in SARS-CoV infections, in which symptoms were milder, without any mortality, and with few hospitalizations [
33].
MERS-CoV shedding was further clarified during the new epidemic in Korea, at the hospital from the first week up to 18 days to 25 days after the onset of symptoms. The viable virus can shed through respiratory secretion from clinically fully recovered patients [
34]. The SARS “viral load” in upper respiratory tract secretions was low in the first 5 days of illness, and then increased progressively, peaking early in the second week [
35]. These results emphasize the need for sufficient isolation based on laboratory results rather than solely on clinical symptoms in patients with coronavirus infection.
6 Antiviral therapy
To date, no approved antiviral therapy is available for MERS-CoV, indicating a much slower response to this potential pandemic than to the avian H7N9 flu. Candidate antiviral agents are identified using three general approaches. First, broad-spectrum antiviral drugs, such as interferon, ribavirin, and cyclophilin inhibitors, which were effective in SARS patients, were determined to test the activity to MERS-CoV [
36–
38].
In vitro studies suggest that the type I interferon exerted an antiviral effect against several cell lines. MERS-CoV was 50 times to 100 times more sensitive to interferon α (IFN α) treatment than SARS-CoV [
36,
37].
In vitro studies also showed significant antiviral effects of ribavirin and interferon α-2b combination therapy [
37]. In a retrospective study, ribavirin and interferon α-2a therapy were significantly associated with improved survival at 14 days but not at 28 days [
38]. The clinical significance of cyclosporine for MERS treatment is likely limited, as the drug’s peak serum level at clinical dosages was below its EC50 for MERS-CoV [
36].
The second approach to identify candidate antivirals targeting MERS involves screening of chemical libraries comprising numerous existing drugs. The advantages of this approach include the commercial availability, known pharmacokinetics, and established safety reports. The first drug identified by this approach was mycophenolic acid, which is an antirejection drug used in transplantation and a broad-spectrum antiviral drug. The EC50 of this drug against MERS-CoV was very low. Therefore, a low dosage may be effective without inducing significant immunosuppression, but this assumption warrants further preclinical evaluation in animal studies [
39]. The other drugs, including lopinavir and chloroquine, still remain in the
in vitro stage.
The third approach to antiviral drug developments is based on the knowledge of genome and structural biology of MERS-CoV. Compared with the drugs identified by the first and second approaches, those identified by this approach were most effective against MERS-CoV. However, this novel drug development needs time and investment. The therapeutic potential of antibodies targeting coronaviruses was well recognized during the SARS outbreak [
40–
42]. To date, monoclonal antibodies targeting different epitopes on the RBD in the S1 subunit of the MERS-CoV S protein have been identified. These monoclonal antibodies bind to RBD with 10-fold to 450-fold higher affinity than the RBD binding affinity to the human DPP4, conferring broader and higher neutralizing activity [
43–
46]. One of the antibodies, m336, neutralizes the virus with exceptional potency and therefore represents a potential drug and vaccine candidate [
47]. HR2P, a synthetic peptide derived from the HR2 domain of MERS-CoV spike protein, specifically binds to the HR1 domain of the viral spike protein and blocks viral fusion, resulting in the inhibition of MERS-CoV replication and its spike protein-mediated cell fusion [
48]. Nevertheless, a major obstacle relates to the difficulty in generating highly potent neutralizing mAbs in a relatively short time during an epidemic. The other challenge relates to the emergence of possible escape mutants.
7 Conclusions
The outbreak of MERS-CoV poses a serious threat to global public health and highlights the imperative for further investigation into the viral epidemiology and pathogenesis, as well as the development of effective therapeutic and prophylactic agents against MERS-CoV infection. First, well-designed large-scale case-control studies are needed to define the transmission chain of MERS-CoV and to enable appropriate intervention by the government. Second, retrospective serum antibody detection should be continued to define its true distribution in humans and animals. Third, systematic surveillance for signs of host adaptation and viral genome variations is very important. Fourth, a randomized control study should be conducted to evaluate the effects of the available antiviral drugs. Finally, animal vaccines limit the animal-to-human transmission [
49], which will alter our approach to drug development against newly emerging infectious diseases. However, with regard to clinical trials, we should note that a traditional sequence of studies in animals followed by phased clinical trials may be very slow during public health emergencies. A common protocol should be launched to rapidly evaluate the promising but unproven therapies in the current fatal emerging infectious disease outbreaks and future epidemics [
50].
Higher Education Press and Springer-Verlag Berlin Heidelberg
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