# Emine Tuğçe AYSOY (MERS-COV VIRUS)

# MERS-COV VIRUS

Given the diversity of animal coronaviruses, it was not unexpected that another human coronavirus was isolated from a patient presenting with severe respiratory illness in June 2012. The 60-year-old man died of kidney and respiratory failure 11 days after his admission to the hospital. He was in a hospital in Jeddah, Saudi Arabia. The new etiologic agent was later named Middle East Respiratory Syndrome coronavirus (MERS-CoV). MERS-CoV is one of six known human coronaviruses to cause respiratory disease in humans with a mortality rate greater than 35% and the first highly pathogenic human coronavirus to emerge since the global fear caused by severe acute respiratory syndrome. This review focuses on current knowledge of MERS-CoV with special reference to genome structure, clinical features, diagnosis and treatment of infection, and vaccine development.

# 1. Genome Structure and Gene Functions

MERS-CoV, a strain C Betacoronavirus (βCoVs), has a positive sense single-stranded RNA (ssRNA) genome of approximately 30 kb in size. MERS-CoV genomes share more than 99% sequence identity indicating low mutation rate and low variance between genomes. MERS-CoV genomes are roughly divided into two parts: the A wing, which contains only a few strains, and the B wing, to which most strains belong. As with other CoV genomes, two-thirds of the first 50 MERS-CoV genomes consist of the replicase complex (ORF1a and ORF1b). The remaining 30 thirds encode the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N), as well as five accessory proteins (ORF3, ORF4a, ORF4b, ORF5 and ORF8b). not required for genome replication but likely involved in pathogenesis. The flanking regions of the genome include 50 and 30. MERS-CoV helper proteins, typical of coronaviruses, do not share homology with any known host or virus proteins except closely related ones.

*Figure 1: Schematic organization of human coronavirus (α and β CoVs) genomes. HCoVs genomes are 26 kb to 32 kb in size. At the 50
-end, overlapping reading frames 1a and 1b (blue) make up two-thirds of the genome. The remaining one third of the genome (expanded region) encodes for the structural (white) and accessory proteins (grey).*

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Mers-CoV structural and accessory protein-coding plasmids transiently transfected into cells showed that, while ORF4b was localised mostly in the nucleus, all of the other proteins (S, E, M, N, ORF3, ORF4a and ORF5) localised to the cytoplasm. Furthermore, studies with MERS-CoV deletion-mutants of ORFs 3 to 5 are attenuated for replication in human airway-derived (Calu-3) cells, and deletion-mutants of ORFs 4a and 4b are attenuated for replication in hepatic carcinoma-derived (Huh-7) cells. This clearly points to important putative roles for the MERS-CoV accessory proteins in viral replication, at least in an in vitro setting. The principal response of mammalian cells to viral infection is the activation of the type I interferon (IFN)-mediated innate immune response through the production of type I IFNs (IFN-α and IFN-β).
On the other hand, evasion of host innate immunity through IFN antagonism is a critical component of viral pathogenesis and is mediated by virus-encoded IFN antagonist proteins.

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Each protein blocks one or more of the key signaling proteins in the IFN and NF-κB pathways to enhance viral replication. Coronaviruses have similarly evolved these mechanisms to inhibit or bypass their hosts' innate immunity at various levels, which ultimately contributes to coronavirus virulence. Various coronavirus proteins have previously been implicated in the disruption of signal transduction events essential for the IFN response, often by interfering with the host's induction of type I interferon. Evidence that MERS-CoV induces type I IFN only weakly and late in infection suggests that MERS-CoV also evolves mechanisms to evade the host immune system. In fact, MERS-CoV M, ORF4a, ORF4b and ORF5 proteins are reported to be potent IFN antagonists.Further studies, using the transient overexpression of MERS-CoV accessory protein ORF4a, ORF4b, and ORF5, show that the MERS-CoV accessory proteins inhibit both type I IFN induction and NF-kappaB signaling pathways. MERS-CoV ORF4a, a double-stranded RNA (dsRNA) binding protein, potentially acts as an antagonist of the antiviral activity of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and the ISRE promoter element signaling pathways. MERS-CoV ORF4b, on the other hand, is an enzyme in the 2H-phosphoesterase (2H-PE) family with phosphodiesterase (PDE) activity. Even though MERS-CoV ORF4b is detected primarily in the nucleus of both infected and transfected cells, the expression levels of cytoplasmic MERS-CoV ORF4b are still sufficient to inhibit activation of RNase L, an interferon-induced potent antiviral activity. MERS-CoV ORF4b is the first identified RNase L antagonist expressed by a human or bat coronavirus and provides a possible MERS-CoV mechanism for evasion of innate immunity by inhibiting the type I IFN and NF-kappaβ signaling pathways.
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**Figure 2:** Molecular structure of MERS-CoV. MERS-CoV

# 2. Clinical Features

The median age of persons with laboratory-confirmed MERS-CoV infection is 49 years (range,<1–94 years); 65% of patients are males.

#### *( Source: https://www.ekachaihospital.com/en/mers-cov/)*

Co-infection of the respiratory tract with at least two viruses is common in hospitalized patients. Similar to other respiratory viruses, MERS-CoV has been found in combination with a second respiratory virus such as influenza A virus respiratory syncytial virus, human parainfluenza virus 3, or human metapneumovirus. MERS-CoV-infected patients requiring mechanical ventilation also exhibited a similar coinfection profile with hospital-acquired bacterial infections, including Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter spp. As yet, little is known about how MERS-CoV damages the airway and disrupts lung barrier function, which favors adhesion and invasion of other pathogens into normally sterile areas within the airway. The first cases of severe neurological syndrome characterized by varying degrees of impaired consciousness, ataxia, focal motor deficits and bilateral hyper-intense lesions were reported from a retrospective study of patients in the intensive care unit.

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Interestingly, MERS-CoV cases have been reported mainly in adults, with children rarely affected. Even so, a recent case study of a MERS-CoV infected a 9-month-old child, newly diagnosed to have infantile nephrotic syndrome, showed complications that resulted in severe respiratory symptoms, multi-organ dysfunction and death. In another study of 11 pediatric cases that tested positive for MERS-CoV, the two symptomatic patients had Down’s syndrome and
cystic fibrosis, respectively, indicating that severe disease could potentially occur in children with serious underlying conditions. Even with these reported pediatric cases, data on infection in children remain scarce, making it difficult to ascertain whether MERS-CoV is really a predominantly adult disease, or whether it often presents differently in children.
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Another subsequent small retrospective study in Saudi Arabia reported that 25.7% of MERS patients developed confusion and 8.6% experienced some form of seizure. To date, other cases of central nervous system involvement have been extensively associated, including four cases involving thrombocytopenia, disseminated intravascular coagulation and platelet dysfunction, a critical illness polyneuropathy, and intracerebral hemorrhage as a result of Bickerstaff encephalitis overlapping with Guillain-Barre syndrome. There have been reports of care unit-derived weakness or other Toxic or infectious neuropathies. The virus has also been detected in feces, serum and urine. Virus shedding peaks about 10 days after the onset of symptoms, but up to 25 days from clinically fully recovered patients, viable viruses can be shed via respiratory secretions. In the healthcare setting, MERS-CoV has been isolated from environmental objects such as bed linens, mattress protectors, IV fluid slings, and X-ray equipment. Another study reported that MERS-CoV can survive for more than two days at 20 ◦C and 40% RH, confirming the risk of contact or fomite transmission in healthcare settings. .
# 3.Diagnosis of Infection

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Since there is no specific, safe antiviral drug or vaccine approved for clinical use in MERS-CoV infections, rapid diagnostic testing is required to manage outbreaks of this virus. Laboratory detection and confirmation of MERS-CoV infections broadly includes molecular detection of MERS-CoV RNA; MERS-CoV antigen detection; or tests to identify a humoral response to prior MERS-CoV infection among humans.
Currently, a positive real-time RT-PCR test targeting at least two different genomic regions is used to confirm MERS-CoV infection, according to the WHO case definition. Of the different assay probes and primer sets used, those targeting ORF1a and upstream of the E gene show the highest sensitivity. In addition, a single positive test result confirmed by gene sequencing can also be considered positive for MERS-CoV infection. Molecular tests can detect nucleic acids derived from MERS-CoV in clinical respiratory, serum, and stool samples. The most appropriate tests here would be those that detect viral antigens or antibodies in the infected host.

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| | Method Used for | 1 Sensitivity/2 Specificity/3 Viral Target Gene |
| --- | --------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| | rtRT-PCR | 1 Sensitivity for upE is 3.4 copies per reaction or 291 copies/mL of sample. 2 No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. 3 Targeting regions upstream of the E gene (upE) or within open reading frame (ORF) 1b, respectively. |
| | qRT-PCR | 1 Sensitivity to widely used upE gene as well as a ORF1a&b was introduced. 2 No false-positive amplifications were obtained with other human coronaviruses or common respiratory viral pathogens or with 336 diverse clinical specimens from non-MERS-CoV cases; specimens from two confirmed MERS-CoV cases were positive with all assay signatures. 3 Two novel signatures used one that targets the MERS-CoV N gene in combination with the up E test. The other a positive test to add to an efficient MERS-CoV kit.|
| |RT-Sequence | Validated-LAMP Assays 1 Could detect 0.02 to 0.2 plaque forming units of MERS-CoV in infected cell culture supernatants. 2 Did not cross-react with common human respiratory pathogens.|
| | RT-LAMP | 1 Capable of detecting as few as 3.4 copies of MERS-CoV RNA; Assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR. 2 No cross-reaction to other respiratory viruses. 3 Assay designed to amplify the MERS-CoV gene. |
| | rt-RPA | 1 Highly sensitive, is able to detect 10 MERS-CoV RNA copies with a more rapid detection time than MERS-RT-PCR. 2 No cross-reaction to other respiratory viruses including HCoVs. 3 Assay designed to amplify the partial nucleocapsid gene of MERS-CoV. |
| | mAb Test | 1 Rapid detection and cost effective ELISA. 2 High specificity used to detect the MERS-CoV nucleocapsid protein. |
| | Immunochromotagraphic tool | 1 Highly sensitive, 2 No cross reactivity with other respiratory pathogens observed in vitro and in silico.3 Detects recombinant MERS-CoV N protein. |
| | Immunofluorescence Assay | 1 Highly sensitive, antigen based detection. 2 Cross reactivity seen with convalescent SARS patient (sera) 3 Assay used both whole virus and S1 portion of the spike protein.|
| | ppNT Assay | 1 Highly sensitive, more sensitive that MNT test. 2 Lack of MERS neutralizing activity indicated high specificity by this assay. No cross reactivity seen with SARS-CoV. 3 Assay was designed for two different genes used: a codon optimized spike gene and a HIV/MERS pseudoparticle was generated. |
| | MNT Test | 1 Highly sensitive; less so than ppNT assay. 2 Highly specific, as SARS-CoV antigen was not detected compared to MERS-CoV. 3 Test designed to detect IgG antibodies generated when using the RBD of the S1 subunit of the spike gene. |
| |Protein Microarray | 1 Highly sensitive assay using protein n microarray technology to detect IgG and IgM antibodies. 2 No cross reactivity seen with sera of patients that had been exposed to four common HCoVs. 3 Assay designed to use the S1 receptor-binding subunit of the spike protein of MERS and SARS as antigens.|
| |One pot RT-LAMP | 1 Capable of detecting four viral copies MERS within 60 min. 2 No cross-reaction to the other acute respiratory disease viruses (influenza type A virus (H1N1 and H3N2), influenza type B virus, HCoV-229E, and human metapneumovirus). 3 Six sets of primers designed specifically to amplify the MERS-CoV genes. |
| | RT-iiPCR assays | 1 Could detect 3.7 × 10−1 plaque forming units (PFU) of MERS-CoV in infected cell culture supernatants and sputum samples.2 Viral nucleic acids extracted from infected cultures that contained HCoV-229E, HCoV-OC43, FIPV, influenza type A and B virus strains yielded negative results, indicating no cross reactivity. 3 Targeting regions upstream of the E gene (upE). |
| |Powerchek MERS Assay | 1 95% limits of detection of assay for the upE and ORF1a were 16.2 copies/µL and 8.2 copies/µL, respectively. 2 No cross reactivity with other respiratory pathogens observed in vitro and in silico. 3 Targeting regions upstream. |
| | acpcPNA-AgNP aggregation assay | 1 Probe designed for targets makes this assay highly specific. Limit of detection found to be 1.53 nM. 2 Cross reactivity with other CoVs was not evaluated. 3 Synthetic oligonucleotides were designed to target MERS. |
| | mCoV-MS | 1 Highly sensitive, multiplex PCR based to target specific genes in HcoVs. 2 Cross reactivity with other respiratory pathogens was not evaluated. 3 Targeting regions upstream of the E gene (upE). |
| | Duplex-RT-PCR method | 1 Highly sensitive, simultaneous detection of MERS and SARS viruses.2 Cross reactivity with other respiratory pathogens was not evaluated. 3 Primers and probes that target the conserved spike S2 region of SARS-CoV, MERS-CoV, and their related bat CoVs were used. |
**Table 1: Detection methods of MERS-CoV.**

# 4. Animal Models
More recently, four transgenic mouse models for MERS-CoV infection have been developed. In the first, a modified adenovirus expressing human DPP4 (huDPP4) is administered intranasally to mice, resulting in huDPP4 expression in all cells of the lung, not just those that naturally express DPP4. In this model, mice show transient human DPP4 expression and mild lung disease. A concern with this model is that constitutively DPP4-expressing cells will become infected, and the role of a broader infection of all cell types may alter pathogenesis. In the second model, a transgenic mouse systemically expressing huDPP4 was generated. In this model, MERS-CoV infection induces high levels of viral RNA and inflammation in the lungs, but unfortunately, significant inflammation and viral RNA are also detected in the brains of infected mice, which represents a non-physiological expression pattern. In the third model, a new transgenic humanized mouse model was generated by replacing the mouse DPP4 coding sequence with the sequence encoding huDPP4, ensuring the correct physiological expression of huDPP4. Mice in this model show lung pathology consistent with radiographic signs of interstitial pneumonia and significant lung disease as seen in humans infected with MERS-CoV. This suggests that this mouse model recapitulates the pathological sequelae seen in MERS-CoV infection in humans. Importantly, unlike that seen in other mouse models of MERS-CoV infection, virus replication and pathology in huDPP4 mice is localized in the lungs and inflammation does not develop in the brain, providing a more physiologically accurate model of human disease. Finally, in 2016 Cockrell et al. established a mouse model that allows MERS-CoV infection but has functional DPP4 immune function. Infecting this DDP4-chimeric mouse with a mouse-adapted MERS-CoV strain mimics MERS-CoV-induced respiratory disease without bystander neurological disease.

*(Source: https://www.sciencedirect.com/science/article/pii/S0042682216303002)*
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# Conclusion
MERS-CoV is a zoonotic disease with bats and dromedary camels playing important parts in its emergence and epidemiology. Camel to human MERS-CoV transmission is well documented but is generally not very efficient. The exact mechanism of transmission is not clear,
including whether other intermediate hosts are involved. Serosurveys in humans across Africa are urgently needed to investigate the possibility of unrecognized MERS-CoV infections in the continent. Furthermore, bats in Eastern Africa should be screened for betacoronaviruses that may provide better understanding of the genetic history of MERS-CoV. Finally, case-control studies of humans with sporadic MERS-CoV infection are urgently needed to identify risk factors and exposures that might explain the chains of transmission from camels and other possible zoonotic or environmental sources of human infections.
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