DOI : 10.9780/2321-3485/1322013/74

Reviews Of Progress ISSN:-2321-3485

ORIGINAL ARTICLE

th

Vol - 3, Issue - 1, May 4 2015

Middle East Respiratory Syndrome-Corona Virus (MERS-CoV): An Overview Sagar Aryal1, Pratiksha Pokhrel Pahadi2, Bikash Rana and Rana Kausar 1

Department of Microbiology, St. Xavier’s College, Kathmandu, Nepal Department of Microbiology, St. Xavier’s College, Kathmandu, Nepal 3 Department of Microbiology, St. Xavier’s College, Kathmandu, Nepal 4 Department of Biochemistry, FUUAST, Karachi, Pakistan

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Abstract: Middle East Respiratory Syndrome-Corona Virus (MERS-CoV) is an emerging virus which causes respiratory infections in animals and humans caused by novel corona virus (CoV). There is presence of Spike Glycoprotein in the virus which gets attached with DPP4 of the target cell during replication. Fever, cough, shortness of breath and gastrointestinal symptoms including diarrhea are some of the major symptoms of this infection having the incubation period of 5.2 days. The first case was seen on 2012 in Saudi Arabia where the patient died of acute respiratory and renal failure. As of 14 October 2014, 896 laboratory-confirmed cases of MERS-CoV have been confirmed including 357 deaths as of 14 October 2014. Transmission can occur from animal to human or from human to human. rRT-PCR is the best diagnostic method for the detection of virus. There is no current treatment or vaccination available for MERS-CoV. KEYWORDS: MERS-CoV, Review, Infections, Outbreaks, Transmission INTRODUCTION: Middle East Respiratory Syndrome (MERS) is the viral respiratory illness which is caused by a corona virus called Middle East Respiratory Syndrome-Corona Virus (MERS-CoV) 6. MERS-CoV was used to be called Novel Corona Virus 1. It was termed by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses which was announced in the “Journal of Virology” on 15 May 2013 2. MERS-CoV is a beta coronavirus belonging to the Coronaviridae, a group of large, enveloped single stranded RNA viruses 1. Infection is primarily zoonotic in nature but can be spread from human to human too 2. According to WHO, as of 11 June 2014, there were 699 laboratory confirmed cases of human infections, including at least 209 deaths 11. According to European Centre for Disease Prevention and Control, as of 14 October 2014, there were 896 laboratory-confirmed cases of MERS-CoV and including 357 deaths 16. Structure of MERS-CoV They measure about 100 to 160 nm. MERS-CoV contains spike glycoprotein (S) which targets the cellular receptor, dipeptidly peptidase 4 (DPP4) which is also known as CD26 5. Glycoprotein also shows an antigenic action. It has neucleocapsid phosphoprotein for RNA-binding and membrane glycoprotein for triple membrane spanning 7. MERS-CoV consists of a core and a receptor binding sub domain in where it

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interacts with DPP4 3,6. Spike glycoprotein consists of a globular SI domain at the N-terminal region, followed by membrane proximal S2 domain, an intracellular domain and also a receptor-binding domain (RBD) 4. MERS-CoV RBD consists of a core subdomain and receptor binding subdomain 3. a. Core Subdomain The core subdomain is a five stranded antiparallel beta (B) sheets (B1, B2, B3, B4 and B9) with two short alpha helicase in the connecting loops. There is presence of 3 disulfide bonds in the core subdomain to maintain the fold 3. b. Receptor Binding Subdomain It is a four stranded antiparallel B sheets (B5, B6, B7 and B8) located between B4 and B9 of the core subdomain. B7 and B9 is connected via a long loop that crosses perpendicular to the B sheet. There is presence of one disulfide bond which connects the long loop with strand B5 3. This supports for the interaction with DPP4.

Figure 1: Structure of MERS-CoV [14] Genome MERS-CoV genomes are phylogenetically categorized into 2 clades, clade A and B clusters. Full genome deep sequencing can be done on nucleic acid extracted straight from PCR-confirmed clinical samples 8. Genome of MERS-CoV is an unsegmented single stranded positive sense RNA having the size of 29.9 kb. Genomes are capped and polyadenylated at 3’ end. Sequence analysis of the MERS-CoV genome identified the emerging virus as being a member of the lineage C of the Betacoronaviridae, with the closest relatives known are bat coronaviruses. RNA are structurally polycistronic but functionally monocistronic. The genome of MERS-CoV encodes both structural proteins as well as non-structural proteins. The 3’ one-third of the genome encodes fours structural proteins – Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) along with the sets of accessory proteins which are required for the formation of viral particles 9. The Polymerase gene, 5’ two third of the RNA genome encodes a large polyprotein (ORF 1a/1b) whose expression is controlled by ribosomal frame shifting 9. It is proteolytically cleaved to generate 15 or 16 nonstructural proteins (nsp’s) which is required for the viral replication as well as the modulation of the antiviral reaction.

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Figure 2: MERS-CoV genomes

Figure 3: A schematic of the complete genome of the first fully sequenced MERS-CoV variant in lineage C of the betacoronaviruses, HCoV 2c EMC/2012.1 [14] Replication The entire cycle of virus replication occurs in the cytoplasm. It involves various steps as follows: 1.Attachment The glycoprotein spike (S) present in the envelope of the virus attached to the DDP4 receptor on the target cell. At the interface a total of 14 residues of the MERS-CoV contact with the 15 residues of the DPP4 with the distance cut off of 3.6A 3. 2. Entry of the genome The hydrophobic core formed between MERS-CoV RBD and DPP4 plays a critical role in mediating the viral binding and entry into the target cell 38. Virus gets into the host cell by receptor mediated endocytosis or by the fusion of viral envelope with the cell membrane. 3. Uncoating and Release After the penetration of the virus particle, uncoating of the genome takes place and released into the cytoplasm. 4. Protein and Genomic RNA Formation MERS-CoV has a single positive stranded RNA genome, which can directly produce their proteins and new genome on the cytoplasm. At first, the viral genomic RNA translocates to produce virusspecific RNA-dependent RNA polymerase. This recognizes and produces viral RNAs. RNA polymerase synthesize the minus (-ve) strand using the positive strand as template. Then this –ve strand RNA serves as template to transcribe full length genomic RNA and sub genomic mRNAs, which are translated into a single polypeptide 15. 5. Nucleocapsid Complex Formation The newly synthesized genomic RNA interacts in the cytoplasm with the Nucleocapsid (N) protein to form the Helical Nucleocapsid. Membrane (M) protein is inserted into the ER and anchored in the Golgi apparatus. Nucleocapsid binds to the M protein at the budding site into the ER lumen. Envelope (E) and Membrane (M) proteins interacts to trigger the budding of virions, enclosing the Nucleocapsid.

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6. Assembly and Maturation Spike Glycoprotein (S) associate with the M protein and are incorporated into the maturing virus particles and virus get fully matured now. 7. Release These viral progeny are finally transported by Golgi vesicles to the cell membrane and released by exocytosis like fusion of vesicles with plasma membrane.

Figure 4: Replication of MERS-CoV [13] Pathogenesis Symptoms of MERS-CoV infections include fever, cough, shortness of breath and gastrointestinal symptoms including diarrhoea 29. Severe illness can cause respiratory failure that requires mechanical ventilation and support in an intensive-care unit. Some patients develop organ failure, especially of the kidneys, or septic shock. Approximately 27% of patients with MERS have died. The virus seems to cause more severe disease in people with weakened immune systems, older peoples, and those with such chronic diseases like diabetes, cancer, and chronic lung disease 31. The average incubation period was found to be 5.2 days 29. In the cell line susceptibility study, MERS-CoV infected human cell lines, with lower respiratory, kidney, intestinal, and liver cells and histiocytes 40. MERS-CoV prevent the secretion of interferon (IFN)-α and IFN-ß and persuade the expression of pro-inflammatory tumor necrosis factor (TNF)-a and Interleukin-6, and inducing the inflammation of surrounding tissue. MERS-CoV infects and replicate in human monocyte–derived macrophages (MDM) and the induction of cytokines in these cells contribute to pathogenesis. Furthermore, in MDM, MERSCoV proliferates the expression of MHC-class I molecule and co-stimulatory leading to an activation of immune responses 36. In the case of MERS-CoV, in vivo target cells contain type II alveolar cells and non-ciliated cells epithelial cells where MERS-CoV is able to infect endothelial cells as well. DPP 4 has many other roles besides being the receptor, in glucose homeostasis, T-cell activation, neurotransmitter purpose, and modulation of cardiac signaling, but the enzymatic function of DPP4 is not essential for viral entry 37,38. When there is entry of MERS-CoV into the host cell, type II transmembrane protease TMPRSS2 activates the spike (S) protein by transforming the mature S protein into two subunits (S1 and S2) and increasing the fusogenicity with the receptor of host cell 32, 35. The existence of both the receptors for MERS-CoV and S cleaving protease determine the potential animal reservoir and the sources of recurring transmission to humans from animals. Since the MERS-CoV receptors in human, horse and camel are similar, screening

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should be done in both camels and horses. The person infected with MERS-CoV doesn’t show only respiratory infections but also acute renal failure 33, 34 . Infection and replication of kidneys with MERS-CoV might therefore not only lead to acute renal failure but also to shedding and transmission of MERS-CoV in urine which leads to new cases not only via airborne transmission but also under favorable conditions via contaminated drinking water 34. Epidemiology As the name suggests, it is mainly distributed in Middle East countries which includes Iran, Jordan, Kuwait, Oman, Qatar, Saudi Arabia, United Arab Emirates and Yemen. It is distributed in Africa and Europe too. This includes Algeria, Egypt and Tunisia in Africa whereas France, Germany, Greece, Italy, the Netherlands and the United Kingdom (UK) in Europe. In Asia, Malaysia and Philippines are affected with MERS-CoV 11,16. It is seen in USA too. There were 896 laboratory-confirmed cases of MERS-CoV reported to the public health authorities worldwide, which includes 357 deaths, as of 14 October 2014 16. MERS-CoV not only affects the human but also infects animals like Camel where the viruses has been isolated from Egypt 17, Saudi Arabia 18,19,22,24, Qatar 21,23, Jordan 25, Oman 26, UAE 27 and Africa 20. Dromedaries Camel is thought to be the possible viral reservoirs 27. Transmission The virus is transmitted by respiratory route in close contact. Transmission to human has been thought to be transmitted from Camels due to high similarities of MERS-CoV carried by human and camels 18 . Airborne nosocomial transmission can occur in the room shared by the patients in the hospitals. There is still the confusion of transmission through body fluids or clinical samples, including stools and a cross transmission with medical devices or hands. The previous studies did not tell about the source of virus, nor did they reveal whether MERS-CoV can be transmitted from human to human or not 28. Later it was found that the virus transmission can probably occur in dialysis units, medical wards and ICUs 29. Outbreaks MERS-CoV was first identified in cell culture taken from the patients who died of pneumonia in Saudi Arabia in 2012. After that, 896 laboratory-confirmed cases of MERS-CoV have been reported, including 357 deaths as of 14 October 2014 16. Very Large number of cases and death has been reported in Saudi Arabia. Table 1 shows the total number of confirmed cases and deaths, by country of reporting, from March 2012 to 13 October 2014 16. Figure 5 and table 1 show the distribution of confirmed cases of MERSCoV reported from March 2012 to 13 October 2014. Many of the cases have been seen in the Middle East Countries like Saudi Arabia, United Arab Emirates, Qatar, Jordan, Oman, Kuwait, Egypt, Yemen, Lebanon and Iran. The latest confirmed case of MERS-CoV infection was seen on a 43-year-old male from Doha, Qatar on 20 October, 2014 30. There were no cases of MERS-CoV infection during Hajj in 2012 or 2013 50.

Figure 5: Distribution of confirmed cases of MERS-CoV reported March 2012–14 October 2014, by reporting country (n=896)

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Table 1: Number of confirmed cases and deaths, by country of reporting, March 2012–13 October 2014 [16] S.No

Country

Cases

Death

Date of most Recent Cases

1

Saudi Arabia

762

324

13/10/2014

2

United Arab Emirates

73

9

11/06/2014

3

Jordan

18

5

23/05/2014

4

Qatar

8

4

12/10/2014

5

United Kingdom

4

3

06/02/2013

6

Iran

5

2

25/06/2014

7

Oman

2

2

20/12/2013

8

Tunisia

3

1

01/05/2013

9

Kuwait

3

1

07/11/2013

10

Germany

2

1

08/03/2013

11

France

2

1

08/05/2013

12

Algeria

2

1

24/05/2014

13

Yemen

1

1

17/03/2014

14

Greece

1

1

08/04/2014

15

Malaysia

1

1

08/04/2014

16

Netherlands

2

0

05/05/2014

17

USA

2

0

01/05/2014

18

Egypt

1

0

22/04/2014

19

Lebanon

1

0

22/04/2012

20

Austria

1

0

29/09/2014

21

Italy

1

0

31/05/2013

22

Philippines

1

0

11/04/2014

23

Total

896

357

Laboratory Diagnosis rRT-PCR can be done from lower respiratory tract specimens such as sputum, endotracheal aspirate, or Broncho-alveolar lavage fluid. Acute and convalescent sera should be taken for serologic testing. If only a single sample is to be obtained, it should be collected at least 14 days after onset of symptoms. A stool sample or rectal swab can also be collected. If a negative result is obtained from a patient with high risk group and suspicion, additional specimens can be taken and examined 41. Specimens should be transported to the laboratory soon after collection. When there is a delay of more than 48 hours for respiratory tract specimens, specimens should be frozen, preferably at -80°C, and transported on dry ice. Serum should be separated from whole blood and then stored and transported at 4°C or frozen to -20°C. Temperature fluctuation should be avoided for the storage of respiratory and serum specimens 42. 1. Cell Culture MERS-CoV can be recovered from Vero and LLC-MK2 cells 1. MERS-CoV strain can also be

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replicated in fully separated human airway bronchial epithelium (HAE) cultures grown at the air-liquid interface (ALI) 54. 2. Polymerase chain reaction and sequencing In a patient with multiple myeloma and MERS-CoV infection high concentrations of MERS-CoV needs to be detected from respiratory specimens by rRT-PCR 45. MERS-CoV was also be detected from nasal secretions, stool, urine and stool, but in low concentrations 46. There are three rRT-PCR assays for routine detection of MERS-CoV 41. Currently described tests are an assay targeting a region upstream of the E protein gene (upE) and assays targeting the open reading frame 1b (ORF 1b) and the open reading frame 1a (ORF 1a) 43,44. In some cases, sequencing should be performed for confirmation. Hemi-nested sequencing amplicons targeting RdRp (present in all corona viruses) and N gene (specific to MERS-CoV) fragments can be generated for confirmation via sequencing. 3. Serology Different serology test have been developed for the detection of MERS-CoV antibodies, including immunofluorescence assays and a protein microarray assay. CDC has developed a two-stage approach, which uses an enzyme-linked immunosorbent assay (ELISA) for screening followed by an indirect immunofluorescence test or microneutralization test for confirmation. Any positive test by a single serologic assay should be confirmed by a neutralization assay. Sensitivity and specificity of antibody tests for MERS-CoV has not been confirmed. According to the WHO, cases with a positive serologic test in the absence of PCR testing or sequencing are considered probable cases if they meet the other elements comprising the case definition of a probable case 41. Treatment There is no treatment or vaccination available for MERS-CoV till now 39. People with MERS can have medical care to help relieve symptoms. In cell culture and animal experiments, combination therapy with interferon (IFN)-alpha-2b and ribavirin seems to be promising by limiting the viral replication 47. Other experimental therapies have been investigated which includes convalescent plasma, monoclonal antibodies, and inhibition of the viral protease 48. Vaccines Currently, there is no vaccine to prevent MERS-CoV infection 39 but some discussion is going on with the partners of CDC for developing the vaccines. One company has developed an experimental candidate MERS-CoV vaccine which is based on the major surface spike protein by means of recombinant nanoparticle technology 51. Other candidate vaccines that are being studied include a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome 52 and a recombinant Modified Vaccine Ankara (MVA) vaccine expressing full-length MERS-CoV spike protein 53. Prevention and Controls CDC routinely advises that people help protect themselves from respiratory illnesses by taking everyday preventive actions 49. v Wash the hands always with soap and water for 20 seconds, and help young children do the same. If soap and water are not available, we should use an alcohol-based hand sanitizer. v Always cover your nose and mouth with a tissue when you cough or sneeze and throw them in the proper place. v Avoid touching the eyes, nose and mouth with unwashed and dirty hands. v Avoid personal contact, such as kissing, or sharing cups or eating utensils, with sick and infected people. v Clean and disinfect the frequently touched surfaces such as toys and doorknobs.

CONCLUSION MERS-CoV is the emerging respiratory diseases that have mostly infection the people of Middle East Countries. Saudi Arabia is the most affected country among all. Since this virus has not treatment and vaccines, we should try to minimize the transmission rate. Travelers should know the general hygiene Website : http://reviewofprogress.org/

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measures, including hand washing before and after touching animals, and avoiding contact with the sick animals. Travelers should also avoid consumption of raw or undercooked animal products. Due to the high risk for severe MERS infections, people with diabetes, kidney failure, or chronic lung disease and people who have weakened immune systems should try to avoid contact with camels and horses. REFERENCES: 1. Zaki, A.M., van Boheemen, S., Bestebroer, T.M., Osterhaus, A.D and Fouchier, R.A, .(2012). Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 367:1814 –1820. 2. de Groot, R.J., Baker, S.C., Baric, R.S., Brown, C.S. and Ziebuhr, J. (2013). Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group. J Virol. 87(14):7790-7922. 3. Wang, N., Shi, X., Jiang, L., Zhang, S. and Wang, X. (2013). Structure of MERS-CoV spike receptorbinding domain complexed with human receptor DPP4. Cell Research 23:986-993. 4. Chen, Y., Li, F., Rajashankar, K.R., Yang, Y. and Agnihothram, S.S. (2013). Crystal Structure of the Receptor-Binding Domain from Newly Emerged Middle East Respiratory Syndrome Coronavirus. J Virol. 87(19): 10777–10783. 5. Mou, H., Raj, V.S., van Kuppeveld, F.J., Rottier, P.J. and Bosch, B.J. (2013). The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies. J Virol. 87(16):9379-83. 6. Lu, G., Hu, Y., Wang, Q., Qi, J. and Gao, G.F. (2013). Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature 500:227–231. 7. Jadav, H.A. (2013). Middle East Respiratory Syndrome - Corona Virus (MERSCoV): A Deadly Killer. IOSR Journal of Pharmacy and Biological Sciences. 8(5):74-81. 8. Cotton, M., Watson, S.J., Kellam, P., Al-Rabeeah, A.A. and Memish, Z.A. (2013). Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study. Lancet. 382(9909):1993-2002. 9. Stephensen, C.B., Casebolt, D.B. and Gangopadhyay, N.N. (1999). Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay. Virus Res. 60(2):181-189. 10. Pyrc, K., Berkhout, B. and van der Hoek, L. (2007). The Novel Human Coronaviruses NL63 and HKU1. J. Virol. 81(7):3051-3057. 11. World Health Organization. Middle East respiratory syndrome coronavirus (MERS-CoV) summary a n d l i t e r a t u r e u p d a t e – a s o f 11 J u n e 2 0 1 4 . Av a i l a b l e f r o m : h t t p : / / w w w. w h o . i n t / e n t i t y / c s r / d i s e a s e / c o r o n a v i r u s _ i n f e c t i o n s / M E R S CoV_summary_update_20140611.pdf 12. Brooks, G.F., Carroll, K.C., Butel, J.S., Morse, S.A. and Mietzner, T.M. (2010). Jawtez, Melnick, & Adelberg’s Medical Microbiology. 25th edition. Mc Graw Hill Companies, Inc. 13. Masters, P.S. (2006). The molecular biology of coronaviruses. Adv Virus Res. 66:193-292. 14. Mackay, I.M. (2014). Middle East respiratory syndrome coronavirus (MERS-CoV). Available from: http://www.uq.edu.au/vdu/VDUMERSCoronavirus.htm 15. de Wilde, A.H., Raj, V.S., Oudshoorn, D., Bestebroer, T.M. and van den Hoogen, B.G. (2013). MERScoronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-alpha treatment. Journal of General Virology. 94:1749–1760. 16. European Centre for Disease Prevention and Control. Severe respiratory disease associated with Middle East respiratory syndrome coronavirus (MERS-CoV). 16 October 2014. Available from: http://ecdc.europa.eu/en/publications/Publications/mers-cov-severe-respiratory-disease-riskassessment-16-october-2014.pdf 17. Chu, D.K.W., Poon, L.L.M., Gomaa, M.M., Shehata, M.M. and Kayali, G. (2014). MERS Coronaviruses in Dromedary Camels, Egypt. Emerging Infectious Diseases. 20(6): 1049-1053. 18. Memish, Z.A., Cotton, M., Meyer, B., Watson, S.J. and Drosten, C. (2014). Human Infection with MERS Coronavirus after Exposure to Infected Camels, Saudi Arabia, 2013. Emerging Infectious Diseases. 20(6): 1012-1015. 19. Hemida, M.G., Chu, D.K.W., Poon, L.L.M., Perera, R.A.P.M. and Peiris, M. (2014). MERS Coronavirus in Dromedary Camel Herd, Saudi Arabia. Emerging Infectious Diseases. 20(7): 1231-1234. 20. Reusken, C.B.E.M., Messadi. L., Feyisa. A., Ularamu. H. and Koopmans, M.P.G. (2014). Geographic Distribution of MERS Coronavirus among Dromedary Camels, Africa. Emerging Infectious Diseases. 20(8): 1370-1374.

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