Virology tidbits

Virology tidbits

Wednesday, 2 April 2014

MERS-CoV and SARS-CoV: two members of a divergent family

Coronavirus infections are commonly associated with relative benign respiratory and enteric diseases in humans, such as the common cold, and with outbreaks among agricultural livestock -chickens, swine or cattle. The outbreak of a novel disease in humans, severe acute respiratory syndrome (SARS), in 2003 however highlighted the potential lethal consequences of
Coronavirus (CoV) induced disease in the human population. In total, SARS-CoV infected 8273 people with a fatality rate of 9.6% (or 775 deaths), with a majority reported from the People’s Republic of China and the Special Administrative Region of Hongkong. In the wake of SARS-CoV, van der Hoek et al. isolated a another novel human Coronavirus, HCoV-NL63, from a seven month old infant; subsequent clinical studies found that HCoV-NL63 in general only causes a mild respiratory disease akin to the previously identified HCoV-OC43 and HCoV-229E isolates, although it might pre-dispose infected patients to bacterial infections with Streptococci and be involved in croup. 

Classification of selected Coronaviruses and their host species (red:Human CoV, green: Bat CoV, blue: Dromedary CoV)
Another relative harmless human coronavirus was isolated in 2005 (HCoV-HKU1) also causing only relative benign symptoms. In the meantime the extensive search for a natural host of SARS-CoV lead to the discovery of a SARS-like Coronavirus in bats from China, Europe, Africa, Brazil, and Mexico, some of these which use the same receptor as SARS-CoV (ACE2). In 2012, a novel Coronavirus emerged in the Saudi Arabia (Middle East Respiratory Syndrome coronavirus; MERS-CoV). So far, infections have been reported not only in Saudi Arabia but also in neighboring countries (Kuwait, Quatar, Oman, Jordan, United Arab Emirates), Tunisia, and Europe (France, Italy, United Kingdom). Most cases however seem not have originated from direct human-to-human transmission but close contact to the source of the infection and there is some speculation that underlying diseases increase the risk of succumbing to MERS-CoV infections. Despite the range of symptoms infections with are associated with Coronavirus infection -ranging from a benign infection of the respiratory and enteric system to the severity of SARS and MERS-CoV associated diseases- the underlying molecular biology does not differ between animal and human CoV.
Genomes of representative "classic" Coronaviruses
Traditionally Coronavirus’ -with a positive ssRNA genome of about 27-32kb in length the largest RNA viruses- were classified in three groups based on their serology and sequence analysis of structural protein genes - namely the Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) genes - as well as the Polymerase. Following the identification of several “novel” CoV however this system was replaced by a revised classification system in which group I CoV became the genus of Alphacoronaviridae (including lineages 1a and 1b), group II became the genus Betacoronaviridae (including lineages 2a,2b, 2c,2d), the group III (avian) CoV, became the genus of Gammacoronaviridae including Beluga Whale CoV) and the newly identified Munia-, Bulbul- and Thrush-CoV were classified with the genus of Deltacoronaviridae – all within the family of Nidovirales.


Replication cycle of the Coronavirus genome
As mentioned above, Coronaviruses a single stranded positive strand RNA genome of about 27-32 kB in length. One of the key functions is the formation not only of nascent viral RNA but also of a nested set of subgenomic RNAs - RNAs of which some are structurally polycistronic but functionally monocistronic. The Polymerase gene itself encodes for two proteins (1a/1b) whose expression is regulated by ribosomal frameshifting. The Coronavirus genome contains cis-acting RNA elements (TAS or Transcription Activating Sites) preceding each gene. All Coronaviruses’ express a set of structural proteins; in addition the genome encodes for additional nonstructural genes which are required for efficient viral replication as well as the modulation of the antiviral response. Others -such as the HE- might be non-essential (at least under laboratory conditions). Mature Coronavirus particles are assembled in double membrane compartments probably at the endoplasmatic reticulum and transported to the cell surface.
In contrast to other RNA viruses, the replication of Coronavirus takes place in the cytoplasm of the infected cell without involvement of the nucleus, although a requirement of the nucleus has been postulated in the early 1980s and the viral N protein is known to localise to the nucleolus.
Viral entry preceded by binding of the Spike protein to the respective receptor, some of which have not been identified while others are known. Following entry, the genome is released into the cytoplasm in a (in the case of SARS-CoV) Cathepsin L and pH dependent manner, similar to Influenza or Ebola virus, although some details are different and vary among the different Coronavirus species.

Zoonotic CoV: differences between SARS-CoV and MERS-CoV

As mentioned above, in the wake of the SARS epidemic in 2003, several novel CoV were identified in bats. Moreover, at least three of the four human CoVs (NL63, 229E and OC43) were postulated to have originated in animal reservoirs and thus have zoonotic origins. The latest human CoV to be zoonotic in origin is MERS-CoV, with a fatality rate at around 60% surpassing SARS-CoV. Patients succumbing to MERS show renal failure, respiratory distress among other symptoms.
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 and -this is important- a potential coronavirus from dromedaries (at this time only MERS-CoV antibodies have been identified as well RNA has been isolated; so far the sequences are identical to human MERS-CoV).
SARS-CoV and MERS-CoV genomes
If a viral particles can be identified in dromedaries then a natural host for MERS-CoV might be identified, presumably allowing the vaccination of camels. Vaccination of camels however might be resisted since the disease does not really make camels sick. One strategy might be to generate genetic modified insect cells which express a fragment of the MERS-CoV spike protein (similar to a SARS-CoV vaccine).
In the absence of an animal model, a pronounced cytopathological effect is visible in infected Vero and human Huh7 cells within 48 hrs p.i., preceded by increased viral RNA synthesis starting at approx. 7 hrs p.i. and the release of nascent viral particles by 10 hrs p.i. . In contrast to SARS-CoV, MERS-CoV is sensitive to pre-treatment of cells with Interferon-α; in SARS-CoV this achieved in part by the orf6 protein which blocks the IFN induced nuclear translocation phosphorylated STAT1. If MERS-CoV however blocks antiviral signaling by mechanisms similar to MHV (Mouse Hepatitis Virus) is not known. Also, stimulation of the Interferon Regulator (IFN)-5 dependent Interferon-β pathway by Cyclosporin A inhibits MES-CoV replication as well (again this in contrast to SARS-CoV). While this is not a cure for the disease these findings provide an important insight into the pathology of the disease. In terms of virus-host interactions, MERS-CoV otherwise behaves similar to other Coronavirus'. MERS-CoV is bound by it's receptor ( Dipetidyl Peptidase 4 ( DPP4) ) on the cell surface, internalized followed by the release of the genome in a Cathepsin B, TMPRSS, and pH-dependent manner, followed by replication of the genome, viral gene expression and assembly of virus particles as outlined above. Interestingly, the MERS-CoV is expressed on a variety of cells including T-lymphocytes as as endothelial and epithelial cells.
Shedding of the receptor following infection with MERS-CoV  has been reported and might as a mechanism to repel neutrophils and to influence the immune response in a negative way. 

Another proposed vaccine would be based on a vaccine against camelpox. Again the lack of an animal model complicates things. The only model available so -rhesus macaques- does not show any symptoms at all if infected with MERS-CoV and is also expensive to use. Stanley Perlman from the University of Iowa however engineered a mouse expressing the human form of the MERS-Cov receptor DPP4) which may solve this problem. Indeed, first studies indicated that these mice once infected with MERS-CoV do exhibit similar symptoms than those observed in humans.

Finally I want to place some remarks on the phenomenon while the annual haji -the annual pilgrimage of devout Muslims to the holy sites of Mecca and Medina which are under the custodianship of the house of Saud- did not result in a an epidemic. The reason might be quite simple in the end; currently the holy month of Ramadan is rather late, well after camels give birth (which is during the winter).

Further reading

Stephensen CB, Casebolt DB, & Gangopadhyay NN (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 research, 60 (2), 181-9 PMID: 10392726 

de Groot, R., Baker, S., Baric, R., Brown, C., Drosten, C., Enjuanes, L., Fouchier, R., Galiano, M., Gorbalenya, A., Memish, Z., Perlman, S., Poon, L., Snijder, E., Stephens, G., Woo, P., Zaki, A., Zambon, M., & Ziebuhr, J. (2013). Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group Journal of Virology, 87 (14), 7790-7792 DOI: 10.1128/JVI.01244-13

Pyrc, K., Berkhout, B., & van der Hoek, L. (2006). The Novel Human Coronaviruses NL63 and HKU1 Journal of Virology, 81 (7), 3051-3057 DOI: 10.1128/JVI.01466-06 

Golda A, Malek N, Dudek B, Zeglen S, Wojarski J, Ochman M, Kucewicz E, Zembala M, Potempa J, & Pyrc K (2011). Infection with human coronavirus NL63 enhances streptococcal adherence to epithelial cells. The Journal of general virology, 92 (Pt 6), 1358-68 PMID: 21325482

Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, Mazet JK, Hu B, Zhang W, Peng C, Zhang YJ, Luo CM, Tan B, Wang N, Zhu Y, Crameri G, Zhang SY, Wang LF, Daszak P, & Shi ZL (2013). Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature, 503 (7477), 535-8 PMID: 24172901 

Chan JF, Chan KH, Choi GK, To KK, Tse H, Cai JP, Yeung ML, Cheng VC, Chen H, Che XY, Lau SK, Woo PC, & Yuen KY (2013). Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation. The Journal of infectious diseases, 207 (11), 1743-52 PMID: 23532101

de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S, Limpens RW, Posthuma CC, van der Meer Y, Bárcena M, Haagmans BL, Snijder EJ, & van den Hoogen BG (2013). MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment. The Journal of general virology, 94 (Pt 8), 1749-60 PMID: 23620378 

Perera RA, Wang P, Gomaa MR, El-Shesheny R, Kandeil A, Bagato O, Siu LY, Shehata MM, Kayed AS, Moatasim Y, Li M, Poon LL, Guan Y, Webby RJ, Ali MA, Peiris JS, & Kayali G (2013). Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 18 (36) PMID: 24079378 

Chu, D., Poon, L., Gomaa, M., Shehata, M., Perera, R., Abu Zeid, D., El Rifay, A., Siu, L., Guan, Y., Webby, R., Ali, M., Peiris, M., & Kayali, G. (2014). MERS Coronaviruses in Dromedary Camels, Egypt Emerging Infectious Diseases, 20 (6) DOI: 10.3201/eid2006.140299

Eckerle I, Müller MA, Kallies S, Gotthardt DN, & Drosten C (2013). In-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during Middle East Respiratory Syndrome (MERS) Coronavirus infection. Virology journal, 10 PMID: 24364985
Perlman S (2013). The Middle East respiratory syndrome--how worried should we be? mBio, 4 (4) PMID: 23963179 Lambeir AM, Durinx C, Scharpé S, & De Meester I (2003). Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV. Critical reviews in clinical laboratory sciences, 40 (3), 209-94 PMID: 12892317 

Raj VS, Mou H, Smits SL, Dekkers DH, Müller MA, Dijkman R, Muth D, Demmers JA, Zaki A, Fouchier RA, Thiel V, Drosten C, Rottier PJ, Osterhaus AD, Bosch BJ, & Haagmans BL (2013). Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature, 495 (7440), 251-4 PMID: 23486063



Barlan A, Zhao J, Sarkar MK, Li K, McCray PB Jr, Perlman S, & Gallagher T (2014). Receptor variation and susceptibility to MERS coronavirus infection. Journal of virology PMID: 24554656 

Zhao J, Li K, Wohlford-Lenane C, Agnihothram SS, Fett C, Zhao J, Gale MJ Jr, Baric RS, Enjuanes L, Gallagher T, McCray PB Jr, & Perlman S (2014). Rapid generation of a mouse model for Middle East respiratory syndrome. Proceedings of the National Academy of Sciences of the United States of America PMID: 24599590

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