Role of Sialic acid binding in Coronavirus attachment and entry

Binding of the viral particle is a crucial step in the establishment of viral infection and subsequent viral replication. In the case of Coronaviridae, the binding of the virus is mediated viral spike protein, a homotrimer composed of subunits that are about 150 kDa in size each. The spike protein itself is composed of two subunits, S1 and S2, the former sufficient for receptor binding and the latter required for the fusion and entry of the virus particle. During the viral replication the S protein is synthesized as a precursor protein and co-translationally glycosylated in the Golgi followed by a cleavage generating the S1 and S2 subunits at a dibasic cleavage site (BBXBB). The S1 subunit contains the receptor-binding site (RBD) followed (in the case of MHV) by a hypervariable region, whereas the S2 subunit contains two heptad repeats (HR1 and 2) as well as the transmembrane region.

Domains of a prototype Coronavirus S potein

Of particular interest is the RBD since blocking peptides or neutralizing antibodies designed to bind the RBD might be used in treating Coronavirus caused diseases, not only in humans (such as SARS or MERS) but also in animals. On the other hand, based on experiments done using the murine Coronavirus (MHV) the heptad repeat domains as well as the putative fusion peptide located within the S2 subdomain may play an important role in the formation of syncytia and thus may contribute to the CPE. Furthermore, the HR might also play a role in the interaction of the RBD with the cellular receptor during viral entry, probably by stabilizing the receptor-RBD complex not only in the case of MHV but also SARS-CoV.

In the past years, however a number of Coronaviruses have been shown to not only contain one but two RBD, one located at the C-terminal end of S1 which is responsible for binding the cellular receptor and an additional one located at the N-terminal end of S1 binding sialic acid. In general, the consensus is that binding to sialic acid by the S1 subunit allows Coronavirus’ to bind to epithelial target cells of the respiratory tract as well the intestine which are normally covered by mucus and thus not readily accessible. This is particular true for members of the Alpha-, Beta-, and Gammacoronaviridae which bind to ciliated intestine and respiratory cells, such as the porcine TGEV and PEDV as well as the enteric feline Coronavirus (FECV) but also for the bovine Coronavirus (BCoV) and the human Coronavirus OC43 (HCoV-OC43) as well as the avian Infectious Bronchitis Virus (IBV). In contrast, MERS-CoV generally does bind and infect primarily non-ciliated bronchial epithelial and alveolar cells of the lower lung and thus might not need sialic acid to bind to DPP4 (although hDPP4 does have sialic acid residues).

                        Feline Enteric Coronavirus (FECV)

Feline intestinal epithelial cells derived from the Ileum and the Colon (illenocytes and coloncytes respectively) pretreated with neuroaminidase exhibit an increase in the efficiency of FECV infection, suggesting that sialic acid might inhibit viral entry. Based on results showing that the pretreatment of porcine TGEV strain Perdue and PEDV with neuroaminidase can unmask the viral sialic acid binding activity, similar experiments confirmed these results for FECV. The application of α2-6-sialyllactose binds and reduces the infectivity of pretreated FECV, demonstrating FECV can bind α2-6-sialic acid. Desialylated cells however were resistant to inhibition of inhibition by α2-6-sialyllactose treatment. FECV therefore does have

a sialic acid binding capacity, which during the passage of the virus through the stomach may be partially masked by virus-associated sialic acids. In the absence of viral enzymes removing virus-associated sialic acids, enzymes within the mucus might remove the sialic acid thus allowing FECV to bind its cellular receptor and thus requiring sialidases for efficient enterocyte infections.

                     Infectious Bronchitis Virus (IBV)

Although the receptor for the avian Infectious Bronchitis Virus is unknown it is known that the treatment of Vero, BHK (Baby Hamster Kidney) as well as primer chicken kidney cells with neuroaminidase -an enzyme which cleaves sialic acid- renders cell lines resistant to infection with IBV strains Beaudette and M41. Moreover, IBV is more sensitive than Sendai or Influenza A virus to pretreatment of cells with neuroaminidase suggesting that IBV requires a higher amount of sialic acid than Influenza A or Sendai and indeed it has been shown that IBV preferentially recognizes α2,3-linked sialic acid as indicated by reacting with lectin. Indeed the infection of the tracheal organ cultures can be inhibited by pretreatment with neuroaminidase. The binding of α2,3-linked sialic acid might be only required for the initial binding of IBV preceding binding to the receptor although the sialic acid binding activity of IBV S protein seems to be more important for viral entry than the sialic acid binding activity of TGEV S protein. This is reflected by the abundance of α2-3 linked sialic acid on susceptible epithelial cells.  

In general, the masking of the viral sialic acid binding site might protect the enteric Coronavirus particles from degradation in the stomach or by gastric mucins. Bacterial and host derived sialidases unmasking these binding site then would allow the virus to attach to the mucin and infect cells of the intestinal tract. In the avian respiratory tract α2-3 linked sialic acid is a common receptor for respiratory viruses such as avian Influenza A. 

So finally what has this to do with emerging Coronaviruses? As I mentioned above so far there is no indication that MERS-CoV S has sialic acid binding activity nor that the primary target cells necessitate this activity. The novel Coronavirus identified in dromedaries however seems to be an enteric Coronavirus and thus the S protein might bind sialic acid.  However, once the genome of DcCoV UAE-HKU23 has been sequenced, we should know more.  One final word about the potential use of neuroaminidase inhibitors which are quite effective in treating Influenza A virus infections: they are not effective against Coronavirus induced infections since Coronaviridae are not dependent on the sialic acid binding to its cognate receptor. 

Further reading

Vlasak R, Luytjes W, Spaan W, & Palese P (1988). Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. Proceedings of the National Academy of Sciences of the United States of America, 85 (12), 4526-9 PMID: 3380803 

Shahwan K, Hesse M, Mork AK, Herrler G, & Winter C (2013). Sialic acid binding properties of soluble coronavirus spike (S1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus. Viruses, 5 (8), 1924-33 PMID: 23896748 

Winter C, Herrler G, & Neumann U (2008). Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent. Microbes and infection / Institut Pasteur, 10 (4), 367-73 PMID: 18396435 

Schmauser B, Kilian C, Reutter W, & Tauber R (1999). Sialoforms of dipeptidylpeptidase IV from rat kidney and liver. Glycobiology, 9 (12), 1295-305 PMID: 10561454

Krempl C, Schultze B, Laude H, & Herrler G (1997). Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus. Journal of virology, 71 (4), 3285-7 PMID: 9060696 

Schwegmann-Weßels, C., Bauer, S., Winter, C., Enjuanes, L., Laude, H., & Herrler, G. (2011). The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus Virology Journal, 8 (1) DOI: 10.1186/1743-422X-8-435 

Desmarets, L., Theuns, S., Roukaerts, I., Acar, D., & Nauwynck, H. (2014). The role of sialic acids in feline enteric coronavirus infections Journal of General Virology DOI: 10.1099/vir.0.064717-0

Are broad spectrum antivirals for Coronavirus infections are just around the corner?

The emergence of a new highly pathogenic virus in animal as well as human populations presents a unique challenge for both veterinarians and physicians alike since vaccines more often than not are not readily available, leaving antiviral treatments the only option to contain an outbreak. Pharmaceuticals however take time to be developed and tested thus the only option available is to identify the antiviral pathways targeted by viral proteins in the hope that existing drugs are available and effective in activating these pathways and thus suppress viral replication. In the meantime, clinicians can only offer supportive care and use serum from convalescent patients - often a scarce commodity and not readily available. As an alternative however pharmaceuticals used and approved for the treatment of other viral diseases or indeed for other diseases might be repurposed.

The recent emergence of both the SARS-CoV in 2002 and MERS-CoV in 2012 have lead to substantial increase in Coronaviruses as a potential human pathogen. Following the emergence of MERS-CoV, the International Respiratory and Emerging Infection Consortium (ISARIC) compiled a list of pharmaceuticals available to physicians based on the experience gained during the SARS-CoV epidemic in 2002/2003, with the most promising drugs being Interferon and Ribavirin, which had been used in combination as well as separate to treat SARS-CoV and pandemic Influenza A/2009 patients. Indeed, both drugs are effective to prevent MERS-CoV replication in a rhesus macaque model but failed to be effective in patients with a severe infection. A screen of chemical library of 1280 pharmaceuticals known to be effective against Influenza A was also assessed for their ability to reduce viral yield and prevent the cytopathic effect following the infection of cells with MERS-CoV confirmed that at least under laboratory conditions MERS-CoV is sensitive to Interferon as well as to two antiretroviral drugs, nefinavir and lopinavir. At first it may seem surprising that two antiretroviral drugs can prevent the replication of a Coronavirus. Both drugs were developed to prevent the replication of HIV by targeting the HIV protease. As discussed before however, the Coronavirus genome encodes for a protease, 3CL^pro which is required for the processing of the orf1ab polyprotein and has been shown sensitive to nefinavir and lopinavir due to the inhibition of the viral 3CL^pro    protease.  Both drugs are non-specific for MERS-CoV and also effective in treating SARS-CoV related infections.

Other targets of antiviral therapy most certainly include preventing viral entry. As discussed in a previous post, monoclonal antibodies against the viral S protein and small molecules binding to the receptor-binding site of the S protein have been shown to be effective to neutralize viral particles. Another possibility is to target the release of the viral genome into the cytoplasm of the cell, which is dependent on a low pH within the endosome. The application of a lysosomotropic agent such as Chloroquine/Hydroxychloroquine (the protonated form of Chloroquine) (an antimalarial drug) or NH4Cl might therefore prevent the fusion of the virus with the endosome by raising the pH. Indeed the application of low doses of Chloroquine to cells infected with SARS-CoV or MERS-CoV as well as Influenza A have shown to prevent viral replication. In addition to prevent the fusion of the viral particle with the endosome, Chloroquine might also prevent the glycosylation of ACE2, the receptor for SARS-CoV and thus prevent binding of the SARS-CoV S1 subunit to its receptor (it remains to be seen if this is the case with DPP4, the receptor for MERS-CoV). The glycosylation of proteins is targeted by inhibiting glycosyltransferases, namely quinone reductase 2, which is involved in the biosynthesis of sialic acid, a component of cellular receptors. Sialic acid moieties are also present within the glycoproteins of HIV-1 glycoproteins, the SARS-CoV receptor ACE2, the MERS-CoV receptor DPP4/CD26, Coronavirus S proteins as well in the receptors for Influenza A thus explaining the broad spectrum activity of Chloroquine. Quinone reductase 2 inhibitors therefore reduce the glycosylation of SARS S proteins although it seems that the reduction has no effect on viral infectivity (or only a marginal effect).

So far however its effectiveness has not been demonstrated in the animal model of MERS and studies with Influenza A have shown that Chloroquine -although effective in cell lines- is not effective in humans thus adding some caution. Apart from being a potential pharmaceutical against a variety of human Coronaviruses, Chloroquine is well tolerated and better known in treating in Malaria patients at therapeutic doses in micro molar concentrations.  Pharmaceuticals effective specifically against MERS-CoV, two pharmaceuticals emerged recently, mycophenolic acid (MPA) and IFN-β, both of which have been discussed previously.      

As outlined previously, the polyprotein 1ab is processed further by auto proteolysis that generates a number of nonstructural proteins varying among the Coronaviridae, which includes not the RNA dependent RNA Polymerase (RdRp) but also an NTPase/Helicase known as nsp 12 and 13 respectively.  In simian Vero E6 cells infected with SARS-CoV these are located within perinuclear double membrane bound vesicles representing replication-transcription complexes containing nascent viral subgenomic RNAs, RdRp as well as viral positive strand RNA and dsRNA intermediates which are resolved by the viral Helicase. Although the precise mechanism and specific function of the Coronavirus Helicase is not known, the replication of SARS-CoV, MERS-CoV, and the murine MHV can effectively inhibited by a small compound, SSYA10-001, targeting the Helicase at amino acid residues K508, R507, and Y277 respectively, thus offering a potential broad spectrum inhibitor of Coronavirus mediated infections and highlighting the importance of the Coronavirus Helicase for viral replication since inhibition of the SARS-CoV Helicase by Bismuth has been shown to inhibit SARS-CoV replication in the past. In addition to its wide spectrum of antiviral activity, SSYA10-001 exhibits only minimal cytotoxicity if applied to cells. The viral RdRp itself can be inhibited by combination of Ribavirin and 5-Flourouracil, the latter being mutagenic and thus sensitizing infected cells to Ribavirin treatment (Ribavirin itself being ineffective). 

Overview of potential and existing antiviral strategies to treat Coronavirus infections

In conclusion, whilst future outbreaks of novel respiratory viruses cannot prevented, pharmaceuticals which are already available might be used in the treatment during a pandemic or an epidemic whilst bioinformatics in conjunction with the identification of ways that viral proteins interact with the host cell might identify effective broad spectrum inhibitors which target highly conserved proteins. A recent screen of potential antiviral pharmaceuticals revealed that even antipsychotic drugs can have an antiviral effect against MERS-CoV, revealing the hidden potential of many drugs already approved.

Further reading

Dyall J, Coleman CM, Hart BJ, Venkataraman T, Holbrook MR, Kindrachuk J, Johnson RF, Olinger GG Jr, Jahrling PB, Laidlaw M, Johansen LM, Lear CM, Glass PJ, Hensley LE, & Frieman MB (2014). Repurposing of clinically developed drugs for treatment of Middle East Respiratory Coronavirus Infection. Antimicrobial agents and chemotherapy PMID: 24841273

Falzarano D, de Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, Brining D, Bushmaker T, Martellaro C, Baseler L, Benecke AG, Katze MG, Munster VJ, & Feldmann H (2013). Treatment with interferon-α2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques. Nature medicine, 19 (10), 1313-7 PMID: 24013700

Falzarano D, de Wit E, Martellaro C, Callison J, Munster VJ, & Feldmann H (2013). Inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin. Scientific reports, 3 PMID: 23594967 

Chan, J., Chan, K., Kao, R., To, K., Zheng, B., Li, C., Li, P., Dai, J., Mok, F., Chen, H., Hayden, F., & Yuen, K. (2013). Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus Journal of Infection, 67 (6), 606-616 DOI: 10.1016/j.jinf.2013.09.029

Kilianski A, & Baker SC (2014). Cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors. Antiviral research, 101, 105-12 PMID: 24269477

Al-Tawfiq JA, Momattin H, Dib J, & Memish ZA (2014). Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome coronavirus: an observational study. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 20, 42-6 PMID: 24406736

Smith EC, Blanc H, Vignuzzi M, & Denison MR (2013). Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics. PLoS pathogens, 9 (8) PMID: 23966862 


Hart BJ, Dyall J, Postnikova E, Zhou H, Kindrachuk J, Johnson RF, Olinger GG Jr, Frieman MB, Holbrook MR, Jahrling PB, & Hensley L (2014). Interferon-β and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays. The Journal of general virology, 95 (Pt 3), 571-7 PMID: 24323636

Coleman CM, Liu YV, Mu H, Taylor JK, Massare M, Flyer DC, Glenn GM, Smith GE, & Frieman MB (2014). Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice. Vaccine, 32 (26), 3169-74 PMID: 24736006

Keyaerts E, Li S, Vijgen L, Rysman E, Verbeeck J, Van Ranst M, & Maes P (2009). Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Antimicrobial agents and chemotherapy, 53 (8), 3416-21 PMID: 19506054

Savarino A, Di Trani L, Donatelli I, Cauda R, & Cassone A (2006). New insights into the antiviral effects of chloroquine. The Lancet infectious diseases, 6 (2), 67-9 PMID: 16439323

Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, Seidah NG, & Nichol ST (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virology journal, 2 PMID: 16115318

van Hemert, M., van den Worm, S., Knoops, K., Mommaas, A., Gorbalenya, A., & Snijder, E. (2008). SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro PLoS Pathogens, 4 (5) DOI: 10.1371/journal.ppat.1000054

Adedeji AO, Singh K, Kassim A, Coleman CM, Elliott R, Weiss SR, Frieman MB, & Sarafianos SG (2014). Evaluation of SSYA10-001 as a Replication Inhibitor of SARS, MHV and MERS Coronaviruses. Antimicrobial agents and chemotherapy PMID: 24841268 

Yang N, Tanner JA, Wang Z, Huang JD, Zheng BJ, Zhu N, & Sun H (2007). Inhibition of SARS coronavirus helicase by bismuth complexes. Chemical communications (Cambridge, England) (42), 4413-5 PMID: 17957304

Towards a MERS-CoV vaccine: the importance of S

For about two or so years Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans and no prophylactic and antiviral therapeutics specifically targeting MERS-CoV have been identified and applied in the field.

One strategy is to generate a MERS-CoV pseudovirus, which can be used to infect target cells and generate an immune response. Such a pseudovirus is generated by inserting the MERS-CoV genome or the protein of interest of into a lentiviral plasmid and transfect this plasmid into a packaging cell line, i.e. a cell line that stably expresses the proteins required for generating lentiviral plasmids. Viral supernatant is then harvested 24-72 hrs post transfection and can be used to transduce target cells. The recombinant virus however is capable of infect these cells but cannot replicate within these - in other words, the virus is replication incompetent. The infection of the target cell with a recombinant virus expressing the S protein of MERS-CoV for instance -in this case antigen presenting cells of the immune system such as T-Lymphocytes, dendritic cells or cells of the respiratory tract- would then allow to induce the production of antibodies which are directed against the viral protein (in this case MERS-CoV S protein). Whilst the concept is used in laboratories worldwide to generate stable cell lines expressing a protein of interest, this technology faces obstacles when used in animals and humans alike, namely the to infect the target cells and the generation of a high antibody titer. Any MERS-CoV pseudovirus therefore must be able to bind the MERS-CoV receptor, hDPP4, and therefore contain the MERS-CoV S protein on its surface, and also be able to generate a high titer of neutralizing antibodies. Indeed a replication incompetent MERS-CoV pseudovirus has been developed and shown to infect a wide variety of human and animal DPP4 expressing cell lines. MERS-CoV pseudoviruses are also used in infectivity assays and to study the contribution of individual viral proteins to viral replication and pathology.

One might ask why not use an attenuated virus strain of MERS-CoV akin to the Polio Vaccine? Indeed such a strain,  a recombinant strain in which the viral E protein has been deleted (rMERS-CoV-ΔE) has been created based on a replicon system developed by Luis Enjuanes from the University of Madrid in 2013.  Interestingly although in cells transfected with cDNA from rMERS-CoV-ΔE the virus replicates, viral particles cannot be detected in the supernatant. If however the E protein is provided in trans, viral infectivity is restored. This however might become a problem in animals and/or humans where different Coronavirus’ can be detected in the same host - in other words, the co-infection with an otherwise benign Coronavirus might lead to the generation of a viable MERS-CoV. Supplementing the E protein in trans however although resulting in the generation of viral particles, these viral particles were propagation defective. Again however studies are required to assess the infectivity and propagation in the presence of other Coronavirus species. These studies need time and one should not forgot that rMERS-CoV-ΔE was first described prior the availably of a transgenic mouse model expressing hDPP4 (which was first described in 2014). Also, no studies regarding the induction of a high titer of neutralizing antibodies have been described yet.

Another strategy involves the Coronavirus S Protein.

                       

                    MERS-CoV S protein as a potential target

The Coronavirus S protein is considered to be the main determinant of cellular tropism; indeed swapping the gene encoding the S protein is sufficient to alter cell tropism. In the case of MERS-CoV, the S gene encodes for a protein of 1353 amino acids in length, which is N glycosylated and assembled into trimers. These trimers constitute the peplomers on the surface of the viral particle that gave the Coronaviridae its name. The S protein combines two functions, binding the host receptor and membrane fusion, which are required for viral entry into the host cell. In the case of MERS-CoV, the former is attributed to the S1 subunit (AA1-751) and the latter to the S2 subunit (AA 752-1353) respectively.  During viral entry, the S protein is cleaved into both subunits by host cell derived proteases such as type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS) in the case of SARS-CoV.

S proteins from different Coronaviridae and their respective domains

Human monoclonal antibodies generated against the MERS-CoV S proteinbcould be used not only as a potential vaccine during an epidemic (in particular local outbreaks) but also upon exposure to MERS-CoV prior to the onset of symptoms. Moreover the identification of potent antibodies targeting the S protein can assist in the development of vaccines targeting specific regions of the S protein and thus aid in the development of specific immunogens. Following the identification of a putative receptor binding domain (RBD) of the viral S protein by sequence comparison with the RBD of SARS-CoV, three potent antibodies with a high affinity for an epitope overlapping with the RBD have been identified by screening an antibody library. One of three monoclonal antibodies identified, m336, neutralized live and pseudotyped MERS-CoV with an exceptional potency of ID₅₀ (half maximal inhibitory concentration) of 0.005 (pseudotyped MERS-CoV) and 0.07  (live MERS-CoV) μg/ml, respectively, by competing with the hDPP4 receptor. In a different study, two more antibodies, MERS-4 and MERS-27, were isolated from an antibody library with an ID₅₀ at nanomolar concentrations as well. Although both antibodies were effective in blocking entry of MERS-CoV, only MERS-4 also inhibited the formation of syncytia mediated by MERS-CoV S protein and DPP4 and thus cell-cell transmission of MERS-CoV.  Highly effective neutralizing antibodies against MERS-CoV S 1 fragment spanning 231 amino acids were also raised in rabbits and successfully tested in HEK-293T cells, further highlighting the importance of the S1 subunit of the MERS-CoV S protein.

Subdomains of MERS-CoV S1 and SARS-CoV S1

Using bioinformatics the information gained from these studies may allow the generation of potent antivirals targeting the RBD of MERS-CoV as well as related bat-CoV and thus not only prevent further outbreaks of MERS but hopefully also the transmission of bat-CoV into the human/animal population. So far however this is not possible although mutagenesis studies identified key residues in the receptor-binding subdomain of the S1 subdomain of MERS-CoV S protein required for binding DPP4 and viral entry. This analysis showed that MERS-CoV RBD consists of core and a receptor-binding subdomain that interacts with the N-terminal domain of DPP4. Accordingly, a truncated fragment of the MERS-CoV S1 containing the RBD fused with human IgG Fc fragment (S377-588-Fc) not only prevents MERS-CoV infection of cell lines but also elicits a high antibody titre in rabbits and mice infected with MERS-CoV akin to an fusion protein generated based on the SARS S1 subunit.

Fc fragments of SARS-CoV and MERS-CoV S1 domains

These studies also confirmed the previous notion that the enzymatic activity of the intracellular domain of the viral receptor is not required for viral entry (similar to SARS-CoV) and indeed inhibitors of DPP4 do not affect virus entry.

Again however, the potential effect of preventing the disease has not been shown in an animal model.

Lastly, we have to consider that the approval process complicates the introduction of vaccine for the use in humans. It might be easier to vaccinate animals such as dromedary camels with a novel vaccine. Such an approach has been undertaken with the introduction of a camel pox vaccine. Since this vaccine is approved for the use in camels, it might be feasible to adopt the vaccine backbone to vaccinate camels against MERS-CoV by inserting parts of the MERS-CoV into the vaccine strain. One of the problems is that MERS-CoV might be more ubiquitous in dromedary camels than we currently know - after all blood samples from camels taken 20 years ago have been shown to contain MERS-CoV antibodies. Secondly, although the transmission of MERS-CoV from camels to humans would be prevented other animals still might be able to transmit the virus into the human population.

What will be the solution in the end? First, we need to identify the mode of transmission and all natural hosts. Second, we need to improve the diagnostics and the isolation procedures. Third, we need to establish how common the infection is among the general population. And lastly, after all we need a vaccine which is not effective but also readily available in future local outbreaks. 

Further reading

Almazán F, DeDiego ML, Sola I, Zuñiga S, Nieto-Torres JL, Marquez-Jurado S, Andrés G, & Enjuanes L (2013). Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate. mBio, 4 (5) PMID: 24023385 

Zhao G, Du L, Ma C, Li Y, Li L, Poon VK, Wang L, Yu F, Zheng BJ, Jiang S, & Zhou Y (2013). A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV. Virology journal, 10 PMID: 23978242

Ying T, Du L, Ju TW, Prabakaran P, Lau CC, Lu L, Liu Q, Wang L, Feng Y, Wang Y, Zheng BJ, Yuen KY, Jiang S, & Dimitrov DS (2014). Exceptionally potent neutralization of MERS-CoV by human monoclonal antibodies. Journal of virology PMID: 24789777

Tang XC, Agnihothram SS, Jiao Y, Stanhope J, Graham RL, Peterson EC, Avnir Y, Tallarico AS, Sheehan J, Zhu Q, Baric RS, & Marasco WA (2014). Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution. Proceedings of the National Academy of Sciences of the United States of America, 111 (19) PMID: 24778221

Jiang L, Wang N, Zuo T, Shi X, Poon KM, Wu Y, Gao F, Li D, Wang R, Guo J, Fu L, Yuen KY, Zheng BJ, Wang X, & Zhang L (2014). Potent Neutralization of MERS-CoV by Human Neutralizing Monoclonal Antibodies to the Viral Spike Glycoprotein. Science translational medicine, 6 (234) PMID: 24778414

Wang N, Shi X, Jiang L, Zhang S, Wang D, Tong P, Guo D, Fu L, Cui Y, Liu X, Arledge KC, Chen YH, Zhang L, & Wang X (2013). Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell research, 23 (8), 986-93 PMID: 23835475

Mou H, Raj VS, van Kuppeveld FJ, Rottier PJ, Haagmans BL, & Bosch BJ (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. Journal of virology, 87 (16), 9379-83 PMID: 23785207

Du L, Kou Z, Ma C, Tao X, Wang L, Zhao G, Chen Y, Yu F, Tseng CT, Zhou Y, & Jiang S (2013). A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines. PloS one, 8 (12) PMID: 24324708

Wong SK, Li W, Moore MJ, Choe H, & Farzan M (2004). A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. The Journal of biological chemistry, 279 (5), 3197-201 PMID: 14670965

Raj, V., Mou, H., Smits, S., Dekkers, D., Müller, M., Dijkman, R., Muth, D., Demmers, J., Zaki, A., Fouchier, R., Thiel, V., Drosten, C., Rottier, P., Osterhaus, A., Bosch, B., & Haagmans, B. (2013). Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC Nature, 495 (7440), 251-254 DOI: 10.1038/nature12005

PAOLETTI, E. (1990). Poxvirus Recombinant Vaccines Annals of the New York Academy of Sciences, 590 (1 Rickettsiolog), 309-325 DOI: 10.1111/j.1749-6632.1990.tb42239.x

Pastoret, P., & Vanderplasschen, A. (2003). Poxviruses as vaccine vectors Comparative Immunology, Microbiology and Infectious Diseases, 26 (5-6), 343-355 DOI: 10.1016/S0147-9571(03)00019-5  

Jones GJ, Boles C, & Roper RL (2014). Raccoonpoxvirus safety in immunocompromised and pregnant mouse models. Vaccine PMID: 24837508