MERS-CoV derived proteases: a potential antiviral target?

As described in past posts of this blog,  an outbreak of more than 650 cases of a severe pneumonia with a high percentage of mortality (up to 50%) has been associated with a novel zoonotic Coronavirus, MERS-CoV or Middle East Respiratory Syndrome Virus. To date, no effective antiviral therapy let alone vaccine to treat MERS-CoV infected patients. Whilst a vaccine would protect individuals from infection with MERS-CoV, antiviral therapy focuses on treating infected individuals and reduces viral spread and mortality associated with MERS-CoV. The window of opportunity in the latter case is thus limited to the period between infection and the onset of symptoms  - in the case of MERS-CoV mediated infections occurs generally with median incubation time of about 5.2-5.5 days, whereas the peak of the viral load can be measured at around day 11 (in the lower respiratory tract) and day 13 (in the urine). 

The emergence of of SARS-CoV in 2002/2003 lead to the identification of a number of potential antivirals which centered around the viral proteases that are required for viral replication. As outlined earlier, the orf1ab of the Coronavirus genome is divided into two large open reading frames, orf1a (which overlaps with orf1b) and orf1b. Both are separated by a ribosome frame shifting sequence (RFS) that allows the translation of two large polyproteins, PP1a (approx.. 500 kDa) and PP1ab (approx. 800 kDa). Not only are these relatively large proteins in size, but in addition they can be further processed by proteolytic auto processing via either the papain-like cysteine protease (PLPRO) or the main Coronavirus 3C chymotrypsin like  protease (MPRO   or 3CLPRO ), a Poliovirus 3C-like protease which is encoded within orf1a/orf1ab  of all members of the Coronaviridae. The proteolytic autoprocessing  leads to formation of a number of non-structural proteins which varies among different members of the Coronaviridae. 

Processing of PP1ab into non structural proteins

Besides its function in generation multiple non-structural proteins, both the PLPRO and  MPRO of SARS-CoV as well as MERS-CoV antagonize the Interferon response by blocking the nuclear translocation and phosphorylation of IFN regulatory factor (IFR-3) thus suppressing the expression of Interferon-β, CCL5, and CXCL10. Furthermore, both SARS-CoV and MERS-CoV PLPRO   act as viral deubiquitinating enzymes, reversing both K48 and K63  linked ubiquitination and ISG15-linked ISGylation and thus downregulate the innate immune response and increase the susceptibility of infected individuals to secondary infections in addition to succumbing to Coronavirus mediated disease. Since all human (and animal) representatives of the Coronaviridae express these proteases, they have been proposed as a potential target for antiviral drugs and compounds targeting SARS-CoV derived PLPRO  and  3CLPRO  have been developed. Unfortunately however these compounds -although effective in limiting SARS-CoV replication- are not effective against the MERS-CoV derived PLPRO  protease due to a amino acid change in the drug binding site. A small-molecule inhibitor however that blocks replication of SARS-CoV (and the murine MHV) by inhibiting 3CLPRO also inhibits the activity of MERS-CoV  derived 3CLPRO. It is however important to note that so far these results reflect only the capability of this inhibitor to inhibit MERS-CoV  derived 3CLPRO   in an experimental system -as experimental studies involving the impact of this inhibitor on viral replication or restoring the antiviral response nor on the pathogenesis of MERS in an animal model have not been conducted yet. These studies, using a replicon based  system to determine the individual contribution of various antiviral proteins for instance, need time and are hampered by current limitations set by the absence of a fully working animal model. Also, it should be noted that MERS-CoV expressed additional proteins which have been implicated in the suppression of the antiviral response such as orf4a, orf4b, and orf5. Any successful antiviral treatment therefore needs to be targeted against different proteins.  Interestingly, MERS-CoV -in contrast to SARS-CoV- seems to target specifically the  Interferon-β mediated immune response, and a combination of Interferon-β and Mycophenolic acid has been shown to reduce MERS-CoV viral titres in vitro whereas the treatment with Ribavirin and Interferon-α is not effective.

Further reading

Al-Abdallat MM, Payne DC, Alqasrawi S, Rha B, Tohme RA, Abedi GR, Al Nsour M, Iblan I, Jarour N, Farag NH, Haddadin A, Al-Sanouri T, Tamin A, Harcourt JL, Kuhar DT, Swerdlow DL, Erdman DD, Pallansch MA, Haynes LM, Gerber SI, & the Jordan MERS-CoV Investigation Team (2014). Hospital-associated outbreak of Middle East Respiratory Syndrome Coronavirus: A serologic, epidemiologic, and clinical description. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America PMID: 24829216 

Memish ZA, Al-Tawfiq JA, Makhdoom HQ, Assiri A, Alhakeem RF, Albarrak A, Alsubaie S, Al-Rabeeah AA, Hajomar WH, Hussain R, Kheyami AM, Almutairi A, Azhar EI, Drosten C, Watson SJ, Kellam P, Cotten M, & Zumla A (2014). Respiratory Tract Samples, Viral Load and Genome Fraction Yield in patients with Middle East Respiratory Syndrome. The Journal of infectious diseases PMID: 24837403

Drosten C, Seilmaier M, Corman VM, Hartmann W, Scheible G, Sack S, Guggemos W, Kallies R, Muth D, Junglen S, Müller MA, Haas W, Guberina H, Röhnisch T, Schmid-Wendtner M, Aldabbagh S, Dittmer U, Gold H, Graf P, Bonin F, Rambaut A, & Wendtner CM (2013). Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection. The Lancet infectious diseases, 13 (9), 745-51 PMID: 23782859

Cauchemez S, Fraser C, Van Kerkhove MD, Donnelly CA, Riley S, Rambaut A, Enouf V, van der Werf S, & Ferguson NM (2014). Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility. The Lancet infectious diseases, 14 (1), 50-6 PMID: 24239323

Perlman S, & Netland J (2009). Coronaviruses post-SARS: update on replication and pathogenesis. Nature reviews. Microbiology, 7 (6), 439-50 PMID: 19430490

Ramajayam, R., Tan, K., & Liang, P. (2011). Recent development of 3C and 3CL protease inhibitors for anti-coronavirus and anti-picornavirus drug discovery Biochemical Society Transactions, 39 (5), 1371-1375 DOI: 10.1042/BST0391371

Zhang, D., & Zhang, D. (2011). Interferon-Stimulated Gene 15 and the Protein ISGylation System Journal of Interferon & Cytokine Research, 31 (1), 119-130 DOI: 10.1089/jir.2010.0110

Yang, X., Chen, X., Bian, G., Tu, J., Xing, Y., Wang, Y., & Chen, Z. (2013). Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease Journal of General Virology, 95 (Pt_3), 614-626 DOI: 10.1099/vir.0.059014-0

Kilianski, A., Mielech, A., Deng, X., & Baker, S. (2013). Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors Journal of Virology, 87 (21), 11955-11962 DOI: 10.1128/JVI.02105-13

Lau, S., Lau, C., Chan, K., Li, C., Chen, H., Jin, D., Chan, J., Woo, P., & Yuen, K. (2013). Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment Journal of General Virology, 94 (Pt_12), 2679-2690 DOI: 10.1099/vir.0.055533-0

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

Al-Tawfiq, J., Momattin, H., Dib, J., & Memish, Z. (2014). Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome coronavirus: an observational study International Journal of Infectious Diseases, 20, 42-46 DOI: 10.1016/j.ijid.2013.12.003

Yang Y, Zhang L, Geng H, Deng Y, Huang B, Guo Y, Zhao Z, & Tan W (2013). The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists. Protein & cell, 4 (12), 951-61 PMID: 24318862

Viruses in bats: reason for concern or nothing to worry about?

Amidst the emergence of novel viruses infecting humans of zoonotic origin, such as MERS-CoV, Ebola or Influenza Virus H7N9, one of the most important questions is to identify the natural host. The emergence of these viruses as human pathogens lead tot he discovery of bats as a favorable animal reservoir for the emergence of novel viruses only second to rodents. In general, bats have a remarkable species diversity with over 1240 known species and a distribution pattern which covers every place on earth with the exception of the polar regions and a few oceanic islands - thus making them important source for viruses to cross the species barrier. Furthermore, bats are hibernating and roosting in diverse places such as caves, tree cavities, rocks, mines, and buildings or under bridges, which are in close proximity with livestock and humans as well. Dissemination and acquisition of pathogens is aided by the capacity to travel long distances, the formation of large colonies and longevity.

Transmission of viral particles from bats to other mammals can occur -as in the case of rabies- via bites or scratches by infected urine and guano. Indeed, one of the most recently identified Coronaviruses was isolated from guano derived from a bat species found in isolated areas of New Zealand. Other sources of transmitting viruses may be the consumption and handling of undercooked bat meat, a practice still common in parts of Asia, Guam and China. 

Besides the ecological and biological traits, bats also possess immunological akin to other mammalians. Similar to other mammalians, some bat species can control viral replication by inducing an antiviral response involving the detection of viral RNA by Pattern Recognition Receptors (PRR) thus as membrane bound Toll-like receptors (TLR) for the detection of ssRNA or dsRNA and/or cytosolic Retinoic Acid Inducible Gene (RIG)-1 like helicases which are homologues of and highly similar to those found in humans or in rodents. The infection of bats with a variety of viruses therefore induces an antiviral response consisting of the activation of the antiviral Interferon response similar to other mammalian species. Indeed, both type I and type III IFN can be found in some bat species whilst absent in others. On exception might be however the infection of bats with DNA viruses since the bat genomes which have been sequenced so far do not encode any members of the PYHIN family of DNA sensing proteins; thus the formation of inflammasomes in these species following viral infection might be impaired. Although this deficiency is not relevant for RNA viruses it might explain the prevalence of bat poxviruses in some bat species. 

Schematic outline of antiviral dsDNA signaling by PYHIN 

In general, asymptomatic or persistent viral shedding in the absence of pathology from bats is reported, indicating that cell mediated immunity might be affected by environmental factors such as hibernation and ecological as well as physiological factors.  

 

                                   Viruses found in bats

Besides Rabies Virus and various members of the Coronaviridae, more than 70 zoonotic viruses have been isolated from various bat species, with the majority non-pathogenic for humans or even to be transmitted to other animals. Zoonotic viruses transmitted from bats include not only Rabies Virus, but also the related Nipah and Hendra Viruses (all of them Lyssaviridae), as well as Ebola Virus and Marburg Virus. In other cases where bats are infected with Alphaviruses, Flaviviruses or Bunya-viruses by arthropods it is not clear if bats are a major natural reservoir and transmit viral particles to other hosts.  

                                        Coronaviridae

Following the SARS epidemic in late 2002 initial results identified palm five cats as the natural reservoir of SARS-CoV which have been sold in areas of China in live animal markets. Subsequent studies however identified bats of the genus Rhinolophus as the natural host of a SARS-like bat CoV. More recently a novel human Coronavirus emerged (named Middle East Respiratory Syndrome (MERS) -CoV), this time in Saudi Arabia, causing more than 500 infections not only in the Middle East but also in Europe and North America with 166 number of deaths.  Sequencing of the MERS-CoV genome revealed that the virus descended from a bat Coronavirus. Furthermore, a highly similar MERS-like CoV (BtCoV/PML/Neo zul/RSA/2011) has been isolated from the guano of bats in South Africa (Vespertilionidae spp.), suggesting that bats might be again the natural reservoir for a highly pathogenic Coronavirus. Alas no route of transmission to humans has been identified, although it might be possible that camels (or dromedaries to be specific) or racehorses might be acting as an intermittent host. Although it seems at first that bats indigenous to South Africa are related to the emergence of MERS-CoV, it should be noted that Eptesicus bobrinskoi -a member of the Vespertilionida spp.- has been reported to be indigenous to Oman back in 1968, a finding controversial at the time, and confirmed in 2006. Whilst bat cell lines are only showing a low affinity for MERS-CoV in vitro, it would be interesting to study MERS-CoV in cell lines derived from Eptesicus bobrinskoi and to compare to bat cells infected with the virus isolated in South Africa. Also it might be worthwhile to study the South African bat-CoV in human as well as in camel and horse cell lines. So far however any studies using bat derived CoV are limited since a replication competent virus is  not available at the moment.

                                               Henipaviridae

In 1994 an outbreak of an acute respiratory illness in a humans and horses in Hendra (a suburb of Brisbane/Australia) lead to the discovery of a novel virus, subsequently named Hendra Virus. This outbreak was followed by an outbreak of a respiratory illness on pig farms in Malaysia and Singapore -affecting humans and pigs alike- in 1999, which lead to he isolation of Nipah virus. Both viruses are classified within the genus of the Henipaviridae, family Paramyxoviridae and the order of the Mononegavirales. As such the genome is a non-segmented negative sense ssRNA of 18.2kb in size, encoding for six genes corresponding to six structural proteins. Besides Hendra and Nipah, the Henipaviridae contain a third member, Ceder Virus (CedPV). In contrast to Hendra and Nipah Virus, CedPV has not been associated with any clinical disease yet.

Schematic of Henipavirus particle

The natural hosts and reservoirs for Hendra and Nipah Virus alike are probably fruit bats of the genus Pteropus spp. including P. alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus, which are indigenous to parts of Asia, Australia, and islands off the coast of East Africa. The importance of bats as a natural reservoir for Henipavirus has been strengthened by recent reports showing that henipavirus-like viruses circulating in fruit bats (Eidolon helvum) in Africa are able to infect a wide range of cell lines including human, simian and bat cell lines.

Since the central determinate of virus tropism are the glycoproteins located on the virus surface, the finding that the glycoprotein from Henipavirus species distinct from those in Southeast Asia and Australia utilizes the same receptor as Nipah Virus might indicate a zoonotic potential. In contrast to Nipah or Hendra Virus however the African variant does not induce syncytia in simian or human cell lines and only limited syncytia formation can be observed in HypNi/1.1, a  bat cell line derived from Hypsignathus monstrosus.

These are just two examples of viruses circulating in bats that have crossed the species barrier in the past and exhibit a tropism for human cells. Other examples which pose a potential risk are Poxviruses or GBV-D, a Flavivirus related to Hepatitis C and GB virus recently isolated from bats in Bangladesh, the role of bats in the emergence of A/H17N10 and A/H18N11 subtypes of Influenza in new world bats, or the isolation of a novel Astrovirus - just to name a few. As these examples show, Virology as a discipline is not dead but still alive! Some of the research cannot be done by Virologists alone, but needs to be done in collaboration with zoologists. One example might be to investigate if the reservoir of the African relative of Nipah Virus, Eidolon helvum, is also native to areas where MERS-CoV is circulating? Indeed, a Bat CoV closely related to MERS-CoV was isolated from Taphozous perforates bats; not only did the colony also contain Eidolon helvum bats (in addition of others) but the colony also lived in close proximity to one patient from Saudi Arabia. Also, the patient reported not have had any contact with bats prior infection suggesting that if he got infected by bats the transmission might be due to contaminated guano and/or urine - in other words, virus shed by bats infected. 

It might be a far-fetched scenario, but could it be also possible that MERS-CoV was transmitted from bats to camels and humans? This scenario is less hypothetical than one might think. It is indeed widely assumed that both the Alpha-and Betacoronaviridae are derived from an (extinct?) BtCoV and MERS-CoV related BtCoV have been found in African and European bat populations. 

Further reading

Calisher, C., Childs, J., Field, H., Holmes, K., & Schountz, T. (2006). Bats: Important Reservoir Hosts of Emerging Viruses Clinical Microbiology Reviews, 19 (3), 531-545 DOI: 10.1128/CMR.00017-06

Smith I, & Wang LF (2013). Bats and their virome: an important source of emerging viruses capable of infecting humans. Current opinion in virology, 3 (1), 84-91 PMID: 23265969

Wynne, J., & Wang, L. (2013). Bats and Viruses: Friend or Foe? PLoS Pathogens, 9 (10) DOI: 10.1371/journal.ppat.1003651

Zhang G, Cowled C, Shi Z, Huang Z, Bishop-Lilly KA, Fang X, Wynne JW, Xiong Z, Baker ML, Zhao W, Tachedjian M, Zhu Y, Zhou P, Jiang X, Ng J, Yang L, Wu L, Xiao J, Feng Y, Chen Y, Sun X, Zhang Y, Marsh GA, Crameri G, Broder CC, Frey KG, Wang LF, & Wang J (2013). Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science (New York, N.Y.), 339 (6118), 456-60 PMID: 23258410 

Schattgen SA, & Fitzgerald KA (2011). The PYHIN protein family as mediators of host defenses. Immunological reviews, 243 (1), 109-18 PMID: 21884171 

Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR, Latz E, & Fitzgerald KA (2009). AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature, 458 (7237), 514-8 PMID: 19158675 

Hoffmann M, Müller MA, Drexler JF, Glende J, Erdt M, Gützkow T, Losemann C, Binger T, Deng H, Schwegmann-Weßels C, Esser KH, Drosten C, & Herrler G (2013). Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses. PloS one, 8 (8) PMID: 24023659 

Ithete NL, Stoffberg S, Corman VM, Cottontail VM, Richards LR, Schoeman MC, Drosten C, Drexler JF, & Preiser W (2013). Close relative of human Middle East respiratory syndrome 
coronavirus in bat, South Africa. Emerging infectious diseases, 19 (10), 1697-9 PMID: 24050621

Barlan A, Zhao J, Sarkar MK, Li K, McCray PB Jr, Perlman S, & Gallagher T (2014). Receptor variation and susceptibility to middle East respiratory syndrome coronavirus infection. Journal of virology, 88 (9), 4953-61 PMID: 24554656 

HARRISON, D. (1968). ON THREE MAMMALS NEW TO THE FAUNA OF OMAN, ARABIA, WITH THE DESCRIPTION OF A NEW SUBSPECIES OF BAT Mammalia, 32 (3) DOI: 10.1515/mamm.1968.32.3.317 

Ithete NL, Stoffberg S, Corman VM, Cottontail VM, Richards LR, Schoeman MC, Drosten C, Drexler JF, & Preiser W (2013). Close relative of human Middle East respiratory syndrome coronavirus in bat, South Africa. Emerging infectious diseases, 19 (10), 1697-9 PMID: 24050621 

Cui J, Eden JS, Holmes EC, & Wang LF (2013). Adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of Middle East respiratory syndrome coronavirus. Virology journal, 10 PMID: 24107353 Luby, S., Gurley, E., & Hossain, M. (2009). Transmission of Human Infection with Nipah Virus Clinical Infectious Diseases, 49 (11), 1743-1748 DOI: 10.1086/647951 

Marsh, G., de Jong, C., Barr, J., Tachedjian, M., Smith, C., Middleton, D., Yu, M., Todd, S., Foord, A., Haring, V., Payne, J., Robinson, R., Broz, I., Crameri, G., Field, H., & Wang, L. (2012). Cedar Virus: A Novel Henipavirus Isolated from Australian Bats PLoS Pathogens, 8 (8) DOI: 10.1371/journal.ppat.1002836 

Muleya W, Sasaki M, Orba Y, Ishii A, Thomas Y, Nakagawa E, Ogawa H, Hang'ombe B, Namangala B, Mweene A, Takada A, Kimura T, & Sawa H (2014). Molecular Epidemiology of Paramyxoviruses in Frugivorous Eidolon helvum Bats in Zambia. The Journal of veterinary medical science / the Japanese Society of Veterinary Science, 76 (4), 611-4 PMID: 24389743 

Chua KB, Koh CL, Hooi PS, Wee KF, Khong JH, Chua BH, Chan YP, Lim ME, & Lam SK (2002). Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes and infection / Institut Pasteur, 4 (2), 145-51 PMID: 11880045 

Lawrence P, Escudero Pérez B, Drexler JF, Corman VM, Müller MA, Drosten C, & Volchkov V (2014). Surface glycoproteins of the recently identified African Henipavirus promote viral entry and cell fusion in a range of human, simian and bat cell lines. Virus research, 181, 77-80 PMID: 24452140 

Kruger, N., Hoffmann, M., Weis, M., Drexler, J., Muller, M., Winter, C., Corman, V., Gutzkow, T., Drosten, C., Maisner, A., & Herrler, G. (2013). Surface Glycoproteins of an African Henipavirus Induce Syncytium Formation in a Cell Line Derived from an African Fruit Bat, Hypsignathus monstrosus Journal of Virology, 87 (24), 13889-13891 DOI: 10.1128/JVI.02458-13 

Epstein JH, Quan PL, Briese T, Street C, Jabado O, Conlan S, Ali Khan S, Verdugo D, Hossain MJ, Hutchison SK, Egholm M, Luby SP, Daszak P, & Lipkin WI (2010). Identification of GBV-D, a novel GB-like flavivirus from old world frugivorous bats (Pteropus giganteus) in Bangladesh. PLoS pathogens, 6 PMID: 20617167 

Chu, D., Poon, L., Guan, Y., & Peiris, J. (2008). Novel Astroviruses in Insectivorous Bats Journal of Virology, 82 (18), 9107-9114 DOI: 10.1128/JVI.00857-08 

Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen X, Recuenco S, Gomez J, Chen LM, Johnson A, Tao Y, Dreyfus C, Yu W, McBride R, Carney PJ, Gilbert AT, Chang J, Guo Z, Davis CT, Paulson JC, Stevens J, Rupprecht CE, Holmes EC, Wilson IA, & Donis RO (2013). New world bats harbor diverse influenza A viruses. PLoS pathogens, 9 (10) PMID: 24130481 

De Benedictis P, Marciano S, Scaravelli D, Priori P, Zecchin B, Capua I, Monne I, & Cattoli G (2014). Alpha and lineage C betaCoV infections in Italian bats. Virus genes, 48 (2), 366-71 PMID: 24242847

Nowotny N, & Kolodziejek J (2014). Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels, Oman, 2013. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 19 (16) PMID: 24786259

Memish, Z., Mishra, N., Olival, K., Fagbo, S., Kapoor, V., Epstein, J., AlHakeem, R., Durosinloun, A., Al Asmari, M., Islam, A., Kapoor, A., Briese, T., Daszak, P., Al Rabeeah, A., & Lipkin, W. (2013). Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia Emerging Infectious Diseases, 19 (11) DOI: 10.3201/eid1911.131172 

Annan A, Baldwin HJ, Corman VM, Klose SM, Owusu M, Nkrumah EE, Badu EK, Anti P, Agbenyega O, Meyer B, Oppong S, Sarkodie YA, Kalko EK, Lina PH, Godlevska EV, Reusken C, Seebens A, Gloza-Rausch F, Vallo P, Tschapka M, Drosten C, & Drexler JF (2013). Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe. Emerging infectious diseases, 19 (3), 456-9 PMID: 23622767