The hand, foot and mouth disease (HFMD) is a common childhood illness that has plagued various countries over many decades. Since there is currently no approved multivalent vaccine available, there is an urgent need to develop effective antivirals against viruses that are known to cause HFMD. In recent years, antiviral research has progressed beyond looking at viral factors as druggable targets to include host factors that are crucial for viral replication. This article hence aims to discuss the pros and cons of developing antivirals that target crucial host factors.
The HFMD is a widespread viral illness in which children below the age of 5 years old are the most susceptible1. The symptoms manifested are usually mild and self-limiting, with infected individuals showing signs of fever, general malaise as well as HFMD characteristic symptoms such as maculopapular or papulovesicular rash, blisters on the palms, soles or buttocks and herpangina2. The main etiological agents of HFMD are the coxsackievirus type A (CVA), enterovirus 71 (EV71) and echovirus (ECHO)3. They are members of the human enterovirus A species within the genus Enterovirus of the Piconaviridae family3. Amongst these viruses known to cause HFMD, EV71 is the most medically important as severe neurological complications such as aseptic meningitis, poliomyelitis-like acute flaccid paralysis and brainstem encephalitis may occur in individuals infected with EV714.
HFMD disease surveillance in Singapore
Since the first identification and characterisation of HFMD in New Zealand in 1957, numerous outbreaks have been occurring in countries including but not limited to the United States, Australia as well as countries in Asia5,6,7,8. In Singapore, despite the strict disease surveillance system and infection control policies implemented to reduce the disease prevalence, the number of HFMD cases still remain persistently high, with the highest number of cases being reported in the year 20169 (Figure 1). While no report has yet to be released by the Ministry of Health (Singapore) on the predominant serotypes that caused HFMD in the year 2016, a report published earlier had indicated that CVA6 was the predominant serotype, followed by EV71 and CVA4 in the year 201510.
The urgent need for an approved antiviral
Although HFMD is generally a mild and self-limiting disease for which patients usually recover within two weeks, the disease still poses a significant public health concern in Singapore due to the risk of developing severe neurological complications in individuals infected with EV712,4. In addition, socioeconomic cost incurred would be exorbitant due to the high number of cases as medical resources have to be diverted into treating the patients and parents have to disrupt their work to take care of their sick child at home. With no approved multivalent prophylactic vaccine against the predominant serotypes that cause HFMD and the drugs that are currently used to treat HFMD patients mainly serve to reduce the symptoms, there is an urgent need to expedite the process of antiviral discovery in order to solve this public health challenge11.
Traditional methods of antiviral development had focused greatly on targeting viral factors to disrupt viral replication. However, in recent years, there has been a change in focus to develop antivirals against crucial host factors that play a role in the viral replication cycle. Hence, in order to facilitate this new era of antiviral research, this article sets out to describe the pros and cons of developing antivirals that target host factors.
Increasing the repertoire of druggable targets
Figure 2A depicts a general replication cycle of human enteroviruses while Figure 2B shows a schematic diagram of a human enterovirus genome as well as its translated polyprotein and cleaved proteins. The enterovirus genome is a single-stranded, positive-sense RNA of approximately 7.4 kb in length12. The genome consists of a 5’ untranslated region which contains the internal ribosome entry site (IRES) to allow for viral translation of a single open reading frame that is downstream of the IRES region12 (Figure 2B). Subsequent cleavage of the translated polyprotein results in the production of four structural proteins and seven non-structural proteins (Figure 2B). Antiviral research on compounds that inhibit viral factors (proteins or IRES) would hence be limited to these few viral targets.
Fortunately, it has always been well-known that viruses hijack cellular machineries for their replication. As such, we would also expand our targets beyond viral factors and look into host factors that are crucial for viral replication. A number of host factors have been elucidated to play a role in the replication of these HFMD-causing viruses, including but not limited to SCARB2, MINK1, FUBP1 as well as proteins that are involved in clathrin-mediated endocytosis13,14,15,16. A transcriptomic analysis conducted by Leong and Chow (2006) identified altered expression in 191 genes when rhadomyosarcoma cells were infected with EV71, suggesting the interplay between these genes and viral replication17. Recently, Wu and colleagues (2016) identified and validated 256 cellular factors that are implicated in EV71 viral replication using genome-wide gene silencing technology18. Hence, an advantage of looking into host factors in antiviral research is that it greatly increases the repertoire of possible targets, increasing the chance and shortening the time required to identify good antiviral(s). Furthermore, a number of approved drugs originally used to treat other diseases that are known to affect the same host proteins or pathways may also be repurposed to treat HFMD. Drug repurposing can speed up the approval process of an effective drug against HFMD as the pharmacokinetics, pharmacodynamics and the safety profiles have already been verified and documented.
Lowering the probability of the emergence of an escape/resistant viral mutant
The replication of an enterovirus genome requires the viral encoded RNA-dependent RNA polymerase (RdRp) which has minimal proofreading activity. As such, enteroviruses tend to exhibit high rates of mutations and as a result, drugs that target viral factors will soon result in the rapid emergence of resistant mutants19. Very often, a single point mutation is sufficient to provide complete resistance to the effects of antivirals targeting viral factors20.
On the other hand, developing an antiviral that targets host factors essential for viral replication would make it harder for the virus to adapt as adaptation would mean that the virus must acquire phenotypes that either abrogate the need for the targeted host factor(s) or acquire phenotypes that give the virus the ability to exploit other similar factor(s) or pathway(s)21. The HSP90 is a chaperone protein that is required by picornaviruses to prevent the degradation of viral proteins via the proteasome pathway and it is also needed in the production of mature capsid22,23. Geldanamycin is a specific inhibitor of HSP90 and it had been shown to exhibit potent antiviral activities against various picornaviruses such as EV71 and poliovirus (PV, within the Enterovirus genus but do not cause HFMD)22,24. Using PV as an example, Geller and colleagues (2007) were unable to identify any resistant mutant against geldanamycin, suggesting that it is indeed harder for the virus to acquire resistance against antivirals that target host factors22.
However, it does not mean that it is impossible for the emergence of resistant mutants against these antivirals. Another small molecule, GW5074, is a cellular kinase inhibitor that exhibits broad-spectrum antiviral activity against enteroviruses such as EV71, coxsackievirus type B3 (CVB3, within the Enterovirus genus but do not cause HFMD) and PV25,26. Arita and colleagues (2008) were unable to isolate any resistant mutants against GW5074 for both EV71 and PV after 12 continuous passages but van der Schaar and colleagues (2012) managed to isolate a CVB3 mutant that was completely resistant to GW507425,26. The latter group identified that a point mutation at the viral 3A protein of either V45A or H57Y completely rescued GW5074 inhibition in CVB3 and they further elucidated that these point mutations allowed the CVB3 mutants to bypass PI4KIIIβ and the need for PI4P lipids for their replication26. Although further research should be conducted to see if similar mutations do result in resistance to GW5074 in HFMD-causing serotypes, the results shown in CVB3 has certainly shed some light on the possibility of the emergence of GW5074 resistant mutants in HFMD-causing serotypes.
Development of a broad-spectrum antiviral against all if not, most of the HFMD-causing serotypes
The ‘host-targeting’ approach in antiviral research is a lucrative way in developing antivirals as viruses within the same genus or even family may exploit the same cellular pathway for their replication. As elaborated in the previous sections, enteroviruses require HSP90 and PI4KIIIβ for efficient viral replication. As such, inhibitors such as geldanamycin and GW5074 can potentially serve as antivirals that have broad-spectrum antiviral activity against most of the known HFMD-causing serotypes. However, it is also important to note that there are many different types of enteroviruses that are known to cause HFMD and due to resource constraints, a selected few serotypes that are of medical importance and prevalence are usually selected in antiviral research or in the identification of host factors necessary for viral replication. As such, further studies should be conducted after the initial identification of an antiviral in these selected few serotypes to further profile the antiviral effects against all of the other HFMD-causing serotypes. This is important even though serotypes that are known to cause HFMD are evolutionary related, slight genetic differences may render the dependence on a particular host factor or pathway to be different amongst the serotypes. An example is camptothecin, which is an inhibitor of TOP1 that had been shown to exhibit antiviral activity against EV71 and CA16 but not ECHO7, all of which are known to cause HFMD27.
In conclusion, antiviral research on crucial host factors required for viral replication may lead to the identification of a broad-spectrum antiviral against serotypes that cause HFMD as related serotypes belonging to the same genus may exploit the same host factors or pathways, but further confirmatory research into the effects of the antiviral on each serotype is necessary.
The downside: “Killing” virus and killing the host?
The survivability of a cell lies in its intricate biochemical processes that are highly regulated. The disruption of any pathways through the inhibition of cellular proteins may disturb cellular homeostasis which can be detrimental. As such, one of the main concerns of antivirals targeting host factors is the cause of unnecessary systemic toxicities. Enviroxime, an inhibitor of PI4KIIIβ, was shown to be a potent antiviral against picornaviruses but the compound was shown to cause undesirable gastrointestinal side effects when taken orally and nasal irritation when administered intranasally26,28,29.
However, this potential problem can be mitigated. In order to facilitate viral replication, these viruses tend to alter the gene expression of the infected cell, upregulating the gene expression of crucial cellular proteins to allow for efficient viral replication, while downregulating those that are detrimental30. As such, studies can be conducted in the development of inhibitors that can inhibit these upregulated proteins or suppressor proteins that repress the expression and activity of these downregulated proteins in an infected cell. This attempts to bring these targets to their “normal” physiological level that is sufficient to block viral replication while maintaining normal cellular function30. Secondly, antivirals that target viral factors do exert toxicities as well and they are most likely to arise from off-target effects30. As such, it boils down to the need for intensive research to identify potential antivirals with large therapeutic windows.
In an urgent need to identify an effective antiviral to curb the HFMD epidemic, it is necessary to expand the scope to look into host factors that are crucial for viral replication. Not only does it increase the repertoire of druggable targets, we can potentially reap the benefits of targeting host factors as stated in the previous sections. Figure 2A shows the replication stages of an enterovirus and researchers have identified host proteins that play a role in every stage of the replication cycle. We are hopeful that this progress in antiviral research to include host factors as possible druggable targets may expedite the discovery and approval of an effective antiviral against viruses that cause HFMD, buying time for the development of a multivalent vaccine that is effective to bring HFMD into history.
About the Authors
Associate Professor Justin Jang Hann Chu is a principal investigator leading the laboratory of Molecular RNA Virology and Antiviral Strategies (MARVAS) in NUS as well as the Collaborative and Translational Unit for HFMD in IMCB, A* STAR. These laboratories focus on the development of vaccines as well as in the identification of antivirals and host factors required for viral replication of several medically important RNA viruses such as the enteroviruses, chikungunya virus, dengue virus and Zika virus.
Mr Tian Sheng Lew is a PhD student in MARVAS laboratory and his research focuses on the identification of host factors that are crucial for EV71 viral replication.
- Huang, X., Wei, H., Wu, S. et al. (2015). Epidemiological and etiological characteristics of hand, foot, and mouth disease in Henan, China, 2008-2013. Sci Rep, 5, 8904. doi:10.1038/srep08904
- Klein, M., & Chong, P. (2015). Is a multivalent hand, foot, and mouth disease vaccine feasible? Hum Vaccin Immunother, 1-17. doi:10.1080/21645515.2015.1049780
- Melnick JL. (1996) Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In: Fields BN, Knipe DM, Howley PM, Chanlock RM, Melnick JL, Monath TP, et al., editors. Field’s virology. 3rd ed. Philadelphia: Lippincott-Raven Publishers; p. 655–712.
- Huang, C. C., Liu, C. C., Chang, Y. C. et al. (1999). Neurologic complications in children with enterovirus 71 infection. N Engl J Med, 341(13), 936-942. doi:10.1056/NEJM199909233411302
- Duff, M. F. (1968). Hand-foot-and-mouth syndrome in humans: coxsackie A10 infections in New Zealand. Br Med J, 2(5606), 661-664.
- Flett, K., Youngster, I., Huang, J. et al. (2012). Hand, foot, and mouth disease caused by coxsackievirus a6. Emerg Infect Dis, 18(10), 1702-1704. doi:10.3201/eid1810.120813
- McMinn, P., Stratov, I., Nagarajan, L. et al. (2001). Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis, 32(2), 236-242. doi:10.1086/318454
- Zhu, Z., Zhu, S., Guo, X. et al. (2010). Retrospective seroepidemiology indicated that human enterovirus 71 and coxsackievirus A16 circulated wildly in central and southern China before large-scale outbreaks from 2008. Virol J, 7, 300. doi:10.1186/1743-422X-7-300
- Ministry of Health, Singapore. (2017). Weekly Infectious Disease Bulletin. Singapore: Author.
- Ministry of Health, Singapore. (2016). Communicable diseases surveillance in Singapore 2015. Singapore: Author.
- Kuo, R. L., & Shih, S. R. (2013). Strategies to develop antivirals against enterovirus 71. Virol J, 10, 28. doi:10.1186/1743-422X-10-28
- van der Linden, L., Wolthers, K. C., & van Kuppeveld, F. J. (2015). Replication and Inhibitors of Enteroviruses and Parechoviruses. Viruses, 7(8), 4529-4562. doi:10.3390/v7082832
- Huang, P. N., Lin, J. Y., Locker, N. et al. (2011). Far upstream element binding protein 1 binds the internal ribosomal entry site of enterovirus 71 and enhances viral translation and viral growth. Nucleic Acids Res, 39(22), 9633-9648. doi:10.1093/nar/gkr682
- Hussain, K. M., Leong, K. L., Ng, M. M. et al. (2011). The essential role of clathrin-mediated endocytosis in the infectious entry of human enterovirus 71. J Biol Chem, 286(1), 309-321. doi:10.1074/jbc.M110.168468
- Leong, S. Y., Ong, B. K., & Chu, J. J. (2015). The role of Misshapen NCK-related kinase (MINK), a novel Ste20 family kinase, in the IRES-mediated protein translation of human enterovirus 71. PLoS Pathog, 11(3), e1004686. doi:10.1371/journal.ppat.1004686
- Yamayoshi, S., Yamashita, Y., Li, J. et al. (2009). Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med, 15(7), 798-801. doi:10.1038/nm.1992
- Leong, W. F., & Chow, V. T. (2006). Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection. Cell Microbiol, 8(4), 565-580. doi:10.1111/j.1462-5822.2005.00644.x
- Wu, K. X., Phuektes, P., Kumar, P. et al. (2016). Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication. Nat Commun, 7, 13150. doi:10.1038/ncomms13150
- Lauring, A. S., Frydman, J., & Andino, R. (2013). The role of mutational robustness in RNA virus evolution. Nat Rev Microbiol, 11(5), 327-336. doi:10.1038/nrmicro3003
- Arellano-Galindo, J., Barron, B.L., Vargas-Infante, Y. et al. (2012). Point Mutations and Antiviral Drug Resistance, Point Mutation, Dr Colin Logie (Ed.), InTech, DOI: 10.5772/33866. Available from: https://www.intechopen.com/books/point-mutation/point-mutations-and-antiviral-drug-resistance
- Plummer, E., Buck, M. D., Sanchez, M. et al. (2015). Dengue Virus Evolution under a Host-Targeted Antiviral. J Virol, 89(10), 5592-5601. doi:10.1128/JVI.00028-15
- Geller, R., Vignuzzi, M., Andino, R. et al. (2007). Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance. Genes Dev, 21(2), 195-205. doi:10.1101/gad.1505307
- Wang, R. Y., Kuo, R. L., Ma, W. C. et al. (2013). Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly. Virology, 443(2), 236-247. doi:10.1016/j.virol.2013.05.001
- Tsou, Y. L., Lin, Y. W., Chang, H. W. et al. (2013). Heat shock protein 90: role in enterovirus 71 entry and assembly and potential target for therapy. PLoS One, 8(10), e77133. doi:10.1371/journal.pone.0077133
- Arita, M., Wakita, T., & Shimizu, H. (2008). Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity. J Gen Virol, 89(Pt 10), 2518-2530. doi:10.1099/vir.0.2008/002915-0
- van der Schaar, H. M., van der Linden, L., Lanke, K. H. et al. (2012). Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication. Cell Res, 22(11), 1576-1592. doi:10.1038/cr.2012.129
- Wu, K. X., & Chu, J. J. (2017). Antiviral screen identifies EV71 inhibitors and reveals camptothecin-target, DNA topoisomerase 1 as a novel EV71 host factor. Antiviral Res, 143, 122-133. doi:10.1016/j.antiviral.2017.04.008
- Hayden, F. G., & Gwaltney, J. M. (1982). Prophylactic activity of intranasal enviroxime against experimentally induced rhinovirus type 39 infection. Antimicrob Agents Chemother, 21(6), 892-897.
- Phillpotts, R. J., Jones, R. W., Delong, D. C. et al. (1981). The activity of enviroxime against rhinovirus infection in man. Lancet, 1(8234), 1342-1344.
- Lin, K., & Gallay, P. (2013). Curing a viral infection by targeting the host: the example of cyclophilin inhibitors. Antiviral Res, 99(1), 68-77. doi:10.1016/j.antiviral.2013.03.020