Available online at www.sciencedirect.com
ScienceDirect Update on enterovirus 71 infection Peng-Nien Huang1,3 and Shin-Ru Shih1,2,3,4 Human enterovirus type 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide. The identified EV71 receptors provide useful information for understanding EV71replication and tissue tropism. Host factors interact with the internal ribosome entry site (IRES) of EV71 to regulate viral translation. However, the specific molecular features of the EV71 genome that determine virulence remain unclear. The EV71 capsid protein VP1 region might contribute to virulence and neurotropism. Transgenic mice expressing the EV71 receptor that were infected with the virus exhibited a disease similar to that observed in infected humans. Antiviral drug and vaccine development is urgently required to prevent EV71 epidemics. Delineating viral host interactions and identifying specific mechanisms that might control the neural tropism of EV71 pathogenesis would be substantial advances. Addresses 1 Research Center for Emerging Viral Infections, Chang Gung University, Taiwan, ROC 2 Graduate Institute of Biomedical Science, Chang Gung University, Taiwan, ROC 3 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taiwan, ROC 4 Clinical Virology Laboratory, Department of Clinical Pathology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, ROC Corresponding author: Shih, Shin-Ru ([email protected], [email protected])
Current Opinion in Virology 2014, 5:98–104 This review comes from a themed issue on Emerging viruses Edited by Chris F Basler and Patrick C Y Woo
1879-6257/$ – see front matter, Published by Elsevier B.V. http://dx.doi.org/10.1016/j.coviro.2014.03.007
Introduction Enterovirus type 71 (EV71) is a member of the human enterovirus species A, and of the family Picornaviridae. In 1969, EV71 was first isolated in California, United States; and was first recognized as a cause of epidemics of handfoot-and-mouth disease (HFMD) in Japan in 1973 [1,2]. EV71, a major pathogen, is responsible for HFMD and has been associated with severe neurological complications and even fatalities in infants and young children worldwide [3,4]. In the 2000s, several countries, including Taiwan, Japan, Malaysia, Singapore, and Vietnam, experienced cyclical epidemics occurring every 2 or 3 years [3]. Since 2008, emerging epidemics of HFMD associated with EV71 have become critical in mainland Current Opinion in Virology 2014, 5:98–104
China [5,6]. In addition, in the past ten years, EV71 was isolated in numerous European and American countries such as France [7,8], Denmark [9], Spain [10], Portugal [11], Brazil [12], Canada [13], and the United States. The geographic distribution of EV71 isolates in the world is summarized in Figure 1, showing that EV71 was identified as an emerging virus worldwide. This review focuses on recent work describing EV71 receptors, host factors involved in viral translation, the EV71 animal model, vaccines, and antiviral agents. The geographic range of EV71 infection is primarily the Asia-Pacific region. However, EV71 infection was also reported in countries in America and Europe, in the past ten years.
Cellular receptors for enterovirus 71 Two human transmembrane proteins, P-selectin glycoprotein ligand-1 (PSGL-1) and scavenger receptor class B, member 2 (SCARB2), have recently been identified as functional receptors for EV71 entry [14,15]. Expression of PSGL-1 enabled EV71 entry and replication, as well as the development of cytopathic effects in unsusceptible mouse L cells [15]. PSGL-1 is a key factor involved in early inflammatory events on immune cells. Post-translational modifications of PSGL-1 regulate PSGL-1 and selectin interaction. Through experiments, a post-translational modification, tyrosine sulfation, at the N-terminal region of PSGL-1 was identified; this modification, is crucial for the binding of PSGL-1 to EV71 and viral replication in lymphocytes [16]. Some EV71 strains did not use PSGL-1 as a receptor to enter cells, suggesting the occurrence of strain-specific replication of EV71 in immune cells [15]. A single amino acid, residue 145 of the viral capsid protein VP1, determines whether a virus binds to the viral receptor PSGL-1 in lymphocytes. When VP1-145G/Q was replaced with E, the PSGL-1-binding strains lost their capacity to bind. Conversely, when VP1145E was replaced with either G or Q nonbinding strains, the strains bound to PSGL-1. The EV71 residue VP1-145 acts as a switch that controls the binding of the virus to PSGL-1 [17]. Unlike PSGL-1, SCARB2 can be used by most EV71 strains as an entry receptor, indicating that SCARB2 plays a crucial role in the entry steps of EV71 infection [14,15]. SCARB2 is widely expressed on various cell types, including neurons, which are directly involved in EV71 infection of the brain. The expression of human SCARB2 permitted typically unsusceptible cell lines to efficiently produce EV71 and develop cytopathic effects [14]. The unsusceptible cells expressing only amino www.sciencedirect.com
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Figure 1
Sweden Norway
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Potugal Spain
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Regions with reported human enterovirus 71 infection.
acids 142–204 of the human SCARB2 sequence were susceptible to EV71. Determination the region in human SCARB2 that is crucial for EV71 binding and infection greatly contributes to the understanding of virus–receptor interactions [18]. The EV71 clinical isolates propagated in human SCARB2 expression cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains [19]. Experiments expressed ectopic PSGL1 or SCARB2 in mouse L929 cells which these two proteins are absent. They used these two cells to compare the function of these two kind receptors. A comparison of the infection efficacies between mouse L cells expressing either SCARB2 or PSGL-1 indicated that L-SCARB2 cells are more susceptible to EV71 infection than LPSGL1 cells. However, L-SCARB2 cells bound with a reduced amount of EV71 compared with L-PSGL1 cells in a virus attachment assay. The binding abilities indicate the 35S-labeled EV71 particles mixed with receptor expressing cells. After the unbound virions were removed by washing, the cells were lysed and the radioactivity was measured as binding capacities. The results indicated that the difference in the binding abilities of the two receptors was not the sole determinant of the infection efficiency. The low infection efficiency of L-PSGL1 cells is due to the inability of PSGL-1 to induce viral uncoating [20]. SCARB2, as an uncoating receptor, contributes considerably to the understanding of the early steps of EV71 infection. The characterization of EV71 receptors www.sciencedirect.com
provides useful information for understanding EV71 replication at a molecular level.
Host factors involved in enterovirus 71 translation EV71 50 untranslated region (50 UTR) RNA contains a type I internal ribosomal entry site (IRES). This region of the EV71 genome is approximately 500 nt. The type I IRES exhibits poor efficiency in initiating viral translation in the absence of certain cellular proteins [21]. IRESdependent viral translation requires numerous trans-acting host factors, known as IRES trans-acting factors (ITAFs). These ITAF proteins play various roles, such as binding to viral RNA across multiple domains, as well as stabilizing the entire IRES in a structure that is suitable for binding canonical translation factors and ribosomal subunits [22]. Several recent reports have stated that ITAFs interact with the EV71 IRES. Lin et al. used streptavidin beads to pull down the host factors interacting with biotinylated EV71 50 UTR RNA. After matrixassisted laser desorption ionization-time of flight mass spectrometry analysis, 12 host factors interacted with the EV71 50 UTR. Some of these host factors were previously observed to interact with the 50 UTR of picornaviruses and to enhance virus replication. Some of these host factors were new ITAFs for RNA viruses [23]. For example, far upstream element binding protein 1 and 2 (FBP1 and FBP2) are novel ITAFs for EV71. An RNA Current Opinion in Virology 2014, 5:98–104
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pulldown assay indicated that FBP2 specifically bound to the EV71 IRES region. Viral protein synthesis and EV71 IRES activity were increased when cells were transfected with the siRNA targeting FBP2. The host factor FBP2 out-competed the polypyrimidine tract-binding protein, the positive-acting ITAF of picornaviral IRESs, in binding to the EV71 IRES. Experiments indicated that FBP2 is a negative regulator of EV71 IRES activity [23]. EV71 infection also triggers the virus-induced proteasome, autophagy, and caspase activity mechanism for the host cells to cleave FBP2, the negative regulator of viralIRES-driven translation. Moreover, the cleavage fragments of FBP2 alter the function of FBP2 from a negative regulator to a positive promoter during viral translation [24]. In contrast to FBP2, FBP1 enhanced the IRES activity of EV71 RNA and viral replication activity [25]. Both FBP1 and FBP2 bind to the region of the EV71 50 UTR containing the linker region (nt 637–745). Competition binding assays indicated that FBP1 and FBP2 competed to bind to the same region [25]. The FBP1 and FBP2 nuclear proteins were observed to relocate from the nucleus to the cytoplasm after EV71 infection [23,25]. However, the opposite effects of these two highly similar host factors on EV71 IRES activity must be further defined. Another nuclear protein, hnRNP A1, was identified as an EV71 ITAF by using a streptavidin beads pulldown assay [23], and is required for EV71 replication because it facilitates viral translation [26]. The mechanism describing how ITAFs regulate EV71 IRES activity is summarized in Figure 2. The new cellular factors associated with EV71 IRES may provide a novel route to new antiviral therapies. Nuclear proteins FBP1, FBP2, and hnRNP A1 relocate from the nucleus to the cytosol during EV71 infection. FBP1, hnRNP A1, and other ITAFs bind to the EV71 50 UTR and play positive roles in EV71 IRES activity. FBP2 has the opposite effect. FBP1 and FBP2 compete to bind to the linker region. EV71 infection also triggers the virusinduced proteasome, autophagy, and caspase activity mechanism to cleave FBP2. The cleaved fragments of FBP2 alter the function of FBP2 from a negative regulator to a positive regulator of IRES activity.
Molecular factor in the enterovirus 71 genome Compared with other non-polio enteroviruses, EV71 is an enterovirus that can increase mortality in children, but the specific molecular features that determine its virulence remain unknown. Most EV71 strains are unable to cause clinically apparent infection in mice. A mouse-adapted EV71 strain that belongs to sub-genogroup B5 was reported to increase virulence in newborn BALB/c mice when the virus exhibited a capsid protein VP1-K244E mutation, indicating that the VP1-K244E mutation is a crucial genetic determinant of mouse adaptation and Current Opinion in Virology 2014, 5:98–104
virulence [27]. Another study reported that the EV71 sub-genogroup C4 expressing the VP1-Q145E mutation was virulent in 5-day-old mice, which exhibited a high mortality rate [28]. Another mouse study by Huang et al. indicated that EV71 capsid proteins of VP2 (149M) and VP1 (145E) mutations enhanced the viral binding and RNA accumulation of EV71, contributing to virusinduced mortality [29]. In a human study, Cordey et al. analyzed the EV71 genome by using blood, stool, bronchoalveolar lavage fluid, and cerebrospinal fluid specimens from an immunocompromised patient. They observed a non-conservative amino acid change in VP1 (L97R) within the BC loop. In vitro cell tropism assays indicated that VP1 (L97R) conferred a replicative advantage in SHSY5Y cells of neuroblastoma origin. This study suggests that the VP1 BC loop region of EV71 may play an essential role in the cell tropism of EV71 infection and the VP1 region may contribute to dissemination and neurotropism [30]. In summary, a major mutation in the EV71 VP1 capsid protein can generate a mouseadaptive and virulent EV71 strain. The emergence of mutations at critical regions of the EV71 genome can produce new phenotypes and neurovirulence in humans.
Enterovirus 71 infection and disease animal models In the past decade, epidemiological and clinical analyses have revealed that the major target of EV71 infection is the central nervous system (CNS) [31]. Several studies have indicated that EV71 circulation in the blood might be the mechanism of dissemination from the body to the CNS [32]. Studies involving rhesus monkeys have demonstrated that EV71 infection causes the virus to injure the CNS at various rates when the virus is administered through different routes. In one study, intracerebral inoculation resulted in hemorrhages, pulmonary edemas, and neuron impairment, whereas intravenous and respiratory inoculations caused a direct infection of the CNS, accompanied by clear inflammation of the lung tissue [33]. This study similarly suggested that the rhesus monkey animal model may be suitable for studying the pathogenesis of EV71 [33]. Although most of the mouse studies show CNS involvement, a controversial result indicated that EV71 primarily replicated in skeletal muscle tissues, causing severe necrotizing myositis. In the aforementioned study, lesions in the CNS and other tissues were not observed in 2-week-old EV71-infected mice. Necrotizing myositis of respiratory-related muscles caused severe restrictive hypoventilation and subsequent hypoxia, which may explain the fatality of 2-week-old EV71-infected mice [34]. The mouse age and strains used for challenge may cause the discrepancy. Previous studies have used rhesus monkeys and neonatal mice that are susceptible to EV71 strains. The rhesus monkey model exhibits ethical and economic problems, whereas, mice that are older than a few weeks lose their susceptibility to EV71. Researchers have generated transgenic mice www.sciencedirect.com
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Figure 2
Negative ITAF FBP2 proteasome, autophagy, caspase Other ITAFs
Cleavage of FBP2 Positive ITAF
Linker IRES hnRNP A1
Positive ITAF
Viral RNA FBP1
IRES activity ↑
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Viral protein ↑
Cytoplasm FBP2
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Overview of the host nuclear proteins behaves as ITAFs to regulate EV71 viral translation.
expressing the EV71 receptor human SCARB2 (hSCARB2). Transgenic mice infected with EV71 exhibited ataxia, paralysis, and death. The pathological features in these transgenic mice were similar to those of EV71 encephalomyelitis in humans and experimentally infected monkeys. Experiments have suggested that the hSCARB2 transgenic mouse model may be a useful animal model for studying EV71 infection and an attractive platform for EV71 vaccine and drug testing [35,36]. Conversely, another study that used transgenic mice indicated that the expression of human PSGL-1 failed to increase the infectivity of EV71. The results indicated that PSGL-1 alone may be insufficient for infection with EV71 [37]. The hSCARB2 transgenic mice can be used to experimentally evaluate the neurovirulence of EV71 with greater statistical significance in the future. However, an ideal model system has not been achieved because the ideal animal model for EV71 infection reproduces the crucial features of the human disease, including neurological symptoms, cardiac dysfunction, and pulmonary edema.
Anti-enterovirus 71 drug research Currently, no effective antiviral drug against EV71 exists. Several recent studies have demonstrated the anti-EV71 www.sciencedirect.com
activity of natural products and other compounds [38,39]. Kappa carrageenan from seaweed, for example, exhibits anti-EV71 behavior. This natural product was reported to reduce plaque formation and prevent viral replication before and during viral absorption of EV71. A virus binding assay indicated that kappa carrageenan can bind to EV71 and disrupt the virus–receptor interaction [39]. Plevka et al. studied the structure of EV71 with the WIN 51711capsid-binding inhibitor. They found that the inhibitor replaced natural pocket factor within the viral protein 1 pocket without changing the structure of the capsid [40,41]. Another research team analyzed the EV71 inhibitors 3-(4-pyridyl)-2-imidazolidinone derivatives structure. They used quantum mechanics to identify additional potentially beneficial substitutions, enhanced ligand docking and synthesized two candidates. They demonstrated that one ligand is an order of magnitude more potent than the previously reported inhibitor 3-(4pyridyl)-2-imidazolidinone [42]. These researches indicated that structural analysis could be a new way to the development of anti-EV71 capsid-binding drugs [40–42]. The monoclonal antibody E18 was prepared by immunizing mice with empty particles of EV71. The researchers indicated that binding of E18 to EV71 causes the virus Current Opinion in Virology 2014, 5:98–104
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to change its conformation and to eject much of viral genome. The E18 antibody mediated virus neutralization by the induction of viral genome release. This study demonstrated that E18 antibody has potential for an anti-EV71 therapy [43]. A six-amino-acid peptide known as LVLQTM was previously shown to inhibit rhinovirus 2A protease. This research indicated that the peptide is an effective substrate against EV71 2A protease. The LVLQTM peptide considerably inhibited the eIF4G cleavage activity of 2A protease during EV71 replication in HeLa cells. This study expanded the development of new antiviral drugs against EV71 from small molecules to peptide drugs [44]. In another study, a synthetic peptide was developed that could be a potential antiviral compound that inhibits EV71. Ninety-five synthetic peptides that overlap the EV71 VP1 capsid protein were tested for use in an antiviral drug against EV71. The peptide was observed to reduce considerably the cytopathic effects of all representative EV71 strains [45]. Deng et al. identified the 50 UTR of the EV71 genome as highly conserved and an appropriate target of siRNA to inhibit viral replication effectively. Transfection of 20 -modified siRNAs targeting the 50 UTR delayed and reduced the cytopathic effects on EV71-infected cells. These 20 -modified siRNAs provide a novel source for studying the effects of RNAi-based antiviral EV71 drugs on EV71 infection [46]. Numerous opportunities appear to be available for developing effective antiviral drugs against EV71.
EV71 vaccines are currently undergoing a phase III clinical trial and the EV71 vaccine will be available in the near future, further study on the mechanism of EV71 infection is still necessary. EV71 can be regarded as a general model for other picornaviruses. Research has indicated certain factors that regulate EV71 replication. However, specific factors that contribute to EV71 associated neural pathogenesis remain unclear. Thus, dissecting viral host interaction and identifying specific mechanisms that may control the neural tropism of EV71 infection is an urgent need. Such studies would facilitate understanding of the neuropathogenesis of EV71 infection, as well as that of several other neurotropic RNA viruses. Antiviral agents against neurotropic RNA viruses with a broader spectrum may be developed based on such investigations.
Acknowledgements This work was supported by funding from the National Science Council of the Republic of China, Taiwan (NSC-102-2325-B-182-015) and Chang Gung Memorial Hospital (CMRPD1A0673).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Schmidt NJ, Lennette EH, Ho HH: An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 1974, 129:304-309.
2.
Hagiwara A, Tagaya I, Yoneyama T: Epidemic of hand, foot and mouth disease associated with enterovirus 71 infection. Intervirology 1978, 9:60-63.
3.
Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH: Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 2010, 10:778-790.
4.
Weng KF, Chen LL, Huang PN, Shih SR: Neural pathogenesis of enterovirus 71 infection. Microbes Infect 2010, 12:505-510.
5.
Tan X, Huang X, Zhu S, Chen H, Yu Q, Wang H, Huo X, Zhou J, Wu Y, Yan D et al.: The persistent circulation of enterovirus 71 in People’s Republic of China: causing emerging nationwide epidemics since 2008. PLoS ONE 2011, 6:e25662.
6.
Xing W, Liao Q, Viboud C, Zhang J, Sun J, Wu JT, Chang Z, Liu F, Fang VJ, Zheng Y et al.: Hand, foot, and mouth disease in China, 2008–12: an epidemiological study. Lancet Infect Dis 2014, 6:308-318.
7.
Schuffenecker I, Mirand A, Antona D, Henquell C, Chomel JJ, Archimbaud C, Billaud G, Peigue-Lafeuille H, Lina B, Bailly JL: Epidemiology of human enterovirus 71 infections in France, 2000–2009. J Clin Virol 2011, 50:50-56.
8.
Kassab S, Saghi T, Boyer A, Lafon ME, Gruson D, Lina B, Fleury H, Schuffenecker I: Fatal case of enterovirus 71 infection and rituximab therapy, France, 2012. Emerg Infect Dis 2013, 19:1345-1347.
9.
Badran SA, Midgley S, Andersen P, Bottiger B: Clinical and virological features of enterovirus 71 infections in Denmark, 2005 to 2008. Scand J Infect Dis 2011, 43:642-648.
Enterovirus 71 vaccine development An EV71 vaccine is urgently required to control and prevent epidemics of EV71. However, no vaccines against EV71 exist [47,48]. These two research groups generated the inactivated alum-adjuvant EV71 vaccine, which was administered in a randomized, double-blind, placebo-controlled, phase III trial and proved to provide high efficacy, satisfactory safety, and sustained immunogenicity [49,50–52]. Virus-like particle (VLP)-based vaccines have been investigated. EV71 VLPs produced in a baculovirus expression system were highly immunogenic in mice and monkeys [53–55]. In the studies, the researchers generated a platform for producing of VLPs for EV71 in Saccharomyces cerevisiae by co-expressing the P1 and 3CD genes of EV71. These VLPs exhibited a protein composition similar to that of the EV71 empty particles produced by EV71-infected cells. In vivo challenge experiments have indicated that immune sera induced by VLPs conferred protection in neonatal mice against a lethal EV71 challenge. In summary, these studies have indicated that a VLP from yeast is another potential vaccine candidate against EV71 infection [56]. In the near future, we believe that a vaccine against EV71 infection will be available.
Conclusion In the past 15 years, EV71 has circulated and emerged as a major cause of viral encephalitis and HFMD in the AsianPacific region. Although the inactivated whole-virus Current Opinion in Virology 2014, 5:98–104
10. Cabrerizo M, Tarrago D, Munoz-Almagro C, Del Amo E, DominguezGil M, Eiros JM, Lopez-Miragaya I, Perez C, Reina J, Otero A et al.: Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain. Clin Microbiol Infect 2013, 20:O150-O156. www.sciencedirect.com
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11. Venancio P, Oliveira M, Silva R, Conceicao C, Brito MJ: First case of severe enterovirus 71 infection in Portugal. Pediatr Infect Dis J 2013, 32:581-582. 12. Lamarao LM, Maciel AM, Gomes MdL: First isolation of enterovirus 71 (EV-71) from Northern Brazil. Braz J Infect Dis 2003, 7:278-281. 13. Merovitz L, Demers AM, Newby D, McDonald J: Enterovirus 71 infections at a Canadian center. Pediatr Infect Dis J 2000, 19:755-757. 14. Yamayoshi S, Yamashita Y, Li J, Hanagata N, Minowa T, Takemura T, Koike S: Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med 2009, 15:798-801. This paper found that SCARB2 as an EV71 and CVA16 receptor contributes to a better understanding of the pathogenicity of these viruses. 15. Nishimura Y, Shimojima M, Tano Y, Miyamura T, Wakita T, Shimizu H: Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat Med 2009, 15:794-797. This paper found that Stable PSGL-1 expression allowed EV71 entry and replication, and development of cytopathic effects in nonsusceptible cells. 16. Nishimura Y, Wakita T, Shimizu H: Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection. PLoS Pathog 2010, 6:e1001174. 17. Nishimura Y, Lee H, Hafenstein S, Kataoka C, Wakita T, Bergelson JM, Shimizu H: Enterovirus 71 binding to PSGL-1 on leukocytes: VP 1–145 acts as a molecular switch to control receptor interaction. PLoS Pathog 2013, 9:e1003511.
generates a mouse-virulent phenotype. J Gen Virol 2012, 93:1935-1940. 29. Huang SW, Wang YF, Yu CK, Su IJ, Wang JR: Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement. Virology 2012, 422:132-143. 30. Cordey S, Petty TJ, Schibler M, Martinez Y, Gerlach D, van Belle S, Turin L, Zdobnov E, Kaiser L, Tapparel C: Identification of sitespecific adaptations conferring increased neural cell tropism during human enterovirus 71 infection. PLoS Pathog 2012, 8:e1002826. This paper show that the VP1 BC loop region of EV71 plays a critical role in cell tropism and neurotropism in the immunocompromised patient 31. Lin TY, Hsia SH, Huang YC, Wu CT, Chang LY: Proinflammatory cytokine reactions in enterovirus 71 infections of the central nervous system. Clin Infect Dis 2003, 36:269-274. 32. Dolin R: Enterovirus 71 — emerging infections and emerging questions. N Engl J Med 1999, 341:984-985. 33. Zhang Y, Cui W, Liu L, Wang J, Zhao H, Liao Y, Na R, Dong C, Wang L, Xie Z et al.: Pathogenesis study of enterovirus 71 infection in rhesus monkeys. Lab Invest 2011, 91:1337-1350. 34. Xiu JH, Zhu H, Xu YF, Liu JN, Xia XZ, Zhang LF: Necrotizing myositis causes restrictive hypoventilation in a mouse model for human enterovirus 71 infection. Virol J 2013, 10:215.
18. Yamayoshi S, Koike S: Identification of a human SCARB2 region that is important for enterovirus 71 binding and infection. J Virol 2011, 85:4937-4946.
35. Fujii K, Nagata N, Sato Y, Ong KC, Wong KT, Yamayoshi S, Shimanuki M, Shitara H, Taya C, Koike S: Transgenic mouse model for the study of enterovirus 71 neuropathogenesis. Proc Natl Acad Sci U S A 2013, 110:14753-14758. This paper suggests that this transgenic mouse could represent a useful animal model for the study of EV71 infection.
19. Yamayoshi S, Iizuka S, Yamashita T, Minagawa H, Mizuta K, Okamoto M, Nishimura H, Sanjoh K, Katsushima N, Itagaki T et al.: Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71. J Virol 2012, 86:5686-5696.
36. Lin YW, Yu SL, Shao HY, Lin HY, Liu CC, Hsiao KN, Chitra E, Tsou YL, Chang HW, Sia C et al.: Human SCARB2 transgenic mice as an infectious animal model for enterovirus 71. PLoS ONE 2013, 8:e57591.
20. Yamayoshi S, Ohka S, Fujii K, Koike S: Functional comparison of SCARB2 and PSGL1 as receptors for enterovirus 71. J Virol 2013, 87:3335-3347. This paper compared the EV71 infection efficiencies of cells that expressed SCARB2 and PSGL-1 and the abilities of SCARB2 and PSGL1 to bind to the virus.
37. Li YP, Liang ZL, Gao Q, Huang LR, Mao QY, Wen SQ, Liu Y, Yin WD, Li RC, Wang JZ: Safety and immunogenicity of a novel human enterovirus 71 (EV71) vaccine: a randomized, placebocontrolled, double-blind, Phase I clinical trial. Vaccine 2012, 30:3295-3303.
21. Thompson SR, Sarnow P: Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved. Virology 2003, 315:259-266. 22. Pilipenko EV, Viktorova EG, Guest ST, Agol VI, Roos RP: Cellspecific proteins regulate viral RNA translation and virusinduced disease. EMBO J 2001, 20:6899-6908. 23. Lin JY, Li ML, Shih SR: Far upstream element binding protein 2 interacts with enterovirus 71 internal ribosomal entry site and negatively regulates viral translation. Nucleic Acids Res 2009, 37:47-59. 24. Chen LL, Kung YA, Weng KF, Lin JY, Horng JT, Shih SR: Enterovirus 71 infection cleaves a negative regulator for viral internal ribosomal entry site-driven translation. J Virol 2013, 87:3828-3838.
38. Chang CW, Leu YL, Horng JT: Daphne Genkwa sieb. Et zucc. Water-soluble extracts act on enterovirus 71 by inhibiting viral entry. Viruses 2012, 4:539-556. 39. Chiu YH, Chan YL, Tsai LW, Li TL, Wu CJ: Prevention of human enterovirus 71 infection by kappa carrageenan. Antiviral Res 2012, 95:128-134. 40. Plevka P, Perera R, Yap ML, Cardosa J, Kuhn RJ, Rossmann MG: Structure of human enterovirus 71 in complex with a capsidbinding inhibitor. Proc Natl Acad Sci U S A 2013, 110:5463-5467. 41. Plevka P, Perera R, Cardosa J, Kuhn RJ, Rossmann MG: Crystal structure of human enterovirus 71. Science 2012, 336:1274. 42. De Colibus L, Wang X, Spyrou JA, Kelly J, Ren J, Grimes J, Puerstinger G, Stonehouse N, Walter TS, Hu Z et al.: Morepowerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules. Nat Struct Mol Biol 2014, 21:282-288.
25. Huang PN, Lin JY, Locker N, Kung YA, Hung CT, Huang HI, Li ML, Shih SR: 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 2011, 39:9633-9648.
43. Plevka P, Lim PY, Perera R, Cardosa J, Suksatu A, Kuhn RJ, Rossmann MG: Neutralizing antibodies can initiate genome release from human enterovirus 71. Proc Natl Acad Sci U S A 2014, 111:2134-2139.
26. Lin JY, Shih SR, Pan M, Li C, Lue CF, Stollar V, Li ML: hnRNP A1 interacts with the 50 untranslated regions of enterovirus 71 and Sindbis virus RNA and is required for viral replication. J Virol 2009, 83:6106-6114.
44. Falah N, Montserret R, Lelogeais V, Schuffenecker I, Lina B, Cortay JC, Violot S: Blocking human enterovirus 71 replication by targeting viral 2A protease. J Antimicrob Chemother 2012, 67:2865-2869.
27. Zaini Z, Phuektes P, McMinn P: Mouse adaptation of a subgenogroup B5 strain of human enterovirus 71 is associated with a novel lysine to glutamic acid substitution at position 244 in protein VP1. Virus Res 2012, 167:86-96.
45. Tan CW, Chan YF, Sim KM, Tan EL, Poh CL: Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1. PLoS ONE 2012, 7:e34589.
28. Zaini Z, McMinn P: A single mutation in capsid protein VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71
46. Deng JX, Nie XJ, Lei YF, Ma CF, Xu DL, Li B, Xu ZK, Zhang GC: The highly conserved 50 untranslated region as an effective target
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towards the inhibition of enterovirus 71 replication by unmodified and appropriate 20 -modified siRNAs. J Biomed Sci 2012, 19:73.
content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines. Vaccine 2011, 29:9668-9674.
47. Xu J, Qian Y, Wang S, Serrano JM, Li W, Huang Z, Lu S: EV71: an emerging infectious disease vaccine target in the Far East? Vaccine 2010, 35(28):6-3521.
52. Li R, Liu L, Mo Z, Wang X, Xia J, Liang Z, Zhang Y, Li Y, Mao Q, Wang J et al.: An inactivated enterovirus 71 vaccine in healthy children. N Engl J Med 2014, 370:829-837.
48. Qiu J: Enterovirus 71 infection: a new threat to global public health? Lancet Neurol 2008, 7:868-869.
53. Chung YC, Ho MS, Wu JC, Chen WJ, Huang JH, Chou ST, Hu YC: Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge. Vaccine 2008, 26:1855-1862.
49. Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, Zhang YT, Yao X, Chu K, Chen QH et al.: Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebocontrolled, phase 3 trial. Lancet 2013, 381:2024-2032. This paper assessed the efficacy, safety, immunogenicity, antibody persistence, and immunological correlates of an inactivated alum-adjuvant EV71 vaccine in phase III trial 50. Zhu FC, Liang ZL, Li XL, Ge HM, Meng FY, Mao QY, Zhang YT, Hu YM, Zhang ZY, Li JX et al.: Immunogenicity and safety of an enterovirus 71 vaccine in healthy Chinese children and infants: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet 2013, 381:1037-1045. 51. Liang Z, Mao Q, Gao Q, Li X, Dong C, Yu X, Yao X, Li F, Yin W, Li Q et al.: Establishing China’s national standards of antigen
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54. Lin YL, Yu CI, Hu YC, Tsai TJ, Kuo YC, Chi WK, Lin AN, Chiang BL: Enterovirus type 71 neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles. Vaccine 2012, 30:1305-1312. 55. Chung YC, Huang JH, Lai CW, Sheng HC, Shih SR, Ho MS, Hu YC: Expression, purification and characterization of enterovirus-71 virus-like particles. World J Gastroenterol 2006, 12:921-927. 56. Li HY, Han JF, Qin CF, Chen R: Virus-like particles for enterovirus 71 produced from Saccharomyces cerevisiae potently elicits protective immune responses in mice. Vaccine 2013, 31:3281-3287.
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ScienceDirect Update on enterovirus 71 infection Peng-Nien Huang1,3 and Shin-Ru Shih1,2,3,4 Human enterovirus type 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide. The identified EV71 receptors provide useful information for understanding EV71replication and tissue tropism. Host factors interact with the internal ribosome entry site (IRES) of EV71 to regulate viral translation. However, the specific molecular features of the EV71 genome that determine virulence remain unclear. The EV71 capsid protein VP1 region might contribute to virulence and neurotropism. Transgenic mice expressing the EV71 receptor that were infected with the virus exhibited a disease similar to that observed in infected humans. Antiviral drug and vaccine development is urgently required to prevent EV71 epidemics. Delineating viral host interactions and identifying specific mechanisms that might control the neural tropism of EV71 pathogenesis would be substantial advances. Addresses 1 Research Center for Emerging Viral Infections, Chang Gung University, Taiwan, ROC 2 Graduate Institute of Biomedical Science, Chang Gung University, Taiwan, ROC 3 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taiwan, ROC 4 Clinical Virology Laboratory, Department of Clinical Pathology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, ROC Corresponding author: Shih, Shin-Ru ([email protected], [email protected])
Current Opinion in Virology 2014, 5:98–104 This review comes from a themed issue on Emerging viruses Edited by Chris F Basler and Patrick C Y Woo
1879-6257/$ – see front matter, Published by Elsevier B.V. http://dx.doi.org/10.1016/j.coviro.2014.03.007
Introduction Enterovirus type 71 (EV71) is a member of the human enterovirus species A, and of the family Picornaviridae. In 1969, EV71 was first isolated in California, United States; and was first recognized as a cause of epidemics of handfoot-and-mouth disease (HFMD) in Japan in 1973 [1,2]. EV71, a major pathogen, is responsible for HFMD and has been associated with severe neurological complications and even fatalities in infants and young children worldwide [3,4]. In the 2000s, several countries, including Taiwan, Japan, Malaysia, Singapore, and Vietnam, experienced cyclical epidemics occurring every 2 or 3 years [3]. Since 2008, emerging epidemics of HFMD associated with EV71 have become critical in mainland Current Opinion in Virology 2014, 5:98–104
China [5,6]. In addition, in the past ten years, EV71 was isolated in numerous European and American countries such as France [7,8], Denmark [9], Spain [10], Portugal [11], Brazil [12], Canada [13], and the United States. The geographic distribution of EV71 isolates in the world is summarized in Figure 1, showing that EV71 was identified as an emerging virus worldwide. This review focuses on recent work describing EV71 receptors, host factors involved in viral translation, the EV71 animal model, vaccines, and antiviral agents. The geographic range of EV71 infection is primarily the Asia-Pacific region. However, EV71 infection was also reported in countries in America and Europe, in the past ten years.
Cellular receptors for enterovirus 71 Two human transmembrane proteins, P-selectin glycoprotein ligand-1 (PSGL-1) and scavenger receptor class B, member 2 (SCARB2), have recently been identified as functional receptors for EV71 entry [14,15]. Expression of PSGL-1 enabled EV71 entry and replication, as well as the development of cytopathic effects in unsusceptible mouse L cells [15]. PSGL-1 is a key factor involved in early inflammatory events on immune cells. Post-translational modifications of PSGL-1 regulate PSGL-1 and selectin interaction. Through experiments, a post-translational modification, tyrosine sulfation, at the N-terminal region of PSGL-1 was identified; this modification, is crucial for the binding of PSGL-1 to EV71 and viral replication in lymphocytes [16]. Some EV71 strains did not use PSGL-1 as a receptor to enter cells, suggesting the occurrence of strain-specific replication of EV71 in immune cells [15]. A single amino acid, residue 145 of the viral capsid protein VP1, determines whether a virus binds to the viral receptor PSGL-1 in lymphocytes. When VP1-145G/Q was replaced with E, the PSGL-1-binding strains lost their capacity to bind. Conversely, when VP1145E was replaced with either G or Q nonbinding strains, the strains bound to PSGL-1. The EV71 residue VP1-145 acts as a switch that controls the binding of the virus to PSGL-1 [17]. Unlike PSGL-1, SCARB2 can be used by most EV71 strains as an entry receptor, indicating that SCARB2 plays a crucial role in the entry steps of EV71 infection [14,15]. SCARB2 is widely expressed on various cell types, including neurons, which are directly involved in EV71 infection of the brain. The expression of human SCARB2 permitted typically unsusceptible cell lines to efficiently produce EV71 and develop cytopathic effects [14]. The unsusceptible cells expressing only amino www.sciencedirect.com
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Figure 1
Sweden Norway
Canada
Denmark Netherlands Hungary France
United Kingdom
United States
Potugal Spain
South Korea
Bulgaria
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China
Taiwan Thailand
Vietnam Cambodia Brunei Malaysia
Brazil
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Reported Enterovirus 71 Infection No Reported Enterovirus 71 Infection
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Regions with reported human enterovirus 71 infection.
acids 142–204 of the human SCARB2 sequence were susceptible to EV71. Determination the region in human SCARB2 that is crucial for EV71 binding and infection greatly contributes to the understanding of virus–receptor interactions [18]. The EV71 clinical isolates propagated in human SCARB2 expression cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains [19]. Experiments expressed ectopic PSGL1 or SCARB2 in mouse L929 cells which these two proteins are absent. They used these two cells to compare the function of these two kind receptors. A comparison of the infection efficacies between mouse L cells expressing either SCARB2 or PSGL-1 indicated that L-SCARB2 cells are more susceptible to EV71 infection than LPSGL1 cells. However, L-SCARB2 cells bound with a reduced amount of EV71 compared with L-PSGL1 cells in a virus attachment assay. The binding abilities indicate the 35S-labeled EV71 particles mixed with receptor expressing cells. After the unbound virions were removed by washing, the cells were lysed and the radioactivity was measured as binding capacities. The results indicated that the difference in the binding abilities of the two receptors was not the sole determinant of the infection efficiency. The low infection efficiency of L-PSGL1 cells is due to the inability of PSGL-1 to induce viral uncoating [20]. SCARB2, as an uncoating receptor, contributes considerably to the understanding of the early steps of EV71 infection. The characterization of EV71 receptors www.sciencedirect.com
provides useful information for understanding EV71 replication at a molecular level.
Host factors involved in enterovirus 71 translation EV71 50 untranslated region (50 UTR) RNA contains a type I internal ribosomal entry site (IRES). This region of the EV71 genome is approximately 500 nt. The type I IRES exhibits poor efficiency in initiating viral translation in the absence of certain cellular proteins [21]. IRESdependent viral translation requires numerous trans-acting host factors, known as IRES trans-acting factors (ITAFs). These ITAF proteins play various roles, such as binding to viral RNA across multiple domains, as well as stabilizing the entire IRES in a structure that is suitable for binding canonical translation factors and ribosomal subunits [22]. Several recent reports have stated that ITAFs interact with the EV71 IRES. Lin et al. used streptavidin beads to pull down the host factors interacting with biotinylated EV71 50 UTR RNA. After matrixassisted laser desorption ionization-time of flight mass spectrometry analysis, 12 host factors interacted with the EV71 50 UTR. Some of these host factors were previously observed to interact with the 50 UTR of picornaviruses and to enhance virus replication. Some of these host factors were new ITAFs for RNA viruses [23]. For example, far upstream element binding protein 1 and 2 (FBP1 and FBP2) are novel ITAFs for EV71. An RNA Current Opinion in Virology 2014, 5:98–104
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pulldown assay indicated that FBP2 specifically bound to the EV71 IRES region. Viral protein synthesis and EV71 IRES activity were increased when cells were transfected with the siRNA targeting FBP2. The host factor FBP2 out-competed the polypyrimidine tract-binding protein, the positive-acting ITAF of picornaviral IRESs, in binding to the EV71 IRES. Experiments indicated that FBP2 is a negative regulator of EV71 IRES activity [23]. EV71 infection also triggers the virus-induced proteasome, autophagy, and caspase activity mechanism for the host cells to cleave FBP2, the negative regulator of viralIRES-driven translation. Moreover, the cleavage fragments of FBP2 alter the function of FBP2 from a negative regulator to a positive promoter during viral translation [24]. In contrast to FBP2, FBP1 enhanced the IRES activity of EV71 RNA and viral replication activity [25]. Both FBP1 and FBP2 bind to the region of the EV71 50 UTR containing the linker region (nt 637–745). Competition binding assays indicated that FBP1 and FBP2 competed to bind to the same region [25]. The FBP1 and FBP2 nuclear proteins were observed to relocate from the nucleus to the cytoplasm after EV71 infection [23,25]. However, the opposite effects of these two highly similar host factors on EV71 IRES activity must be further defined. Another nuclear protein, hnRNP A1, was identified as an EV71 ITAF by using a streptavidin beads pulldown assay [23], and is required for EV71 replication because it facilitates viral translation [26]. The mechanism describing how ITAFs regulate EV71 IRES activity is summarized in Figure 2. The new cellular factors associated with EV71 IRES may provide a novel route to new antiviral therapies. Nuclear proteins FBP1, FBP2, and hnRNP A1 relocate from the nucleus to the cytosol during EV71 infection. FBP1, hnRNP A1, and other ITAFs bind to the EV71 50 UTR and play positive roles in EV71 IRES activity. FBP2 has the opposite effect. FBP1 and FBP2 compete to bind to the linker region. EV71 infection also triggers the virusinduced proteasome, autophagy, and caspase activity mechanism to cleave FBP2. The cleaved fragments of FBP2 alter the function of FBP2 from a negative regulator to a positive regulator of IRES activity.
Molecular factor in the enterovirus 71 genome Compared with other non-polio enteroviruses, EV71 is an enterovirus that can increase mortality in children, but the specific molecular features that determine its virulence remain unknown. Most EV71 strains are unable to cause clinically apparent infection in mice. A mouse-adapted EV71 strain that belongs to sub-genogroup B5 was reported to increase virulence in newborn BALB/c mice when the virus exhibited a capsid protein VP1-K244E mutation, indicating that the VP1-K244E mutation is a crucial genetic determinant of mouse adaptation and Current Opinion in Virology 2014, 5:98–104
virulence [27]. Another study reported that the EV71 sub-genogroup C4 expressing the VP1-Q145E mutation was virulent in 5-day-old mice, which exhibited a high mortality rate [28]. Another mouse study by Huang et al. indicated that EV71 capsid proteins of VP2 (149M) and VP1 (145E) mutations enhanced the viral binding and RNA accumulation of EV71, contributing to virusinduced mortality [29]. In a human study, Cordey et al. analyzed the EV71 genome by using blood, stool, bronchoalveolar lavage fluid, and cerebrospinal fluid specimens from an immunocompromised patient. They observed a non-conservative amino acid change in VP1 (L97R) within the BC loop. In vitro cell tropism assays indicated that VP1 (L97R) conferred a replicative advantage in SHSY5Y cells of neuroblastoma origin. This study suggests that the VP1 BC loop region of EV71 may play an essential role in the cell tropism of EV71 infection and the VP1 region may contribute to dissemination and neurotropism [30]. In summary, a major mutation in the EV71 VP1 capsid protein can generate a mouseadaptive and virulent EV71 strain. The emergence of mutations at critical regions of the EV71 genome can produce new phenotypes and neurovirulence in humans.
Enterovirus 71 infection and disease animal models In the past decade, epidemiological and clinical analyses have revealed that the major target of EV71 infection is the central nervous system (CNS) [31]. Several studies have indicated that EV71 circulation in the blood might be the mechanism of dissemination from the body to the CNS [32]. Studies involving rhesus monkeys have demonstrated that EV71 infection causes the virus to injure the CNS at various rates when the virus is administered through different routes. In one study, intracerebral inoculation resulted in hemorrhages, pulmonary edemas, and neuron impairment, whereas intravenous and respiratory inoculations caused a direct infection of the CNS, accompanied by clear inflammation of the lung tissue [33]. This study similarly suggested that the rhesus monkey animal model may be suitable for studying the pathogenesis of EV71 [33]. Although most of the mouse studies show CNS involvement, a controversial result indicated that EV71 primarily replicated in skeletal muscle tissues, causing severe necrotizing myositis. In the aforementioned study, lesions in the CNS and other tissues were not observed in 2-week-old EV71-infected mice. Necrotizing myositis of respiratory-related muscles caused severe restrictive hypoventilation and subsequent hypoxia, which may explain the fatality of 2-week-old EV71-infected mice [34]. The mouse age and strains used for challenge may cause the discrepancy. Previous studies have used rhesus monkeys and neonatal mice that are susceptible to EV71 strains. The rhesus monkey model exhibits ethical and economic problems, whereas, mice that are older than a few weeks lose their susceptibility to EV71. Researchers have generated transgenic mice www.sciencedirect.com
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Figure 2
Negative ITAF FBP2 proteasome, autophagy, caspase Other ITAFs
Cleavage of FBP2 Positive ITAF
Linker IRES hnRNP A1
Positive ITAF
Viral RNA FBP1
IRES activity ↑
Positive ITAF
Viral protein ↑
Cytoplasm FBP2
Nucleus FBP1 hnRNP A1
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Overview of the host nuclear proteins behaves as ITAFs to regulate EV71 viral translation.
expressing the EV71 receptor human SCARB2 (hSCARB2). Transgenic mice infected with EV71 exhibited ataxia, paralysis, and death. The pathological features in these transgenic mice were similar to those of EV71 encephalomyelitis in humans and experimentally infected monkeys. Experiments have suggested that the hSCARB2 transgenic mouse model may be a useful animal model for studying EV71 infection and an attractive platform for EV71 vaccine and drug testing [35,36]. Conversely, another study that used transgenic mice indicated that the expression of human PSGL-1 failed to increase the infectivity of EV71. The results indicated that PSGL-1 alone may be insufficient for infection with EV71 [37]. The hSCARB2 transgenic mice can be used to experimentally evaluate the neurovirulence of EV71 with greater statistical significance in the future. However, an ideal model system has not been achieved because the ideal animal model for EV71 infection reproduces the crucial features of the human disease, including neurological symptoms, cardiac dysfunction, and pulmonary edema.
Anti-enterovirus 71 drug research Currently, no effective antiviral drug against EV71 exists. Several recent studies have demonstrated the anti-EV71 www.sciencedirect.com
activity of natural products and other compounds [38,39]. Kappa carrageenan from seaweed, for example, exhibits anti-EV71 behavior. This natural product was reported to reduce plaque formation and prevent viral replication before and during viral absorption of EV71. A virus binding assay indicated that kappa carrageenan can bind to EV71 and disrupt the virus–receptor interaction [39]. Plevka et al. studied the structure of EV71 with the WIN 51711capsid-binding inhibitor. They found that the inhibitor replaced natural pocket factor within the viral protein 1 pocket without changing the structure of the capsid [40,41]. Another research team analyzed the EV71 inhibitors 3-(4-pyridyl)-2-imidazolidinone derivatives structure. They used quantum mechanics to identify additional potentially beneficial substitutions, enhanced ligand docking and synthesized two candidates. They demonstrated that one ligand is an order of magnitude more potent than the previously reported inhibitor 3-(4pyridyl)-2-imidazolidinone [42]. These researches indicated that structural analysis could be a new way to the development of anti-EV71 capsid-binding drugs [40–42]. The monoclonal antibody E18 was prepared by immunizing mice with empty particles of EV71. The researchers indicated that binding of E18 to EV71 causes the virus Current Opinion in Virology 2014, 5:98–104
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to change its conformation and to eject much of viral genome. The E18 antibody mediated virus neutralization by the induction of viral genome release. This study demonstrated that E18 antibody has potential for an anti-EV71 therapy [43]. A six-amino-acid peptide known as LVLQTM was previously shown to inhibit rhinovirus 2A protease. This research indicated that the peptide is an effective substrate against EV71 2A protease. The LVLQTM peptide considerably inhibited the eIF4G cleavage activity of 2A protease during EV71 replication in HeLa cells. This study expanded the development of new antiviral drugs against EV71 from small molecules to peptide drugs [44]. In another study, a synthetic peptide was developed that could be a potential antiviral compound that inhibits EV71. Ninety-five synthetic peptides that overlap the EV71 VP1 capsid protein were tested for use in an antiviral drug against EV71. The peptide was observed to reduce considerably the cytopathic effects of all representative EV71 strains [45]. Deng et al. identified the 50 UTR of the EV71 genome as highly conserved and an appropriate target of siRNA to inhibit viral replication effectively. Transfection of 20 -modified siRNAs targeting the 50 UTR delayed and reduced the cytopathic effects on EV71-infected cells. These 20 -modified siRNAs provide a novel source for studying the effects of RNAi-based antiviral EV71 drugs on EV71 infection [46]. Numerous opportunities appear to be available for developing effective antiviral drugs against EV71.
EV71 vaccines are currently undergoing a phase III clinical trial and the EV71 vaccine will be available in the near future, further study on the mechanism of EV71 infection is still necessary. EV71 can be regarded as a general model for other picornaviruses. Research has indicated certain factors that regulate EV71 replication. However, specific factors that contribute to EV71 associated neural pathogenesis remain unclear. Thus, dissecting viral host interaction and identifying specific mechanisms that may control the neural tropism of EV71 infection is an urgent need. Such studies would facilitate understanding of the neuropathogenesis of EV71 infection, as well as that of several other neurotropic RNA viruses. Antiviral agents against neurotropic RNA viruses with a broader spectrum may be developed based on such investigations.
Acknowledgements This work was supported by funding from the National Science Council of the Republic of China, Taiwan (NSC-102-2325-B-182-015) and Chang Gung Memorial Hospital (CMRPD1A0673).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Schmidt NJ, Lennette EH, Ho HH: An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 1974, 129:304-309.
2.
Hagiwara A, Tagaya I, Yoneyama T: Epidemic of hand, foot and mouth disease associated with enterovirus 71 infection. Intervirology 1978, 9:60-63.
3.
Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH: Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 2010, 10:778-790.
4.
Weng KF, Chen LL, Huang PN, Shih SR: Neural pathogenesis of enterovirus 71 infection. Microbes Infect 2010, 12:505-510.
5.
Tan X, Huang X, Zhu S, Chen H, Yu Q, Wang H, Huo X, Zhou J, Wu Y, Yan D et al.: The persistent circulation of enterovirus 71 in People’s Republic of China: causing emerging nationwide epidemics since 2008. PLoS ONE 2011, 6:e25662.
6.
Xing W, Liao Q, Viboud C, Zhang J, Sun J, Wu JT, Chang Z, Liu F, Fang VJ, Zheng Y et al.: Hand, foot, and mouth disease in China, 2008–12: an epidemiological study. Lancet Infect Dis 2014, 6:308-318.
7.
Schuffenecker I, Mirand A, Antona D, Henquell C, Chomel JJ, Archimbaud C, Billaud G, Peigue-Lafeuille H, Lina B, Bailly JL: Epidemiology of human enterovirus 71 infections in France, 2000–2009. J Clin Virol 2011, 50:50-56.
8.
Kassab S, Saghi T, Boyer A, Lafon ME, Gruson D, Lina B, Fleury H, Schuffenecker I: Fatal case of enterovirus 71 infection and rituximab therapy, France, 2012. Emerg Infect Dis 2013, 19:1345-1347.
9.
Badran SA, Midgley S, Andersen P, Bottiger B: Clinical and virological features of enterovirus 71 infections in Denmark, 2005 to 2008. Scand J Infect Dis 2011, 43:642-648.
Enterovirus 71 vaccine development An EV71 vaccine is urgently required to control and prevent epidemics of EV71. However, no vaccines against EV71 exist [47,48]. These two research groups generated the inactivated alum-adjuvant EV71 vaccine, which was administered in a randomized, double-blind, placebo-controlled, phase III trial and proved to provide high efficacy, satisfactory safety, and sustained immunogenicity [49,50–52]. Virus-like particle (VLP)-based vaccines have been investigated. EV71 VLPs produced in a baculovirus expression system were highly immunogenic in mice and monkeys [53–55]. In the studies, the researchers generated a platform for producing of VLPs for EV71 in Saccharomyces cerevisiae by co-expressing the P1 and 3CD genes of EV71. These VLPs exhibited a protein composition similar to that of the EV71 empty particles produced by EV71-infected cells. In vivo challenge experiments have indicated that immune sera induced by VLPs conferred protection in neonatal mice against a lethal EV71 challenge. In summary, these studies have indicated that a VLP from yeast is another potential vaccine candidate against EV71 infection [56]. In the near future, we believe that a vaccine against EV71 infection will be available.
Conclusion In the past 15 years, EV71 has circulated and emerged as a major cause of viral encephalitis and HFMD in the AsianPacific region. Although the inactivated whole-virus Current Opinion in Virology 2014, 5:98–104
10. Cabrerizo M, Tarrago D, Munoz-Almagro C, Del Amo E, DominguezGil M, Eiros JM, Lopez-Miragaya I, Perez C, Reina J, Otero A et al.: Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain. Clin Microbiol Infect 2013, 20:O150-O156. www.sciencedirect.com
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11. Venancio P, Oliveira M, Silva R, Conceicao C, Brito MJ: First case of severe enterovirus 71 infection in Portugal. Pediatr Infect Dis J 2013, 32:581-582. 12. Lamarao LM, Maciel AM, Gomes MdL: First isolation of enterovirus 71 (EV-71) from Northern Brazil. Braz J Infect Dis 2003, 7:278-281. 13. Merovitz L, Demers AM, Newby D, McDonald J: Enterovirus 71 infections at a Canadian center. Pediatr Infect Dis J 2000, 19:755-757. 14. Yamayoshi S, Yamashita Y, Li J, Hanagata N, Minowa T, Takemura T, Koike S: Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med 2009, 15:798-801. This paper found that SCARB2 as an EV71 and CVA16 receptor contributes to a better understanding of the pathogenicity of these viruses. 15. Nishimura Y, Shimojima M, Tano Y, Miyamura T, Wakita T, Shimizu H: Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat Med 2009, 15:794-797. This paper found that Stable PSGL-1 expression allowed EV71 entry and replication, and development of cytopathic effects in nonsusceptible cells. 16. Nishimura Y, Wakita T, Shimizu H: Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection. PLoS Pathog 2010, 6:e1001174. 17. Nishimura Y, Lee H, Hafenstein S, Kataoka C, Wakita T, Bergelson JM, Shimizu H: Enterovirus 71 binding to PSGL-1 on leukocytes: VP 1–145 acts as a molecular switch to control receptor interaction. PLoS Pathog 2013, 9:e1003511.
generates a mouse-virulent phenotype. J Gen Virol 2012, 93:1935-1940. 29. Huang SW, Wang YF, Yu CK, Su IJ, Wang JR: Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement. Virology 2012, 422:132-143. 30. Cordey S, Petty TJ, Schibler M, Martinez Y, Gerlach D, van Belle S, Turin L, Zdobnov E, Kaiser L, Tapparel C: Identification of sitespecific adaptations conferring increased neural cell tropism during human enterovirus 71 infection. PLoS Pathog 2012, 8:e1002826. This paper show that the VP1 BC loop region of EV71 plays a critical role in cell tropism and neurotropism in the immunocompromised patient 31. Lin TY, Hsia SH, Huang YC, Wu CT, Chang LY: Proinflammatory cytokine reactions in enterovirus 71 infections of the central nervous system. Clin Infect Dis 2003, 36:269-274. 32. Dolin R: Enterovirus 71 — emerging infections and emerging questions. N Engl J Med 1999, 341:984-985. 33. Zhang Y, Cui W, Liu L, Wang J, Zhao H, Liao Y, Na R, Dong C, Wang L, Xie Z et al.: Pathogenesis study of enterovirus 71 infection in rhesus monkeys. Lab Invest 2011, 91:1337-1350. 34. Xiu JH, Zhu H, Xu YF, Liu JN, Xia XZ, Zhang LF: Necrotizing myositis causes restrictive hypoventilation in a mouse model for human enterovirus 71 infection. Virol J 2013, 10:215.
18. Yamayoshi S, Koike S: Identification of a human SCARB2 region that is important for enterovirus 71 binding and infection. J Virol 2011, 85:4937-4946.
35. Fujii K, Nagata N, Sato Y, Ong KC, Wong KT, Yamayoshi S, Shimanuki M, Shitara H, Taya C, Koike S: Transgenic mouse model for the study of enterovirus 71 neuropathogenesis. Proc Natl Acad Sci U S A 2013, 110:14753-14758. This paper suggests that this transgenic mouse could represent a useful animal model for the study of EV71 infection.
19. Yamayoshi S, Iizuka S, Yamashita T, Minagawa H, Mizuta K, Okamoto M, Nishimura H, Sanjoh K, Katsushima N, Itagaki T et al.: Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71. J Virol 2012, 86:5686-5696.
36. Lin YW, Yu SL, Shao HY, Lin HY, Liu CC, Hsiao KN, Chitra E, Tsou YL, Chang HW, Sia C et al.: Human SCARB2 transgenic mice as an infectious animal model for enterovirus 71. PLoS ONE 2013, 8:e57591.
20. Yamayoshi S, Ohka S, Fujii K, Koike S: Functional comparison of SCARB2 and PSGL1 as receptors for enterovirus 71. J Virol 2013, 87:3335-3347. This paper compared the EV71 infection efficiencies of cells that expressed SCARB2 and PSGL-1 and the abilities of SCARB2 and PSGL1 to bind to the virus.
37. Li YP, Liang ZL, Gao Q, Huang LR, Mao QY, Wen SQ, Liu Y, Yin WD, Li RC, Wang JZ: Safety and immunogenicity of a novel human enterovirus 71 (EV71) vaccine: a randomized, placebocontrolled, double-blind, Phase I clinical trial. Vaccine 2012, 30:3295-3303.
21. Thompson SR, Sarnow P: Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved. Virology 2003, 315:259-266. 22. Pilipenko EV, Viktorova EG, Guest ST, Agol VI, Roos RP: Cellspecific proteins regulate viral RNA translation and virusinduced disease. EMBO J 2001, 20:6899-6908. 23. Lin JY, Li ML, Shih SR: Far upstream element binding protein 2 interacts with enterovirus 71 internal ribosomal entry site and negatively regulates viral translation. Nucleic Acids Res 2009, 37:47-59. 24. Chen LL, Kung YA, Weng KF, Lin JY, Horng JT, Shih SR: Enterovirus 71 infection cleaves a negative regulator for viral internal ribosomal entry site-driven translation. J Virol 2013, 87:3828-3838.
38. Chang CW, Leu YL, Horng JT: Daphne Genkwa sieb. Et zucc. Water-soluble extracts act on enterovirus 71 by inhibiting viral entry. Viruses 2012, 4:539-556. 39. Chiu YH, Chan YL, Tsai LW, Li TL, Wu CJ: Prevention of human enterovirus 71 infection by kappa carrageenan. Antiviral Res 2012, 95:128-134. 40. Plevka P, Perera R, Yap ML, Cardosa J, Kuhn RJ, Rossmann MG: Structure of human enterovirus 71 in complex with a capsidbinding inhibitor. Proc Natl Acad Sci U S A 2013, 110:5463-5467. 41. Plevka P, Perera R, Cardosa J, Kuhn RJ, Rossmann MG: Crystal structure of human enterovirus 71. Science 2012, 336:1274. 42. De Colibus L, Wang X, Spyrou JA, Kelly J, Ren J, Grimes J, Puerstinger G, Stonehouse N, Walter TS, Hu Z et al.: Morepowerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules. Nat Struct Mol Biol 2014, 21:282-288.
25. Huang PN, Lin JY, Locker N, Kung YA, Hung CT, Huang HI, Li ML, Shih SR: 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 2011, 39:9633-9648.
43. Plevka P, Lim PY, Perera R, Cardosa J, Suksatu A, Kuhn RJ, Rossmann MG: Neutralizing antibodies can initiate genome release from human enterovirus 71. Proc Natl Acad Sci U S A 2014, 111:2134-2139.
26. Lin JY, Shih SR, Pan M, Li C, Lue CF, Stollar V, Li ML: hnRNP A1 interacts with the 50 untranslated regions of enterovirus 71 and Sindbis virus RNA and is required for viral replication. J Virol 2009, 83:6106-6114.
44. Falah N, Montserret R, Lelogeais V, Schuffenecker I, Lina B, Cortay JC, Violot S: Blocking human enterovirus 71 replication by targeting viral 2A protease. J Antimicrob Chemother 2012, 67:2865-2869.
27. Zaini Z, Phuektes P, McMinn P: Mouse adaptation of a subgenogroup B5 strain of human enterovirus 71 is associated with a novel lysine to glutamic acid substitution at position 244 in protein VP1. Virus Res 2012, 167:86-96.
45. Tan CW, Chan YF, Sim KM, Tan EL, Poh CL: Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1. PLoS ONE 2012, 7:e34589.
28. Zaini Z, McMinn P: A single mutation in capsid protein VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71
46. Deng JX, Nie XJ, Lei YF, Ma CF, Xu DL, Li B, Xu ZK, Zhang GC: The highly conserved 50 untranslated region as an effective target
www.sciencedirect.com
Current Opinion in Virology 2014, 5:98–104
104 Emerging viruses
towards the inhibition of enterovirus 71 replication by unmodified and appropriate 20 -modified siRNAs. J Biomed Sci 2012, 19:73.
content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines. Vaccine 2011, 29:9668-9674.
47. Xu J, Qian Y, Wang S, Serrano JM, Li W, Huang Z, Lu S: EV71: an emerging infectious disease vaccine target in the Far East? Vaccine 2010, 35(28):6-3521.
52. Li R, Liu L, Mo Z, Wang X, Xia J, Liang Z, Zhang Y, Li Y, Mao Q, Wang J et al.: An inactivated enterovirus 71 vaccine in healthy children. N Engl J Med 2014, 370:829-837.
48. Qiu J: Enterovirus 71 infection: a new threat to global public health? Lancet Neurol 2008, 7:868-869.
53. Chung YC, Ho MS, Wu JC, Chen WJ, Huang JH, Chou ST, Hu YC: Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge. Vaccine 2008, 26:1855-1862.
49. Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, Zhang YT, Yao X, Chu K, Chen QH et al.: Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebocontrolled, phase 3 trial. Lancet 2013, 381:2024-2032. This paper assessed the efficacy, safety, immunogenicity, antibody persistence, and immunological correlates of an inactivated alum-adjuvant EV71 vaccine in phase III trial 50. Zhu FC, Liang ZL, Li XL, Ge HM, Meng FY, Mao QY, Zhang YT, Hu YM, Zhang ZY, Li JX et al.: Immunogenicity and safety of an enterovirus 71 vaccine in healthy Chinese children and infants: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet 2013, 381:1037-1045. 51. Liang Z, Mao Q, Gao Q, Li X, Dong C, Yu X, Yao X, Li F, Yin W, Li Q et al.: Establishing China’s national standards of antigen
Current Opinion in Virology 2014, 5:98–104
54. Lin YL, Yu CI, Hu YC, Tsai TJ, Kuo YC, Chi WK, Lin AN, Chiang BL: Enterovirus type 71 neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles. Vaccine 2012, 30:1305-1312. 55. Chung YC, Huang JH, Lai CW, Sheng HC, Shih SR, Ho MS, Hu YC: Expression, purification and characterization of enterovirus-71 virus-like particles. World J Gastroenterol 2006, 12:921-927. 56. Li HY, Han JF, Qin CF, Chen R: Virus-like particles for enterovirus 71 produced from Saccharomyces cerevisiae potently elicits protective immune responses in mice. Vaccine 2013, 31:3281-3287.
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