8. Enterovirus 71 Encephalitis LUAN-YIN CHANG, SHIN-RU SHIH, LI-MIN HUANG, AND TZOU-YIEN LIN 8.1. Virology Enterovirus 71 (EV71) was first described in 1974 after it was isolated from patients in California (Schmidt et al., 1974) and has been associated with sporadic cases or outbreaks of a wide spectrum of diseases, including hand, foot, and mouth disease (HFMD), herpangina, aseptic meningitis, encephalitis, cerebellar ataxia, poliomyelitis-like syn- drome, and even fatal disease. Since the poliovirus has been eradicated, EV71 has become one of the most important enteroviruses known to cause fatalities and sequelae in children. Therefore, understanding its virology, epidemiology, clinical manifestations, treatment management, diagnosis, and prevention is important. 8.1.1. Virological Classification of Enterovirus 71 Enterovirus 71 is an enterovirus. Enteroviruses belong to Picornaviridae, a family of single-stranded, positive-sense RNA viruses. They can be classified into four groups: enteroviruses (e.g., poliovirus), rhinoviruses (e.g., human rhinovirus), cardioviruses (e.g., Mengo virus), and aphthovirus (e.g., foot-and-mouth virus). The rhinovirus and enterovirus genera are most closely related; the cardioviruses fall halfway between the two closely related ones and the foot-and-mouth virus, FMDV. Serologic studies, using neutralization tests with antisera against enteroviruses, have identified more than 67 human enterovirus serotypes: poliovirus serotypes 1–3, 23 coxsackie A virus serotypes, 6 coxsackie B virus serotypes, 31 echovirus serotypes, and the numbered enterovirus serotypes (enteroviruses 68–71). 295 296 L-Y. Chang et al. 8.1.2. General Characteristics of Enterovirus Most enteroviruses replicate in primate cells. The specific receptor on the surface of the host cell can be used to identify the host range and tis- sue tropism. The specific receptors of poliovirus and rhinovirus have been identified as belonging to the superimmunoglobulin family of proteins. About 90% of all rhinoviruses use intercellular adhesion molecule-1 (ICAM-1; an immunoglobulin-like molecule with five domains) as a receptor (Greve et al., 1989), whereas polioviruses use PVR (CD155, an immunoglobulin-like molecule with three domains) (Mendelsohn et al., 1989). Echoviruses use integrin very late antigen, VLA-2 (an integrin-like molecule) (Bergelson et al., 1992 ) or decay accelerating factor (DAF; CD55) as their receptors. Coxsackievirus B uses coxsackie-adenovirus receptor (CAR) and enterovirus 70 uses DAF as its receptor. The specific receptor for enterovirus 71 remains unidentified. 8.1.3. Virion Structure of Enterovirus Structurally, enterovirus is a small, nonenveloped, icosahedrally sym- metrical spherical particle ~30 nm in diameter. The virus has a single- stranded, positive-sense RNA and simple protein capsid. The protein capsid has 60 copies of each of the structural proteins VP1, VP2, VP3, and VP4 (Rossmann et al., 1985 ). The protein capsid protects viral RNA from nucle- ase cleavage, recognizes the receptors on the surfaces of the specific host cells, and displays antigenicity. VP1-3, located on the virion surface, are the neutralization epitopes. Sequence comparisons and phylogenetic reconstructions suggest that VP1 contains serotype-specific information, which can be used to identify viruses and study evolution. Because enterovirus serotypic differentiation is based on neutralization and the VP1 sequence correlates with neutral- ization type, it is logical to assume that molecular diagnosis could be used to target the VP1 coding region and should be able to produce typing results that correlate with the serotype identified by neutralization with type-specific antisera (Rigonan et al., 1998 ). VP4, which is located within the capsid and anchors the RNA genome to the capsid, interacts with the N termini of VP1 to enhance the genomic stability. Each of the proteins, VP 1–3, contains different amino acid sequences, but all share an eight-stranded, antiparallel β-barrel motif with a similar tertiary structure. Interestingly, a special structure, the hydrophobic pocket, is located beneath the canyon floor on the surface of VP1. According to the “canyon hypothesis” (Rossmann, 1989a, 1989b), cell attachment occurs at a depression on the surface, which is inaccessi- ble to neutralizing antibody. This depression can be said to shield the 8. Enterovirus 71 Encephalitis 297 invading virus from immune surveillance. Such depressions have been found on the surface of HRV14, poliovirus, and Mengo virus, and corrob- orative evidence supports their existence in the case of HRV. 8.1.4. Replication Cycle of Enterovirus Enterovirus infection begins with the virus attaching itself to a single receptor unit on the host cells. This binding is reversible. After attach- ment, the virion undergoes an irreversible conversion to form the 135S, or A, particle. The 135S particle has a lower sedimentation coefficient than the native virion particle (135S vs. 160S for the poliovirus), and the anti- genic and proteolytic properties of the 135S particle change and become different from those of the unattached virus. Upon conversion, cell attach- ment is mediated by externalized VP4 and the N termini of VP1. The externalized N termini of VP1 form an amphipathic helix and is inserted into the cell membrane to produce a pore through which the viral RNA can leave the capsid. This stage is called the uncoating stage. Later, the 135S particle releases its RNA during its conversion to the 80S (or H) par- ticle, and the viral RNA can be used as the translation template to synthe- size viral proteins and to amplify viral positive-sense RNA genome (Belnap et al., 2000; Huang et al., 2000). During advanced infection, viral capsid proteins VP 1–4 form the capsid, and the progeny viral RNAs are packaged into mature virion. The newly synthesized viruses can infect neighboring cells by disrupting the old cells and causing their release. Picornavirus replication is rapid, and a cycle is completed in 5–10 h. After ~1–2 h of initial infection/attachment, the host cellular macro- molecular synthesis sharply declines in a process called “shutoff,” and chromatin marginates. From 2.5 to 3 h postinfection, viral proteins begin synthesis and cytoplasm vacuolates. At 6 h postinfection, newly synthe- sized viral RNA and proteins assemble in the cytoplasm. The infected cell lyses and releases the virus particle at 6–10 h after being infected. 8.1.5. Genotypes and Neurovirulence of EV71 In order to understand the virological basis of neurovirulence, Shih et al. (2000b) analyzed the nucleotide sequence of VP1, which is impor- tant for serotypic specificity, and the 5′-non-coding region (5′-NCR), which is important for efficient replication. Phylogenic analysis of both VP1 and 5′-NCR of nine EV71 isolates derived from specimens of fatal patients and seven isolates derived from uncomplicated HFMD patients of the 1998 epidemic showed that all but one isolate could be categorized as being of the genotype C. The one distinct isolate (TW/1743/98) from a 298 L-Y. Chang et al. case of uncomplicated HFMD belonged to genotype B and was clustered along with one strain (TW/253/86) that was isolated from the southern Taiwan in 1986 (Shih et al., 2000b). Shih et al. (2000b) further analyzed complete sequences of two selected EV71 strains from the spinal cord of a fatal case and from vesi- cles of a patient with a mild case of HFMD, both from the 1998 epidemic. There was a high degree of identity (97–100%) in nucleotide sequence throughout the entire genome, except in the focal regions of 3C encoding viral protease and 3′-NCR, where the nucleotide homology was 90–91%. Wang et al. (2002) also found that most EV71 isolates from the 1998 epidemic belonged to genotype C, whereas only one-tenth of the isolates were genotype B. However, most isolates from years 1999, 2000, and 2001 belonged to genotype B (Wang et al., 2002). Both B and C geno- types of EV71 can cause severe clinical illness. Shimizu et al. (1999), analyzing EV71 from fatal and nonfatal cases of HFMD epidemics in Malaysia, Japan, and Taiwan, found the isoloates to be geographically and temporally heterogeneous. Cardosa et al. (2003) studied the molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region. Phylogenetic analysis of the VP4 and VP1 genes of recent EV71 strains indicates that several geno- types of the virus have been circulating in the Asia-Pacific region since 1997. The first of these recent outbreaks, described in Sarawak (Malaysian Borneo) in 1997, was caused by genotype B3. The large out- break in Taiwan in 1998 was mainly caused by genotype C2, and in Perth (Western Australia) in 1999, the viruses belonging to genotypes B3 and C2 cocirculated. Singapore, Taiwan, and Sarawak had EV71 epidemics in 2000, caused predominantly by viruses belonging to genotype B4, though during that epidemic only Taiwan suffered a large number of fatalities. An epidemic of HFMD in Korea was found to be caused by EV71, which constituted a new genotype C3. Two studies reported that 3C and 2A protease of EV71 can induce apoptotic cell death in neuronal or non-neuronal cells (Kuo et al., 2002; Li et al., 2002), and these two proteases may to some extent account for viral neurovirulence. However, the most important viral neurovirulence or virulence has not been clarified and needs further investigation. 8.2. Transmission and Incubation Period 8.2.1. Route of Transmission Human beings are the only natural hosts of enteroviruses. Enterovirus can infect humans through gastrointestinal cells as well as the respiratory tract. Enterovirus is usually transmitted through fecal–oral 8. Enterovirus 71 Encephalitis 299 contact and sometimes through droplet transmission. Furthermore, enterovirus can live for more than 24 h in stainless-steel containers, and such contaminated objects may be one way that it is transmitted. It can live in low-pH conditions (pH = 3) and is resistant to 70% ethanol and ether.