**4. Pathogenesis**

The enterovirus is a small, non-enveloped spherical particle around 30nm in diameter. The viral genomic RNA is encapsidated within the capsid shell comprising the VP1-4 capsid pro‐ teins. The capsid proteins are arranged into a symmetrical icosahedral lattice. These capsid proteins recognize receptors on host cells and demonstrate antigenicity. The viral genome is translated into a polyprotein around 250kDa which then undergoes cleavage via the viral proteases. The viral non-structural proteins 2A-C and 3A-D are essential for the replication

Phylogenetic studies of enterovirus 71 have identified 3 genotypes and numerous subtypes. The 3 genotypes are A, B and C, whereas the subtypes are classified numerically. Increased neurovirulence have been attributed to certain subtypes, such as genotype C1 [5]. Still, the

Herpes simplex virus holds the dubious honor of being the commonest cause of acute focal encephalitis, and is thus the presumptive diagnosis in patients with viral encephalitis. How‐ ever, prospective studies have shown that about 9% have a different etiology and may be due to enteroviruses [6, 7]. Enteroviruses have a worldwide distribution, but recent out‐ breaks of EV71 have been centered in Asia, particularly East and Southeast Asia [8-16]. En‐ terovirus 71 is not the only enterovirus that involves the central nervous system (CNS). In a Canadian survey of enteroviral infections of the CNS from 1973 to 1981, coxsackie-virus A9, B1, B2, B3 and B5, echoviruses type 6, 7, 9, 11, 30, poliovirus type 2 were isolated as well [17]. The incidence of encephalitis specifically in enterovirus infections is reported to be at

Clinically evident infection occurs mainly in children with few cases reported in adults [19]. There is a male preponderance [19]. In children, the infection usually presents as hand, foot and mouth disease (HFMD). Yet from the late 1990s onwards, increasingly severe cases caused by enterovirus have been documented, particularly involving EV71. In adults, there have been a few case reports occurring after immunosuppressive therapy such as rituximab [20]. Rituximab is a chimeric anti-CD20 monoclonal antibody that can cause profound B-cell lymphopenia and antibody deficiency. There are 3 main different clinical neurological com‐ plications of EV71 infection; 1.flaccid paralysis and encephalitis [3, 21], 2.HFMD and menin‐ goencephalitis [22-24] and 3.HFMD or herpangina and rhombencephalitis with neurogenic

The incidence of CNS complications in enterovirus infection has been reported to range from 2-10% [28]. Even so, according to a prospective study of 773 children [5] and retrospec‐ tive study of 423 patients [19], it can go as high as to 19-42%, respectively. Of the 773 chil‐ dren, EV71 was isolated in 277 (41%) and out of the 277 children, a further 28 had coinfections with a second virus (other enteroviruses, adenovirus and unidentified virus) [5]. Coxsackie A virus was isolated in 85 patients and out of these, 4 had coinfections as well. Other enteroviruses, adenoviruses or unidentified viruses were isolated in 58 [5].

exact pathogenesis for the variation in disease presentation is unknown.

of the virus within infected cells [4].

3% [18], with the majority presenting meningitis.

pulmonary edema [16, 25-27].

**3. Epidemiology**

264 Encephalitis

The reservoir of human pathogenic enterovirus is humans and transmission of enteroviruses occurs through the fecal-oral route via droplets or in utero [28]. Infection starts in the gastro‐ intestinal system with proliferation in the pharynx or intestinal lymph nodes before dissemi‐ nating to the rest of the body.

*In vitro* studies of EV71 show that the virus binds to DLD-1 intestinal cells which express sialic acid (SA) linked glycan on the cell surface [29]. Decreasing O-linked glycans or gly‐ colipids on the cell surface decreased EV71 infection of DLD-1 intestinal cells but this was not reproducible on decreasing N-linked glycans. SA linked glycans isolated from human milk also inhibited EV71 infection of DLD-1 intestinal cells [29], suggesting poten‐ tial therapeutic use.

The first step for a virus to infect the CNS is to cross the blood brain barrier (BBB). The BBB serves as a physical barrier, consisting of endothelial cells joined to each other by tight junc‐ tions and surrounded by foot processes of astrocytes, preventing access to the CNS. The me‐ ninges, choroid plexus and ependymal cells lining the ventricles also prevent access. Within the CNS are also dendritic cells and macrophages that detect pathogens and contribute to the host defense response. In utero, the BBB has not fully matured and viruses crossing into the placental circulation can also result in CNS infection.

Several RNA viruses causing neurological symptoms e.g. poliovirus, enter the CNS through axonal transport from the peripheral nervous system (PNS), circumventing the blood brain barrier. Coxsackie-virus B3 on the other hand targets nestin+ myeloid cells which subse‐ quently migrate through ependymal cell layer of the BBB into the CNS [30]. Other enterovi‐ ruses such as EV71 cross the BBB by binding to receptors e.g. P-selectin glycoprotein ligand-1, infecting cells (leucocytes and lymphocytes) that normally cross the BBB [31], hitchhiking their way into the CNS. Enterovirus 71 and coxsackie-viruses have also been shown to bind to scavenger receptor class B member 2 (SCARB2) found on fibroblasts and GPI-anchored protein decay-accelerating factor found on epithelial cells in the CNS, gaining entry into the CNS [28, 32]. SCARB2 participates in membrane transportation and the re-or‐ ganization of endosomal and lysosomal compartments [33]. The coxsackievirus and adeno‐ virus receptor (CAR) also facilitates viral entry in a caveolin-dependent or independent manner [32, 34] while human poliovirus receptor, an adhesion molecule, is used by human poliovirus in a caveolin independent manner but dynamin-dependent manner to gain entry [32, 35]. The receptors and varying method of entry in different cell types may explain for the tropism to a certain degree. Human poliovirus receptors are found in high levels in the anterior horn cells of the spinal cord, accounting for the predilection of poliovirus for infec‐ tion anterior horn cells [36]. However, there are other factors that contribute to the tropism of the viruses such as cell proliferation. It has been reported that coxsackievirus 3B targets neural progenitor and stem cells and viral replication increases markedly during cell divi‐ sion and when the cells are arrested at the G1 or G1/S phase while viral replication is reduced in quiescent cells in the G0/G2/M phase [37].

In a case-control study of 78 children who had EV71 infection, of which 31 children devel‐ oped meningoencephalitis, expression of CD40-ligand on T cells was significantly lower in cases than in controls in the acute phase, but not in the convalescent phase of the disease [46]. CD40 ligand expression on T cells is recognized as a marker of T and B cell interaction [47]. Thus, a decrease in expression may suggest a decrease in stimulation of B cells, anti‐ body class switching and antibody production. Yet, there was no significant difference in lymphocyte proliferation between cases and controls. In cases with meningoencephalitis, it was also noted that interleukin 4 production was significantly lower in cases than in con‐ trols during the acute phase, suggesting a decrease response from Th2 cells which stimulate the humoral immune system [46]. This suggests that a compromised immune status precipi‐

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In the same study, significant polymorphism of the cytotoxic T lymphocyte antigen-4 (CTLA-4) was noted, with cases having more G/G genotype at position 49 of exon 1 than in controls. CTLA-4 is involved in T cell anergy and apoptosis, and different polymorphisms

In enterovirus infection, autophagocytosis is subverted and induced in infected neurons, al‐ lowing for intracellular replication of viral particles before host cell death occurs. Autopha‐ gy is usually a protective process that occurs in cells to sequester and breakdown unwanted organelles or protein aggregates. In poliovirus [48] and coxsackievirus B4 [49] infections, however, it has been induced to assist in virus replication instead. It is postulated that enter‐ ovirus utilizes the autophagosome membrane for viral replication and increase in viral repli‐ cation is associated with autophagy induction [49]. The exact mechanism by which

Non-structural 3C protein of EV71 has been reported to block polyadenylation of host mes‐ senger RNA while non-structural 2A protein impedes the host cap-dependent translation and simultaneously stabilizes polysomes enhancing translation of viral messenger RNA [50]. Non-structural 2A protein thus boosts viral protein synthesis, and it has been shown to be necessary for host cell apoptosis as well [51]. Accordingly, it is assumed that by deactivat‐ ing the host cell translation, enteroviruses may directly cause apoptotic cell death in neu‐ rons. Enteroviruses bring about both anti-apoptotic (non-structural 3A and 2B proteins) and pro-apoptotic effects (VP2, non-structural 2A and 3C proteins) on the host cell [52]. Viral non-structural protein 2B is also known to be a viroporin that increases permeability of host

The highest rates of infections occur in young children below 4 years of age, with fatal infections most commonly occurring at the ages of 6-11 months [13, 16, 53]. This age pe‐ riod is also associated with a decline in maternal antibodies within the child. Enterovirus 71 infects human peripheral blood monocytes, and it is postulated that in the presence of sub-neutralizing amounts of anti-EV71 antibodies, infectivity is enhanced [54]. This is al‐ so known as antibody-dependent enhancement (ADE), widely described in dengue infec‐ tion as well as other viral infections such as human immunodeficiency virus infection [55]. Heterotypic non-neutralizing antibodies bind to the virions, forming EV71-antiEV71 antibody complexes, which subsequently bind to Fc-R on human monocytes and there‐

tates the fulminant progression of EV infections.

cell resulting in eventual cell death.

has been linked to infectious and autoimmune conditions [46].

autophagocytosis increases viral replication is currently unclear.

The human immune system consists of adaptive and innate immunity, both of which uti‐ lizes pattern recognition receptors (PRR) e.g. Toll-like receptor and RIG-I-like receptors that detect viral nucleic acids and initiate host defence [38], including modulating the release of chemokines, cytokines and interferons [39]. An appropriate host response to viral infection requires a complex interplay between the innate and adaptive immune system.

After entry into the CNS, glial cells which constitute part of the CNS innate immune system, detect the intracellular viral nucleic acid, and stimulate the release of IFN-1, causing apopto‐ sis and inhibit viral replication. It is, however, important to note that collateral damage in‐ curred upon activation of cytolytic T cells during an adaptive immune response within the CNS may be more damaging to neurons than the infection. Furthermore, both greater cyto‐ kine induced tissue destruction due to higher systemic levels of proinflammatory cytokines like IL-6, IL-1β, and TNF [40] and the pervasive infiltration of leukocytes into the CNS exac‐ erbate the neuropathology linked to enteroviruses [41]. Due to this, the host may contain im‐ mune response towards viral infection within the CNS.

Even so, viruses have also evolved to escape host defence by producing viral proteins that inhibit host anti-viral response. For example, EV71 produces protein 3C which inhibits RIG-I like receptors and thus blocks host IFN-1, and protein 2C which inhibits IkB kinase beta phosphorylation, consequently blocking the TNF alpha activated NκB signaling pathway [42, 43]. *In vitro* studies also showed that protein 2C stimulates neuronal apoptosis via acti‐ vation of the Abl-Cdk5 pathway [44]. Interestingly, *in vitro* studies of coxsackievirus A16 which is less neurovirulent than EV71 [45] may suggest that coxsackievirus A16 stimulates Abl to a smaller degree and does not stimulate Cdk5 [44]. Viruses can also dodge the adap‐ tive immune system by binding to dendritic cell specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). The DC-SIGN is a receptor present on macrophages and dendritic cells which recognizes and binds to pathogen associated molecular patterns (PAMPs) on viruses, bacteria and fungi. The binding itself stimulates phagocytosis and re‐ sults in pathogen entry into dendritic cells and T-cells. Intracellular entry is not only mediat‐ ed via DC-SIGN but also by other receptors such as CD36 and CD163. This results in suboptimal T and NK cell response as viral proteins inhibit IFN-1 synthesis and escape im‐ mune surveillance.

Neurological complications are reported at higher frequencies in younger children. The ex‐ act pathogenesis remains unknown, but research has elucidated a number of differences be‐ tween patients who developed such complications and those who do not.

In a case-control study of 78 children who had EV71 infection, of which 31 children devel‐ oped meningoencephalitis, expression of CD40-ligand on T cells was significantly lower in cases than in controls in the acute phase, but not in the convalescent phase of the disease [46]. CD40 ligand expression on T cells is recognized as a marker of T and B cell interaction [47]. Thus, a decrease in expression may suggest a decrease in stimulation of B cells, anti‐ body class switching and antibody production. Yet, there was no significant difference in lymphocyte proliferation between cases and controls. In cases with meningoencephalitis, it was also noted that interleukin 4 production was significantly lower in cases than in con‐ trols during the acute phase, suggesting a decrease response from Th2 cells which stimulate the humoral immune system [46]. This suggests that a compromised immune status precipi‐ tates the fulminant progression of EV infections.

the tropism to a certain degree. Human poliovirus receptors are found in high levels in the anterior horn cells of the spinal cord, accounting for the predilection of poliovirus for infec‐ tion anterior horn cells [36]. However, there are other factors that contribute to the tropism of the viruses such as cell proliferation. It has been reported that coxsackievirus 3B targets neural progenitor and stem cells and viral replication increases markedly during cell divi‐ sion and when the cells are arrested at the G1 or G1/S phase while viral replication is reduced

The human immune system consists of adaptive and innate immunity, both of which uti‐ lizes pattern recognition receptors (PRR) e.g. Toll-like receptor and RIG-I-like receptors that detect viral nucleic acids and initiate host defence [38], including modulating the release of chemokines, cytokines and interferons [39]. An appropriate host response to viral infection

After entry into the CNS, glial cells which constitute part of the CNS innate immune system, detect the intracellular viral nucleic acid, and stimulate the release of IFN-1, causing apopto‐ sis and inhibit viral replication. It is, however, important to note that collateral damage in‐ curred upon activation of cytolytic T cells during an adaptive immune response within the CNS may be more damaging to neurons than the infection. Furthermore, both greater cyto‐ kine induced tissue destruction due to higher systemic levels of proinflammatory cytokines like IL-6, IL-1β, and TNF [40] and the pervasive infiltration of leukocytes into the CNS exac‐ erbate the neuropathology linked to enteroviruses [41]. Due to this, the host may contain im‐

Even so, viruses have also evolved to escape host defence by producing viral proteins that inhibit host anti-viral response. For example, EV71 produces protein 3C which inhibits RIG-I like receptors and thus blocks host IFN-1, and protein 2C which inhibits IkB kinase beta phosphorylation, consequently blocking the TNF alpha activated NκB signaling pathway [42, 43]. *In vitro* studies also showed that protein 2C stimulates neuronal apoptosis via acti‐ vation of the Abl-Cdk5 pathway [44]. Interestingly, *in vitro* studies of coxsackievirus A16 which is less neurovirulent than EV71 [45] may suggest that coxsackievirus A16 stimulates Abl to a smaller degree and does not stimulate Cdk5 [44]. Viruses can also dodge the adap‐ tive immune system by binding to dendritic cell specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). The DC-SIGN is a receptor present on macrophages and dendritic cells which recognizes and binds to pathogen associated molecular patterns (PAMPs) on viruses, bacteria and fungi. The binding itself stimulates phagocytosis and re‐ sults in pathogen entry into dendritic cells and T-cells. Intracellular entry is not only mediat‐ ed via DC-SIGN but also by other receptors such as CD36 and CD163. This results in suboptimal T and NK cell response as viral proteins inhibit IFN-1 synthesis and escape im‐

Neurological complications are reported at higher frequencies in younger children. The ex‐ act pathogenesis remains unknown, but research has elucidated a number of differences be‐

tween patients who developed such complications and those who do not.

requires a complex interplay between the innate and adaptive immune system.

in quiescent cells in the G0/G2/M phase [37].

266 Encephalitis

mune response towards viral infection within the CNS.

mune surveillance.

In the same study, significant polymorphism of the cytotoxic T lymphocyte antigen-4 (CTLA-4) was noted, with cases having more G/G genotype at position 49 of exon 1 than in controls. CTLA-4 is involved in T cell anergy and apoptosis, and different polymorphisms has been linked to infectious and autoimmune conditions [46].

In enterovirus infection, autophagocytosis is subverted and induced in infected neurons, al‐ lowing for intracellular replication of viral particles before host cell death occurs. Autopha‐ gy is usually a protective process that occurs in cells to sequester and breakdown unwanted organelles or protein aggregates. In poliovirus [48] and coxsackievirus B4 [49] infections, however, it has been induced to assist in virus replication instead. It is postulated that enter‐ ovirus utilizes the autophagosome membrane for viral replication and increase in viral repli‐ cation is associated with autophagy induction [49]. The exact mechanism by which autophagocytosis increases viral replication is currently unclear.

Non-structural 3C protein of EV71 has been reported to block polyadenylation of host mes‐ senger RNA while non-structural 2A protein impedes the host cap-dependent translation and simultaneously stabilizes polysomes enhancing translation of viral messenger RNA [50]. Non-structural 2A protein thus boosts viral protein synthesis, and it has been shown to be necessary for host cell apoptosis as well [51]. Accordingly, it is assumed that by deactivat‐ ing the host cell translation, enteroviruses may directly cause apoptotic cell death in neu‐ rons. Enteroviruses bring about both anti-apoptotic (non-structural 3A and 2B proteins) and pro-apoptotic effects (VP2, non-structural 2A and 3C proteins) on the host cell [52]. Viral non-structural protein 2B is also known to be a viroporin that increases permeability of host cell resulting in eventual cell death.

The highest rates of infections occur in young children below 4 years of age, with fatal infections most commonly occurring at the ages of 6-11 months [13, 16, 53]. This age pe‐ riod is also associated with a decline in maternal antibodies within the child. Enterovirus 71 infects human peripheral blood monocytes, and it is postulated that in the presence of sub-neutralizing amounts of anti-EV71 antibodies, infectivity is enhanced [54]. This is al‐ so known as antibody-dependent enhancement (ADE), widely described in dengue infec‐ tion as well as other viral infections such as human immunodeficiency virus infection [55]. Heterotypic non-neutralizing antibodies bind to the virions, forming EV71-antiEV71 antibody complexes, which subsequently bind to Fc-R on human monocytes and there‐ fore, enhancing infectivity. *In vitro* experiments have shown that the addition of immune sera from patients increased EV71 infection of THP-1 cells, a leukemia cell line of macro‐ phage lineage with monocytic markers, whereas addition of Fc-RI (CD64) significantly in‐ hibited the infection [54].

with agammaglobulinemia [67, 68]. In fact, the first case of enteroviral meningoencephalitis

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Enteroviruses can cause a wide spectrum of clinical diseases, including but not limited to the common cold, gastroenteritis, hand, foot and mouth disease (HFMD), herpangina, myo‐ carditis, severe neonatal sepsis-like disease, hepatoadrenal failure, aseptic meningitis, acute flaccid paralysis, meningoencephalitis, encephalitis, neurogenic pulmonary edema, pulmo‐ nary hemorrhage and shock induced sudden death especially in the young age group [13, 62, 70]. The neurological presentations include aseptic meningitis, benign intracranial hyper‐ tension, acute flaccid paralysis, opsoclonus-myoclonus syndrome, Guillain-Barre syndrome, transverse myelitis, encephalitis, cerebellitis, brainstem encephalitis, rhombencephalitis and

Clinical manifestations can be classified according to 5 grades. In grade I, patients demon‐ strate clinical signs of HFMD and/or herpangina with erythematous vesicles on palms, soles, elbows and trunk and oral ulcers on mucosa of lips as well as palate. The majority of pa‐ tients will display grade I symptoms as seen in the 1998 Taiwan epidemic where 5506 out of 5632 patients were classified to have grade I symptoms [61]. In grade II, patients suffer from fever, photophobia, vomiting, headache, and abdominal pain. Patients who initially exhibit grade II symptoms may subsequently progress onto grade III. The disease may take a fulmi‐ nant course in patients younger than 2 years of age, deteriorating directly to grade IV in a short period of time. In grade III, patients may demonstrate lethargy, apathy, drowsiness, tachycardia, cranial nerve involvement (VI-XII), myoclonic jerks, monoparesis or hemipare‐ sis, conjugate gaze disturbances, dyspnea and ataxia. Patients with grade III symptoms that are younger than 2 years of age usually progress to grade IV while older patients tend to recover completely after 1-2 weeks. In grade IV, patients experience hypothermia, pulmona‐ ry edema, respiratory failure, neurogenic shock and semicoma. In the last stage, grade V, there is pulmonary hemorrhage, respiratory distress syndrome, cardiorespiratory failure, coma and death. According to symptomatology, encephalitis is suspected in grade III-IV.

HFMD and herpangina are generally mild, self-limiting illnesses that occur in infants and young children. The culprit virus for HFMD and herpangina is usually coxsackie-virus A16 or EV71 [8, 14, 19]. Despite this, a small percentage of patients can rapidly decompensate and die within days. In cases where neurological complications occur, the culprit viruses isolated are

Patients who had CNS complications were usually younger and more likely to have symp‐ toms of fever, vomiting, breathlessness and signs of shock that includes cold peripheries and poor urinary output [5]. The exact symptoms and signs depend on the extent of CNS in‐ volvement. For example, in EV71 encephalitis, clinical signs of lethargy and cranial nerve palsies such as conjugate gaze disturbance, dyspnea and tachycardia suggest involvement of the brain stem, [61] and this is further substantiated by the magnetic resonance imaging.

usually EV71 and coxsackie-virus A16 [19] in some instances other echovirus 7 [77, 78].

was reported in a patient with agammaglobulinemia [69].

**7. Clinical signs and symptoms**

encephalomyelitis [13, 25, 26, 71-76].

Furthermore, patients who suffer from neurogenic pulmonary edema have been reported to have lower absolute monocyte counts, CD4, CD8 and NK cells counts as compared to pa‐ tients who had autonomic nerve system abnormalities and uncomplicated brainstem ence‐ phalitis [56].
