Investigations on the immunopathology of enterovirus 71 2

CHAPTER 1 LITERATURE REVIEW Table 1.3 Neurological syndromes associated with EV71 infection Frequency Purely neurological manifestations Acute flaccid paralysis anterior myelitis Frequ

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CHAPTER 1 LITERATURE REVIEW

1.3 Clinical features

1.3.1 Mucocutaneous and respiratory

HFMD is usually a mild childhood exanthema that is characterized by 3-4 days of fever, followed by the formation of papulovesicular rashes on the buccal mucosa, tongue, gums and

palate, as well as on the palms, soles and buttocks (Ho et al, 1999; Shah et al, 2003) Other

frequently encountered symptoms include poor appetite, vomiting and lethargy In addition

to HFMD, EV71 was also identified as a cause of herpangina which is an illness characterized by an abrupt onset of fever and sore throat, associated with the development of raised papular lesions on the mucosa of the anterior pharyngeal folds, tonsils, soft palate and uvula HFMD is moderately contagious; spreading is through direct contact with nose and

throat discharges, saliva, fluid from blisters, or the stool of the infected patients (Lin et al,

2002) This disease may be caused by a number of different enteroviruses like coxsackievirus (CV) group A4-A7, CV-A9, CV-A10, CV-A24, CV-B2 to B5, echoviruses 1, 4, 11, 18 and EV18 However, the two major etiological agents of this disease are CA16 and EV71, with

the latter being associated with significant mortality (Huang et al, 1999; Lin et al, 2002)

While older children commonly display a classic course of HFMD, those aged 2 years and

younger develop more widespread and atypical rashes (Chang et al, 2004)

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1.3.2 Neurological and systemic manifestations

HFMD and herpangina are common clinical syndromes of EV71 infection, but they are usually mild and self-limiting In some occasions, EV71 infection can lead to severe neurological manifestations ranging from aseptic meningitis to acute flaccid paralysis and brainstem encephalitis (BE), which is associated with systemic features, such as severe PE and shock (McMinn, 2002) In a large prospective clinical study of several epidemics occurring over 7 years in Sarawak, a subset (10–30%) of children hospitalized with EV71-related HFMD also developed CNS complications, while these neurological complications

were not presented in children with CVA16 infections (Ooi et al, 2007) The common

presentations of CNS complications include brainstem and/or cerebellar encephalitis, accounting for 58% of neurological manifestations, followed by aseptic meningitis (36%) and BE with cardiorespiratory dysfunction (4%) Table 1.2 shows a brief description of the various neurological syndromes associated with EV71 infection

EV71-associated BE is usually clinically associated with a constellation of manifestations including myoclonic jerks, tremors, ataxia, limb weakness and cranial nerve palsies (Huang

et al, 1999) In severe cases, these neurological symptoms can progress to include seizures,

altered consciousness, and increased intracranial pressure Neurogenic pulmonary oedema (PE), hemorrhage and acute respiratory distress syndrome (ARDS) might sometimes ensue

BE, and are believed to be the main cause of mortality (Huang et al, 1999; Chan et al, 2000; 2003) Without intensive care, most children affected in this way will die before reaching hospital or within 24 hour (h) of admission In the few studies where BE, magnetic resonance imaging (MRI) and post-mortem findings have correlated well, the diencephalon, pons, cerebellum and medulla, including brainstem respiratory and vasomotor centers are

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frequently destructed and inflamed, coupled with the presence of EV71 antigens detected within the neurons (Shen et al, 1999; Wong et al, 2000)

Acute flaccid paralysis (AFP), the primary presenting feature in several neurological syndromes caused by EV71, may occur with encephalitis or as an isolated entity (McMinn, 2002) The virus antigens and pathology is typically limited to the spinal cord central gray matter, with an affinity for anterior horn cells, as in poliovirus-associated poliomyelitis However, EV71 induced AFP appears to be milder and has a higher incidence of recovery compared to poliovirus infection

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Table 1.3 Neurological syndromes associated with EV71 infection

Frequency

Purely neurological manifestations

Acute flaccid paralysis (anterior myelitis) Frequent

Neurological and systemic manifestation

Brainstem encephalitis with cardiorespiratory failure Frequent

Manifestations indicative of immune-mediated mechanisms

Reproduced from Ooi et al., 2010 with permission

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1.3.3 Pathological observations

Inflammation of the CNS has long been proposed as the underlying pathogenic mechanism

of EV71-associated encephalitis, aseptic meningitis, BE and cardiopulmonary collapse (Lin

et al, 2003; Shekhar et al, 2005) Studies on EV71 patients have shown that EV71-induced

inflammation occurs predominantly in the grey matter of the spinal cord and the whole medulla oblongata, including the dorsal nucleus of the vagus, tractus solitarius, the nucleus,

and reticular formation (Fig 1.4 A-C) (Shen et al, 1999; Wong et al, 2000) In addition, the

hypothalamus, subthalamic and dentate nuclei, and to a lesser degree motor cortex of the

cerebrum are also involved (Lum et al, 1998; Huang et al, 1999; Hsueh et al, 2000; Wong et

al, 2008) No inflammatory changes were observed in the cerebellar cortex, thalamus, basal

ganglia, peripheral nerve and autonomic ganglia (Fig 1.4D)

Encephalitis induced by EV71 is generally similar to that observed in acute encephalitis caused by other viruses Typically, widespread histopathological changes such as perivascular cuffing by mononuclear cells, variable oedema, neuronophagia and extensive

amount of microglia nodules are observed in the specific regions of CNS (Yang et al, 2009) Yang et al reported that these inflammatory lesions included abundant amount of a

distinctive neutrophil-like infiltrate mostly made up of CD68+ and CD15- cells Using various detection methods other studies have also detected EV71 viral genome and antigens

in the neurons, neuronal processes and associated inflammatory cells although no virus

inclusion has been observed (Shieh et al, 2001; Yang et al, 2009)

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Figure 1.4 Distribution of inflammation in brain sections of EV71 patients (A) Whole

brain coronal sections of cerebral hemispheres with distribution of inflammation indicated by black dots (>half-field inflamed) and blue dots (<half-field inflamed) Inflammation was most intense around the hypothalamus area and least intense in the cerebral cortex, including the motor cortex (arrows) (B) Whole brain coronal sections of cerebral hemispheres (at the level of the splenium) with distribution of inflammation indicated by black and blue dots (C) Horizontal section of cerebellum and midpons showing the distribution of inflammation (dots) Inflammation was most intense in the tegmentum and dentate nucleus The cerebellar cortex and anterior pontine nuclei areas were uninflamed (D) Schematic diagram indicating

the anatomy of human brain Modified from Wong et al (2008) with permission

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Patients infected with EV71 reportedly died from pulmonary oedema (PE) (Lum et al, 1998; McMinn et al, 2001) However, its actual pathogenesis remained unclear Most case reports

support that neurogenic mechanisms secondary to brainstem inflammation seem to contribute

to PE, but other reports argue that cardiac dysfunction, increased vascular permeability and

cytokine storm may also contribute to the clinical manifestation (Solomon et al, 2010) (Fig

1.5) In patients with PE, extensive inflammation and viral particles were only detected in the

spinal cord and brain regions, but not in the lung and heart of the patients (Shieh et al, 2001)

However, an echocardiographic study in EV71 patients with brainstem encephalitis showed

that cardiac function was impaired despite the lack of evidence of viral myocarditis (Fu et al,

2004) Consequently, two children treated with a cardiac assist device survived, whereas the

others did not survive (Fu et al, 2003) Another histological analysis from six fatal cases and

one survivor revealed characteristic features of catecholamine-associated cardiotoxic effects

in the cardiac ventricular tissue biopsy samples, suggesting that cardiac dysfunction may also

contribute to PE (Fu et al, 2004; Chen & Schifferli, 2007) In conclusion, the development of

PE could be a result of interplay between neurogenic and cardiac dysfunction as well as the effects of the systemic inflammatory response on the vascular endothelium The key to further decipher the exact mechanisms for PE in EV71 encephalitis relies on the development

of more sophisticated in vivo models, including those in mice and non-human primates to

accurately recapitulate the important features of severe EV71 disease, such as neuroinvasion with inflammatory changes and severe systemic features, such as PE

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Figure 1.5 The postulated pathology of EV71-associated acute PE SVR=systemic

vascular resistance SBP=systemic blood pressure HR=heart rate LV=left ventricular

Reproduced from Solomon et al., 2010 with permission

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1.4 Pathogenesis

The pathogenesis underlying EV71 infection is well recognized as a result from the interplay

of multiple factors contributed by both host and virus In the host, factors such as age, immune status and genetic background may contribute to the disease susceptibility Virus genome is also likely to contribute to the strain virulence and the disease outcome Risk factors are certainly involved in the wide-ranging clinical phenotypes in infected individuals, but a thorough understanding of the pathogenesis is urgently required to identify them

1.4.1 Viral determinants of virulence

The features of the clinical presentation in the major outbreaks of EV71 appear to be changing throughout the decades The epidemic in Europe had the highest percentage of

paralysis (21%) but few dermatological features (Chumakov et al, 1979; Nagy et al, 1982)

On the contrary, those reported in the Asia-Pacific after the 1980s had both dermatological

and neurological features (Cardosa et al, 1999; Huang et al, 1999; Prager et al, 2003; Zhang

et al, 2010) It is therefore suggested that different subtype of EV71 might exert different

pathologic effects However, the factors that determine whether EV71 infection will be asymptomatic or lead to dermatological or neurological disease are still largely unknown because direct comparison of epidemiological data have been hindered by differences in study designs and viral diagnostic capabilities

So far, the strongest data that support a key role of viral determinants in the pathogenesis of

severe neurological disease have been obtained with a mouse-adapted EV71 strain (Arita et

al, 2008; Chua et al, 2008) Results in this study pointed to the EV71 capsid region as a

major determinant of viral infectivity whereby mutant strains harbouring a point mutation at

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titers and triggered increased apoptosis Synergistic double mutations at both amino acid positions enhanced viral binding and RNA accumulation in infected neuronal cell line Consistently, the dual substitution mutant increased the mortality rate in neonatal mice significantly, thus implying that the molecular composition of the virus is critical to the

pathogenesis of EV71 (Huang et al, 2012)

Another evidence that different EV71 strains can exert differential pathological effects was obtained with a study in an established monkey model Hashimoto and Hagiwara (1983) showed that subcutaneous inoculation of EV71 isolates from patients induced paralytic illness in cynomolgus monkeys In the same study, the researchers also revealed the presence

of two forms of the virus: temperature resistant (tr) and sensitive (ts) forms of an EV71 BrCr clinical isolate Whereas the tr strain was isolated from a HFMD-associated encephalitis case and grew in cell culture at both 35°C and 39.5°C, the ts variant was isolated from cases with

uncomplicated HFMD only and grew at 35°C Infection of cynomolgus monkeys showed

that the tr strains are more neurovirulent than ts strains (Hashimoto & Hagiwara, 1983) A

later study revealed that several molecular determinants at the viral VP1, VP2 and 2C regions

conferred ts and attenuated phenotypes to EV71, indicating that certain genetic variation in the viral genome may indeed contribute to its neurovirulence and pathogenicity (Arita et al,

2005)

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1.4.2 Host genetic factors

Genetic factors have been hypothesized to predispose an individual to increase susceptibility

to EV71 has become an emerging area of research The cytotoxic T-lymphocyte antigen 4 (CTLA-4) protein, also known as CD152, is an important regulator of T-cell cytotoxicity and plays a role in the regulation of immune response (Walker & Sansom, 2011) In an earlier study, children with EV71 meningoencephalitis were found to possess a higher frequency of G/G allele at position 49 of CTLA-4 gene (58.1%) than those without meningoencephalitis

(29.8%) (Yang et al, 2001) However, a subsequent study found no such association (Chang

et al, 2008) Nevertheless, this study raised the possibility that genetic makeup may

contribute and significantly skew the outcome of disease in EV71 infection

The human leukocyte antigen (HLA), the major histocompatibility complex (MHC) in humans, and disease associations have been widely studied across the populations world-wide and was found to be important in prediction of disease susceptibility and resistance Although the actual role of HLA in EV71 infection remains unknown, one study with 219 patients with confirmed EV71 infection revealed that HLA-A33 significantly influences

susceptibility to EV71 infection (Chang et al, 2008) HLA-A33 is found more frequently in Asian populations (17-35%) than in Caucasian populations (0-1%), which may explain the frequent outbreaks of EV71 in Asian countries (Middleton et al, 2003) In the same study, researchers noticed an apparent correlation between HLA-A2 and the development of EV71-related cardiopulmonary failure Meanwhile, a polymorphism of another immune-regulatory gene, TNF-á promoter type II, a key mediator of fever and inflammatory response to infection, has also been associated with EV71 susceptibility in a univariate analysis but was not associated with clinical severity (Chang et al, 2008) Cytokines have been implicated in

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with 178 Chinese patients, the IFN-ã +874 A allele was observed with significantly greater frequency in patients with EV71 encephalitis (76.2%) compared with HFMD patients without complications (61.1%) Similarly, the interleukin-10 (IL-10) +1082 A allele was observed with significantly greater frequency in patients with EV71 encephalitis (86.2%) compared with HFMD patients without complications (77.0%) Together, these studies demonstrated that host genetic determinants are associated with susceptibility to severe EV71 disease Further investigations into the mechanisms underlying the role of host genetic polymorphisms in the context of EV71 infection could provide important insights into the pathogenesis of the virus

1.4.3 Immunopathogenesis

Most EV71 infections are controlled by the immune system which resolves the infection with minimal impact or without causing any substantial tissue damage The immune system protects the host from viral pathogens by both modulating the release of chemokines and inflammatory cytokines for lymphocyte recruitment, and by limiting initial virus replication

as well as subsequent viral spread by IFN dependent immunity Studies have shown that

certain Type I IFN subtypes displayed potent antiviral activity against EV71 in vitro, while

the production of Type I IFN is critical for the survival of mice infected with the virus (Liu et

al, 2005; Yi et al, 2011) T- and B- lymphocytes also play non-overlapping roles in the

adaptive immune response to EV71 infection in the CNS; infiltration of CD4+ T cells, CD8+

T cells and B lymphocytes within the CNS has been shown following EV71 infection (Lin et

al, 2009) Exacerbation of viral-induced morbidity may occur in mice deficient in T- and B-

lymphocytes, indicating that all 3 species of lymphocytes play a critical, non-redundant role

to combat infection

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Although the immune response is critical for virus clearance, there are indications that it may also cause substantial tissue damage in certain circumstances Pallansch and Ross (2007) suggested several reasons for considering a role for immune-mediated pathology in EV-induced diseases: (a) Inflammatory pathology is often seen with EV infection; (b) the success

of immunosuppressive drugs as treatment for EV infection; and (c) the close resemblance between EV-induced diseases and human diseases that are considered to have an autoimmune contribution Several hypotheses have been proposed to elucidate the mechanism of EV71-associated immunopathogenesis, including cytokine and chemokine-induced bystander damage, lymphocyte depletion, virus spread using immune target cell (Trojan horse hypothesis) and finally antibody-dependent enhancement (ADE)

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1.4.3.1 Cytokine and chemokine-induced bystander damage

Several studies have recently documented immune factor changes in EV71 patients in order

to elucidate the relationship between EV71-induced inflammation and complications Upon retrospective analysis of these data, it was proposed that overwhelming virus replication combined with the induction of massive pro-inflammatory cytokines could be responsible for the pathogenicity of EV71 High levels of interleukin-1â (IL-1â), IL-6, IL-10, IL-13, IFN-ã and tumor necrosis factor alpha (TNF-á) in the serum and cerebral spinal fluid (CSF) from EV71-infected patients have been consistently reported (Lin et al, 2002; Wang et al, 2003;

2007) In particular, when compared with healthy individuals and patients with encephalitis but no PE, the CSF levels of IL-1â, IL-6, and TNF-á were found to be significantly elevated

in patients suffering from both PE and encephalitis, demonstrating a strong correlation between pro-inflammatory cytokine production and clinical severity in EV71 infections (Lin

et al, 2002)

Furthermore, administration of intravenous immunoglobulin (IVIG), a critical treatment upon diagnosis of neuro-dysregulation in EV71-infected patients, could effectively reduce the level of pro-inflammatory cytokines such as IL-6 and IL-8 during the early phase of EV71-associated autonomic nervous system (ANS) dysregulation and prevent further progression to

PE (Wang et al, 2006) IVIG contains natural anti-cytokine antibodies such as antibodies

against IL-1, IL-6, and IFNs that modulate the cytokine cascade (Abe et al, 1994) As there were no significant changes in neutralizing antibody titers against EV71 before and after the administration of IVIG in the recovered patients, the changes in concentrations of pro- and anti-inflammatory cytokines, as well as of soluble cytokine receptor and receptor antagonists are likely to be responsible for the therapeutic effect of IVIG Collectively, these

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a prominent role in the overwhelming disease process induced upon EV71 infection Subsequently, further studies were conducted to investigate the role of pro-inflammatory cytokines in EV71 pathogenesis in mice Particularly, researchers have shown that systemic exogenous treatment of IL-6, IL-13 and IFN-ã could provoke mild PE and exacerbate pulmonary abnormality of EV71-infected mice, thus implicating a crucial role of pro-inflammatory cytokines in the immunopathogenesis of EV71 (Huang et al, 2011)

Relatively little study was done on immune factors other than cytokines Yet overexpression

of the chemokine cascade in the CNS compartment was shown to play an important role in the elicitation of EV71-associated brainstem encephalitis (Wang et al, 2008) Serum levels of IL-8, IFN-ã-induced protein 10 (IP-10), monocyte chemo-attractant protein 1 (MCP-1), and monokine induced by IFN-ã (MIG) were found to be similar or mildly higher in patients with both encephalitis and PE than in those with encephalitis only The levels of IP-10 and IL-8 were also significantly higher in the CSF from patients with both encephalitis and PE compared to uninfected individuals Together, these studies offer strong support to the hypothesis that EV71-associated neurological complications result from the synergistic effect between immunological disarray and CNS damage

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1.4.3.2 Lymphocyte depletion

The effector functions of lymphocytes are essential for viral clearance during EV71 infection

(Lin et al, 2009) However, a study with 73 EV71-infected patients revealed that the amounts

of natural killer (NK) cells, CD4+ and CD8+ T-lymphocytes were decreased in patients with

PE, despite an increase in white blood cell (WBC) counts (Wang et al, 2003) The

inadequacy in mounting EV71-specific immune responses coincided with the onset of PE, which usually manifests one to two days after hospitalization, suggesting that lymphocyte depletion may contribute to the pathogenesis of PE Interestingly, this depletion was only

limited to cells involved in cellular immune response as Yang et al (2001) detected similar

titers of EV71-specific neutralizing antibody in children with and without meningoencephalitis in the acute and convalescent stages, indicating that altered cellular but not humoral response may be linked to EV71 pathogenesis

1.4.3.3 Virus spread using immune target cell

The ability of certain viruses and bacteria to infect migratory immune cells for efficient dissemination within a host has been well established A classical example is measles virus: once inhaled, the virus first establishes its replication within the DCs residing in the respiratory tract, which traffic the virus to bronchus-associated lymphoid tissue (BALT) or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination

of the virus (Rima & Duprex, 2011)

As a strict intracellular pathogen, the benefits to virus dissemination by targeting migratory immune cells may be immense Firstly, the migratory immune cells such as monocytes and lymphocytes are programmed to circulate from the site of inflammation or injury in response

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to early infection, a strategic location for the virions to “hitch-hike” onto these cells as they are temporarily attached to intra-junctional proteins such as the cellular receptors on

epithelial cells (Lekkerkerker et al, 2006) Furthermore, activated immunocytes tend to be

highly mobile and proliferative, and each virus may induce the expression of unique

chemokine profiles to attract their preferred immune cells (Clay et al, 2007) The inherent

characteristics of the activated immune cells could inevitably maximize viral replication and viral spread especially to highly inaccessible areas, such as the CNS, as they undergo diapedesis through the tight junctions (McGavern & Kang, 2011) Finally, neutralizing antibodies are thought to play a major role in enterovirus clearance, and virions travelling in

the immunocytes might be temporarily shielded from antibodies upon dissemination (Yu et

al, 2000) As a result, virus evolution may have optimized this sophisticated strategy to

exploit normal haematological and lymphatic routes of immune surveillance in order to maximize viral spread using immune target cell, a strategy known as ‘Trojan horse’ entry Supporting this hypothesis, evidences for viral targeting of lymphocytes or monocytes for spread into primary target organs and maximal viral replication has been described for

several enteroviruses such as PV and CVB3 (Eberle et al, 1995; Mena et al, 1999)

Being closely related to PV and CVB3, EV71 has long been suspected to disseminate in a

“Trojan horse” manner EV71 is capable of replicating in immune cells, including DCs, macrophages, T-lymphocytes and freshly isolated human peripheral blood mononuclear cell

(PBMC) (Chen & Yeh, 2009; Lin et al, 2009) Consistent with the “Trojan horse”

hypothesis, EV71-infected DCs gain increase viability as they undergo maturation while

releasing cytokines that stimulate T-cell proliferation (Lin et al, 2009) In addition, live EV71

increased secretion of two pro-inflammatory cytokines, TNF-α and macrophage migration inhibitory factor (MIF) by T-lymphocytes and macrophages, whose functions are mainly to

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induce T cells activation and pro-inflammatory cytokine production in macrophages

(Calandra et al, 1994) Although no study has been dedicated to address the migratory pattern

of the virus-infected immune cells, the growing number of studies demonstrating EV71 infection of immune cells suggests that further investigation of the “Trojan horse” model may be warranted

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1.4.3.4 Antibody-dependent enhancement

The concept of antibody-dependent enhancement (ADE) hypothesis was first made famous

by (Hawkes, 1964) to explain the severe manifestation of dengue hemorrhagic fever (DHF) occurring in Thai children The ADE hypothesis postulates that the pre-existence of heterotypic, sub-neutralizing antibodies derived from a maternal or a previous primary infection, could bind to the virion and enhance a secondary viral entry through the interaction between virus-antibody complexes and Fc receptors (FcR) on FcR-bearing cells, particularly

on monocytes Following that case study, the ADE phenomenon has been described for influenza virus, human immunodeficiency virus (HIV) type 1, CVB and EV71 (Tamura et al,

1994; Beck et al, 2008; Sauter & Hober, 2009; Han et al, 2011) The ADE phenomenon in EV71 is less studied and was only recently reported by Wang et al (2010), who demonstrated

that EV71 infection was enhanced in the human monocytic cell line (THP-1) by the presence

of sub-neutralizing titres of anti-EV71 antibodies Similar to the dengue virus ADE model, further analysis revealed that the enhancement of EV71 virus entry is mediated partially by

FcR To verify the ADE effect in vivo and to elucidate the correlation between ADE and

disease severity, a secondary infection model was later established in suckling mice by Han and colleagues (2011) In this study, primary asymptomatic EV71 infection was shown to increase the mortality of the secondary heterologous EV71 infection, hence confirming the

enhancement effect of pre-existing immune effectors including antibodies Overall, these in vitro and in vivo findings have successfully shed new light to the pathogenesis of severe

EV71 infection

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1.5 Control of viral infections

1.5.1 Virus surveillance and social distancing

Over the past 12 years EV71 has become the cause of regular major epidemics across the Asia-Pacific region, and is now believed to be the most important threat to the world among the enteroviruses, now that poliovirus has been largely eradicated in most parts of the world through vaccination With no effective vaccine or antiviral treatment available, the only measure available for disease control is public health approaches Early detection of outbreaks and intervention could minimize the impact of the disease Therefore, many countries in the Asia-Pacific region, including Japan, Malaysia, Singapore, Taiwan, and Vietnam have maintained high vigilance over the disease situation through the implementation of surveillance system for EV71 (Mizuta et al, 2005; Podin et al, 2006; Chen

et al, 2007; Ang et al, 2009) In general, the enteroviruses have a distinct seasonal pattern of

circulation In tropical or subtropical areas, circulations tend to be year round, with more outbreaks in the rainy season (Suzuki et al, 2010) Despite the active surveillance effort in

various affected countries, it remained difficult to predict the epidemic potential of a particular outbreak based on the circulating sub-genogroup due to the rapid evolution of the virus (Tan et al, 2011)

Epidemic control measures are primarily targeted at interrupting virus transmission between individuals and through contacts with contaminated surfaces According to the World Health Organization (WHO) guidelines, member states are recommended to conduct information and education campaigns on good hygiene and basic sanitation, including frequent hand washing, proper disposal of soiled nappies, and then disinfecting them with chlorinated solutions (WHO, 2012) Increased spread and severity of disease are associated with factors

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Hence, authorities are recommended to focus the health education on health care facilities and places where young children congregate including kindergartens, daycare facilities and schools

Transmission reducing measures such as social distancing and case isolation are commonly implemented to prevent the spread of infectious diseases during an epidemic However, the effectiveness of social distancing in controlling the spread of EV71 remained debatable due

to the lack of systematic assessment of such measure Nevertheless, most countries in the region, including Malaysia, Singapore, Taiwan, Hong Kong and China regularly practice social distancing measures such as school closure and cancellation of public functions involving children during outbreaks (Ang et al, 2009; Solomon et al, 2010) In one study in Singapore, closure of preschools coupled with additional control measures such as issuing alert letters to affected institutions, conducting field investigations and mobilizing multi-agency efforts to prevent further transmission of the disease have been shown to be effective

in stemming the spread of infection (Ang et al, 2009) However, others have argued that

transmission of the virus within families rather than the peer-group at school could lead to increased prevalence of severe cases even though the contact rate could be lowered (Chang et

al, 2004) In the particular sero-epidemiological study by Change et al (2004), household

transmission produced a higher rate of clinical symptoms among children (94%) compared with transmission outside the household (29%) This was speculated to be due to the higher viral dose in the inoculums as the virus was transmitted among siblings or family members in the same household Furthermore, the socioeconomic consequences may present a major impediment to adopting this mitigation measure With their children out of school, many parents will stay home from work In one of the most comprehensive simulation study on the

socioeconomic impact of school closure, Sadique et al (2008) reported that closing all

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schools for four weeks in the United Kingdom would cost between 0.1-0.4% of gross domestic product (GDP) The cost was estimated by identifying workers likely to be primary caretakers for children, adding up estimates of their wages and adjusting for workers who cope with a closure by using informal child care, working from home, or making up work in the future (Sadique et al, 2008) The health care workforce is central to the mitigation

strategy However, school closure might incur more pressure on the healthcare system as some caretakers who are forced to stay home will most certainly be healthcare workers who would be treating or vaccinating patients If the health care absenteeism is high, the heath care system’s capacity could be at its minimum when it is most needed Therefore, surveillance for future EV71 outbreaks must be consistently enforced, as this will likely allow in a dynamic, adaptive decision-making of health policies as new data become available during an epidemic

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1.5.2 EV71 Vaccine development

1.5.2.1 Inactivated Virus Vaccines

The formaldehyde-inactivated polio vaccine (IPV) developed by Jonas Salk in 1952 represents one of the first described vaccines that could effectively prevent an enterovirus

infection (Ehrenfeld et al, 2009) More recently, a number of inactivated whole virus vaccine

candidates have reached phase I or phase II clinical trials in China, Taiwan and Singapore (Clinicaltrials.gov)

Killed vaccines are known to be stable, safe and can be given as polyvalent vaccines The IPV developed by Jonas Salk at Pittsburgh is among the most successful and widely used

vaccines ever produced (Ehrenfeld et al, 2009) In 1955, IPV was found to be effective in a

monumental trial involving more than 1.8 million U.S school children Rapid distribution of Salk IPV led to dramatic reduction of paralytic poliomyelitis incidences in developed countries However, multiple injections are required and cell-mediated immunity may not be induced (Nathanson and Langmuir, 1995) Moreover, the current cost of the vaccines as well

as the requirements for trained medical personnel to administer intramuscular injections, and the production scale-up that would be needed to provide this vaccine globally, currently prohibited a worldwide usage of IPV

Sero-epidemiologic studies have indicated that the pre-existing neutralizing antibody to

EV71 is protective against the severe outcomes of infection (Ho et al, 1999; Chang et al, 2002) In a study comparing various vaccine modalities, (Wu et al, 2001) demonstrated that neonatal mice receiving serum from the formalin-inactivated and heat-inactivated neu strain

of EV71, a clinical isolate obtained from the spinal cord of an 8-year-old patient during the

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challenge dose Meanwhile, maternal immunization with inactivated EV71 vaccine was able

to prolong the survival of suckling mice after EV71 lethal challenge These results show the value of the inactivated virus vaccine for the effective control of EV71 A Vero-cell adapted

virulent strain of EV71, YN3-4a was subsequently developed from the neu strain of EV71 and was characterized as suitable for use as an inactivated virus vaccine (Lin et al, 2002)

More recently, a microcarrier culture aimed at large-scale production of inactivated EV71 vaccines was developed and optimized, while further studies have been conducted to develop highly immunogenic virus strains (EV71-075 and EV71-117) using the microcarrier culture

system (Wu et al, 2004; Liu et al, 2007)

1.5.2.2 Live-attenuated Vaccines

Live-attenuated vaccines are live micro-organisms that have been cultivated under conditions which disable their virulent properties They are highly effective in evoking a full range of immune responses and one or two doses are able to provide immunity that can last for a long period of time The use of live-attenuated organism has since proven effective for rabies, yellow fever, poliomyelitis, measles, mumps, rubella and chicken pox (Nathanson & H, 2007) Three monovalent strains of live-attenuated polioviruses developed by Albert Sabin

were licensed for use in the U.S in 1961 and 1962 (Ehrenfeld et al, 2009) The

live-attenuated poliovirus replicates very efficiently in the gut, which is the primary site of viral infection and replication but is unable to replicate efficiently within nervous system tissue (Modlin, 2012) Trivalent oral poliovirus vaccine (OPV) was introduced in 1962 and rapidly replaced IPV for routine use globally because of its superior immunogenicity, the ease of oral administration as opposed to intramuscular injection, the ability to induce mucosal immunity and the public health benefit of transmission of live vaccine viruses from immunized to

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and has been reported to cause complications In some rare incidences, the attenuated virus used in OPV reverts to neurovirulence and caused vaccine-associated paralytic poliomyelitis (VAPP) in vaccines or results in transmissible vaccine-derived poliovirus (VDPV) strains The estimated reversion rate of VAPP is expected to be 2-4 cases per million vaccines per

year, which translates to 25-500 VAPP cases annually (Ehrenfeld et al, 2009)

It was demonstrated that active immunization of 1-day-old mice with avirulent EV71 strain

or CVA16 virus via the oral route triggered the production of anti-EV71 antibodies with neutralizing activities and passive immunization with the CVA16-immune serum prolonged

the survival rate of mice upon lethal EV71 challenge (Wu et al, 2007) Based on the

homology to poliovirus type I vaccine strain, a live attenuated strain of EV71, EV71 (S1-3),

derived from the prototype strain BrCr was developed using genetic manipulations (Arita et

al, 2008) The live attenuated EV71 carries mutations in the 5’UTR, 3Dpol and 3’UTR regions, which resulted in attenuated neurovirulence and limited spread of the virus Intravenous inoculation of cynomolgus monkeys led to the production of antibodies against a broad spectrum of EV71 genotypes that protected against a lethal challenge with virulent

EV71 (Ooi et al, 2007; Arita et al, 2007) However, the vaccine itself caused mild

neurological symptoms, thus indicating that further work on attenuation is required Furthermore, certain issues including the reversion of attenuated viruses into its pathogenic form as well as frequent unpredictable mutations of the viral genome are problems to be solved before the clinical use of live attenuation vaccines

1.5.2.3 Subunit Vaccines

These third generation vaccines comprise purified component from pathogenic organisms,

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are considerably stable and safer for usage (Nathanson, 2007) However, the limited region

of microbial protein means a stronger adjuvant is needed to compensate for the reduced immunogenicity of subunit vaccines In addition, the purification processes might subject the vaccine production to cost more, hence not suitable for mass vaccination campaigns in developing countries

The VP1 capsid protein is immunogenic and contains neutralization epitopes (Ranganathan

et al, 2002; Foo et al, 2007) It is therefore not surprising that VP1 has been used for the

development of DNA vaccines, synthetic peptide vaccines and subunit vaccines against

EV71 (Chiu et al, 2006; Tung et al, 2007; Foo et al, 2007)

In a comparison study of VP1 DNA vaccine and recombinant VP1 protein vaccine, Wu and

colleagues (2001) found that the intraperitoneal injection of VP1 recombinant protein with a

complete Freund’s adjuvant is able to elicit a neutralizing antibody response, enhance T helper cell proliferation, and induce high levels of IL-10 and IFN-ã in mice Consequently, the subunit vaccine could successfully confer protection against lethal EV71 challenge in newborn mice, thus illustrating that VP1 protein contains neutralizing epitopes and represents

a promising subunit vaccine candidate against EV71 infection

Transgenic edible plants and milk products are palatable oral delivery system that has been proposed as an alternative to pro- and eukaryotic cell culture systems The oral vaccines can usually elicit good mucosal as well as systemic immune response, making them particularly

attractive for vaccines against infectious agents intruding via the mucosal route (Lin et al,

2002; Wang & Coppel, 2008) EV71 initiates disease following implantation in the gut mucosa, showing the potential of an oral vaccine for immunization against EV71 infection

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Also, previous success with the OPV that induces a secretory IgA protective mucosal response that is not seen with IPV has prompted extensive research on the EV71 oral VP1 vaccine deliver system for the past decade (Ogra et al., 1968) Transgenic tomato fruit expressing the VP1 protein and a live attenuated recombinant Salmonella vaccine expressing VP1 were both successful in mounting significant humoral and cellular immune responses in

mice upon oral immunization (Chen et al, 2006; Chiu et al., 2006) Oral immunization has

also been investigated with the expression of VP1 protein in the milk of transgenic mice

(Chen et al, 2008; Ooi et al, 2009) Neonatal mice fed with VP1-enriched milk produced

neutralizing antibodies against the virus and were protected upon viral challenge

Generally, intranasal vaccination could achieve stronger immunogenicity than oral administration of the same vaccine but has been far less studied with respect to EV71 (Locht, 2000) Recently, a synthetic peptide SP70 representing a neutralizing linear VP1 epitope of EV71 was successfully identified in eliciting high titers of neutralizing antibodies in Balb/c

mice based on in vitro micro-neutralization assays (Foo et al, 2007) and in vivo passive

protection experiments (Foo et al., 2007) Subsequently, three copies of SP70 were

expressed in the highly attenuated strain of B pertussis, BPZE1 using the filamentous hemagglutinin FHA or the BrkA autotransporter as carrier respectively (Ho et al, 2008) Nasal administration of the live recombinant B pertussis bacteria producing the BrkA-

(SP70)3 chimera triggered a strong systemic anti-SP70 neutralizing antibody response, further supporting the potential of SP70 as a peptide vaccine candidate delivered via the nasal route

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1.5.3 Treatment against EV71

1.5.3.1 Antiviral drugs against EV71 infection

Despite the severity and prevalence of EV71, there is currently no broadly effective preventative treatment available The mainstay of treatment for severe EV71 disease is supportive management Administration of milrinone, a cyclic nucleotide phosphodiesterase inhibitor is routinely used to prevent cardiopulmonary failure, but the efficacy of this

treatment remains unclear (Wang et al, 2006) The ‘WIN’ group of compounds binds within

the pocket that lies beneath the floor of the virus receptor-binding pocket, and has been used

in clinical trials of aseptic meningitis (Pevear et al, 1999) One of the ‘WIN’ compounds –

pleconaril was found to provide significant therapeutic benefit in aseptic meningitis for many enterovirus serotypes, but this drug is notoriously inactive against EV71

Ribavarin is a nucleoside analog that has been reported to have broad-spectrum anti-viral activities It has been shown to have a dose-dependent inhibition against EV71 replication in infected rhabdomyosarcoma (RD), SK-N-SH (neuronal) and N18 (mouse neuronal) cell lines

(Li et al, 2008; Zhang et al, 2012) Ribavarin-treated infected 12-14 day old ICR mice had a

greater survival rate, and exhibited a later onset and less severe hind limb paralysis as compared to control mice Viral loads in tissues of the central nervous system were also

reduced in ribavirin-treated infected mice (Li et al, 2008) However, another study revealed that ribavirin treatment was not effective against EV71 in 1-day old ICR mice (Zhang et al,

2012) Thus, the true efficacy of ribavirn remains controversial and awaits further investigation

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Extracts from plant Paris polyphylla were shown to display dose-dependent inhibition of

EV71 activity in RD cells with an IC50 of 78.46 µg/ml (Wang et al, 2011) Concomitantly the levels of IL-6 production rose with increasing extract doses It was thus proposed that the

inhibitory effects of Paris polyphylla extracts could be mediated by production of elevated

protective IL-6 levels However, the role of IL-6 during EV71 pathogenesis, whether protective or detrimental, is still a matter of debate High levels of IL-6 in the serum and

cerebral spinal fluid from EV71-infected patients have been consistently reported (Lin et al, 2002; Wang et al, 2003; Wang et al, 2007) Our laboratory has also previously reported that

administering anti-IL-6 neutralizing antibodies improved survival rates and clinical scores of

EV71-infected neonates, thereby suggesting a detrimental role of IL-6 (Khong et al, 2011)

On the other hand, IL-6 KO mice displayed increased disease severity and administration of

recombinant Il-6 post-infection improved disease pathology (Poffenberger et al, 2009) Thus the role of increased IL-6 production in the dose-dependent protective effect of Paris polyphylla extracts has yet to be fully elucidated

RNA interference is another anti-EV71 therapeutic approach Recombinant vectors producing short hairpin RNA (shRNA) targeting the VP1 and 3Dpol regions of EV71

inhibited viral protein expression and decreased viral production in HeLa and Vero cells (Lu

et al, 2004) The sequence-specific nature of RNAi however resulted in the selection of resistant mutants (Gitlin et al, 2005) Such drawback could be circumvented by targeting

conserved regions, such as the 3Dpol [57] Small interfering RNA (siRNA) against 3’ untranslated region, 2C, 3Cpro and 3Dpol were able to elicit a dose-dependent inhibition of EV71 viral replication and also delay cytopathic effects in RD cells with siRNA targeting the 3Dpol being the most potent in its inhibitory effect (Sim et al, 2005) Infected 1-day old

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BALB/c mice injected via the oral or i.p route with 3Dpol –specific siRNA or shRNA did not display hind limb paralysis and weight loss as compared to the control untreated group In addition, viral particles and cytopathic damage were not detected in the intestines of treated mice However, the protective effect was mostly seen when treatment was administered at 1 day post-infection Delaying treatment resulted in significantly lower protective effects (Tan

et al, 2007)

Several other therapeutic agents that target on the VP1 protein have been developed on the assumption that its occupancy by suitable compounds will stabilize the virus capsid and prevent uncoating of virus for RNA release, but none of these compounds has come close to commercialization, largely due to the inconsistency and controversial results of the drug candidates in various animal models The capsid function inhibitors reported to possess

antiviral activities against EV71 include bovine lactoferrin (Weng et al, 2005), pyridyl imidazolidinone (Chen et al, 2008), pyridanzinyl oxime ethers (Barnard et al, 2004) and a suramin analog (Arita et al, 2008) Table 1.4 provides a brief summary of the on-going anti-

EV71 drug-based strategies and their putative modes of inhibition

1.5.3.2 Interferon therapy against EV71 infection

IFNs are potent, selective mediators of cellular changes that induce a number of antiviral and immunological effects, all of which collectively could affect the host susceptibility to

enterovirus infection (Kandolf et al, 1985) IFN-á has been shown to be a potent inhibitor of

most enterovirus infections and synergistic protection is observed when used with

capsid-binding compound (Langford et al., 1985) A recent study indicated that type I IFNs might

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shown to improve the survival rate of EV71-challenged mice by decreasing the tissue viral titers (Liu et al, 2005) In addition, treatment with a

exacerbated the disease progression and worsened the survival outcome of the EV71-infected mice To date, there are about 20 different human Type I IFNs identified Although they are highly homologous in their amino acid sequences and share the same receptors, the biologic effect of each IFN is apparently different A further analysis of the treatment revealed that 4

out of 17 type I IFN subtypes exhibit potent antiviral activity against EV71 ( Liu et al, 2005; Jaks et al, 2007; Yi et al, 2011) Together, these studies suggest that an earlier detection of

viral involvement of the central nervous system followed by immediate type I IFN therapy should be considered as an appropriate treatment regimen for EV71

Scientists also spent great efforts to identify effective anti-EV71 compounds or components from herbs or traditional Chinese medicine (TCM) Aloe-emodin is a plant-derived anthraquinone derivative In one study, aloe-emodin was able to induce a several fold

increase in IFN-ã production while showing low toxicity in vitro (Lin et al, 2008)

Pre-treatment of cells with the compound resulted in lowered plaque formation when infected

with EV71 However, no in vivo data is available to evaluate the anti-EV71 activity of emodin thus far

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aloe-CHAPTER 1 LITERATURE REVIEW

1.5.3.3 Intravenous immunoglobin (IVIG)

IVIG is a blood product administered intravenously that contains pooled immunoglobulins extracted from blood donors It is the only approved treatment for viral encephalitis, is routinely used presumptively for severe EV71 infection in many Asian countries (NIH, 1991;

Lin et al, 2002; Chang et al, 2004; Wang et al, 2006; Ooi et al, 2007; Tu et al, 2007)

Retrospective analysis of several EV71 outbreaks suggests a benefit from this treatment if

given early (Chang et al, 2004; Ooi et al, 2009) Particularly, in one study in Malaysia, a

large majority (95%) of children with severe CNS complications survived after receiving IVIG treatment, typically upon displaying signs of severe disease such as tachycardia, poor perfusion or altered consciousness IVIG has therefore been introduced into the national treatment guidelines as part of the stage-based management during EV71 infection in Taiwan

(Wang et al, 2006) The actual therapeutic mechanism of IVIG is unclear and highly

speculative Analysis of cytokine profiles before and after treatment showed substantial reduction in concentrations of some pro-inflammatory cytokines although no change in anti-EV71 neutralizing antibody titre was observed IVIG contains antibodies directed against IL-

1, IL-6 and IFNs that modulate the cytokine cascade (Wang et al, 2006) The changes in

concentrations of pro- and anti-inflammatory cytokines, as well as soluble cytokine receptor and receptor antagonist upon administration might thus be responsible for the therapeutic effect of IVIG However, a randomized, placebo-controlled phase II trial is urgently needed

to address the actual therapeutic effect of this expensive human blood product treatment

Many molecules that block EV71 replication have been reported in the preceding decade

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none of them got final approval for marketing In many cases, toxicity, poor pharamacokinetics and the lack of compelling results posed major issues Moreover, the clinical applications for such treatments called for little if not no adverse effects as treatments for children requires a drug that is extremely safe and well-tolerated In view of the clinical relevance of EV71, the demands as well as the efforts in the field are accelerating The identification of the individual proteins employed by the viruses to disable the host anti-virus response including the innate immune system, is vital to provide information to develop specific therapy to treat EV71 Furthermore, the solving of the crystal structure of EV71 should facilitate the rational design and the development of novel antiviral compounds

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Table 1.4 Anti-EV71 activity of selected compounds

status

Reference Capsid binding

Pyridanzinyl oxime ethers

Bovine lactoferrin 10.5 N/R In vivo, mice Weng et al (2005)

N/R N/R

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status Reference 2C inhibitor

3D pol inhibitor

DTrip22

Aurintricarboxylic acid

0.15 2.9 >100211 In vitro In vitro

Chen et al (2009)

Ho et a., (1999) Hung et al (2010)

N/R 1.521 65.86 µg/ml 100.5ug/ml

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1.6 Animal models

1.6.1 Non-human primate animal model

Animal models offer obvious advantages compared to in vitro systems since they can mirror

the cellular complexity that occur in the human host, making them more accurate and

predictive than in vitro settings for which very limited cell type interactions can be

reproduced However, the lack of an ideal animal model (Table 1.5) has severely impeded the progress of deciphering the mechanisms underlying EV71 pathogenesis, as well as the advancement of vaccine and therapeutic development Experimental infection has been reported in cynomolgus monkeys, African green monkeys and Rhesus monkeys upon

subcutaneous, intraspinal, oral or intracerebral inoculation (Hashimoto et al, 1978; Chumakov et al, 1979; Liu et al, 2011; Zhang et al, 2011) Upon infection, the EV71-infected monkeys generally do not develop any typical neurological complications including flaccid paralysis and ataxia, except from one study of cynomolgus monkeys (Nagata et al,

2002) In the latter report, intraspinal inoculation with several EV71 isolates consistently resulted in neurological manifestations within 1-6 days post-inoculation, irrespective of the inoculated strains Notably, the neurological manifestations included not only pyramidal tract signs such as flaccid paralysis, but also extrapyramidal tract signs such as tremor and ataxia Consistent with the clinical data, viral antigens and virus-related lesions were detected in the spinal cord, brainstem, cerebrum, cerebellar cortex and dentate nuclei Despite the ability to mirror most of the neurological manifestations in patients, the monkeys with brainstem lesions did not develop PE, the clinical symptom that correlates most to fatality in human Aside from the need to further investigate the neuropathogenesis in the primate model, the use of non-human primates is limited by ethical concerns, restricted availability and prohibitively high cost These confounding issues have thus called for an urgent need for

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development of effective vaccines and therapeutic treatments

1.6.2 Mouse models

The mouse, although not a natural host for EV71, has served as an important small animal model that could allow the study of certain aspects of EV71 pathogenesis One-day-old mouse neonates were found to be susceptible to high infectious doses of EV71 (Sasaki et al,

1986; Chen et al, 2004; Foo et al, 2007) The infected pups typically display a range of

clinical symptoms over the course of infection, including hunched back, reduced motility, hind limb paralysis, and eventually death at day 12 post-infection However, in contrast to the typical neuro-manifestations of EV71, no sign of tissue damage was observed in the brain and CNS, while a high level of inflammation and severe tissue damage were apparent in the limb muscle from the infected mice Thus, in this animal model, myositis is believed to contribute to the hind limb paralysis observed implying a myotropism of the virus (Khong et

al, 2011) This restricted tropism as well as the immaturity of the immune system in mouse

neonates has greatly limited the investigations and calls for cautious interpretation of any data generated in this animal model In addition, one-day old mice are not suitable for anti-EV71 drug or vaccine testing with a susceptibility window limited to a couple of days after birth (Lee & Chang, 2010) Antibody-mediated protection could however be assayed indirectly through passive transfer of immune serum or by immunizing the mothers (Wu et

al, 2001) Lastly, the necessity to administer -and thus produce in vitro, high viral doses

combined with the fragility and extremely small size of neonatal mice make this model technically challenging and overall unsatisfactory

In an attempt to circumvent the limitations encountered in the mouse neonate model, (Wang

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et al, 2004) generated a mouse-adapted EV71 strain through 4 serial passages in mouse

brains The mouse-adapted EV71 strain was reported to be more virulent that its parental strain, but its infectivity is still limited to 14-day-old and younger mice Host adaptation is a well-known strategy to improve the infectivity of a virus in a non-natural host However, it represents the disadvantage to bias the natural tropism of the pathogen In particular, viruses and adapting the virus through serial passages in the mouse brain or muscle cells inevitably selects for neurotropic and myotropic variants, respectively (Wang et al, 2004; 2011)

Consequently, the adaptation process may account for some of the observations made with these mouse-adapted virus strains, in particular the tropism-related features- rendering them less relevant in the clinical context (Chen et al, 2007)

With the constant application of drug screening for potential antiviral treatments and the development of vaccines, the animal model of EV71 will continue to be a highly valuable tool While there has been an increased variety and complexity of murine models over time, none of them has been able to reproduce all aspects of EV71 infection in humans, and the choice of a particular animal model must be driven by the hypothesis to be tested In any case, caution should always be applied when translating observations made in an animal model to humans

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Table 1.5 Summary of established animal models for EV71 infection

Virus Strain Species/Age Animal Inoculation Route Inoculation Dose Outcome Clinical Source Non-human Primate Model

Nagata et

al

(2002)

EV71 FY-23/China 08 Rhesus/ 3-3.5 year-old IC, IV aerosol 10CCID4.5-6.5

50 Slight fever Zhang et al (2011)

EV71 FY-23/China 08 Rhesus/ 1-1.5 month-old Intra-tracheal 10CCID4.5

Foo et al

(2007)

Cell Culture-adapted Virus Strain Mouse Model

Neural Cell Culture

10 6 PFU Skin rash, limb paralysis, death Chen et al (2004)

Muscle Cell Culture

Adapted of 0805a ICR/1 day old IP 10

5

TCID 50

Failure to gain weight

Wang et

al

(2011)

Mouse-adapted Virus Strain Model

Mouse Brain Adapted

(NOD/SCID) NOD SCID/ 3-4 week old IC, IV 5x10

Strain M2 ICR/ 7 day old Oral 5x106 PFU Paralysis, death Lin et al (2009)

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1.7 Specific aims

Most of the research efforts have been devoted to understanding the neurotropism and neuropathogenesis of EV71 with the limitations of available animal models, whereas the immunopathogenesis aspect of the viral infection has remained largely unknown Hypotheses have been proposed mainly based on observations and findings made from EV71- infected

patients; and a limited number of experimental evidences generated in vitro have confirmed

some of the proposed hypotheses and have provided some additional information As for many acute viral infections, the role of viral versus immunopathological events in EV71 pathogenesis has been discussed It was proposed that overwhelming virus replication combined with the induction of massive pro-inflammatory cytokines is responsible for the pathogenicity of EV71

Several important findings have suggested that destruction of vasomotor and respiratory centers in the medial, ventral, and caudal medulla by EV71 led to CNS damages commonly

seen in EV71-induced severe disease such as ANS dysregulation and PE (Ho et al, 1999; Huang et al, 1999; Wang et al, 1999; Wang et al, 2002) Similar to that observed in bulbar

poliomyelitis, the CNS damage caused by the virus produced sympathetic hyperactivity,

which subsequently induced a surge of catecholamine and autonomic dysfunction (Wang et

al, 2003; Kao et al, 2004) Catecholamines are among the neurotransmitters that affect

immune responses humorally through systemic production of epinephrine, as well as locally through neuronal release of norepinephrine (Hasko, 2001) The interactions of these neuroendocrine mediators with specific receptor in immune cells establish a functional neuroimmune connection capable of modulating various responses, including cytokine production This supports the possibility that fulminant PE, defined as respiratory distress accompanied by bilateral pulmonary infiltrated, might be an end result of increased

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