Investigations on the immunopathology of enterovirus 71 1

105 CHAPTER 4: EV71 INFECTION OF BONE-MARROW DERIVED DENDRITIC CELLS BMDCS .... 90 Figure 3.4 Histological examination of the muscles, intestines, and spleen from EV71- infected mice eit

INVESTIGATIONS ON THE IMMUNOPATHOLOGY OF

ENTEROVIRUS 71

KHONG WEI XIN

(B Sc (Hons.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Acknowledgements

This thesis could not have been written without Dr Sylvie Alonso, who not only served as

my supervisor but also encouraged and challenged me throughout my academic program Thank you for being such a fantastic teacher and for guiding me patiently throughout the dissertation process, never accepting less than my best effort The impact of your help is significant, and will benefit me for the rest of my life I truly can't thank you enough and will

be forever grateful

Special gratitude to Associate Professor Vincent Chow and Associate Professor Kevin

Tan Thank you for all the much-appreciated advice and guidance

Thank you a million times over to my past and present lab mates from the SA lab I find

myself so fortunate to have such wonderful friends working alongside me Thanks a ton for making the lab a blissful working environment and for returning my endless complains with support and understanding You are a great source of strength to me over the past years

To Michelle, Wenwei, Regina, Vanessa, Zarina and Fiona, thank you for the wonderful

time We made it! Every happy moment we had together has been seared in my memory, which I'll never forget

I'm forever indebted to Jowin, Grace, Andrew, Boon King and Eng Lee, who offered so

much valuable insights to my work, and for always being there, in big ways and smalls

A most loving and special thank you to my family and Adrian Words alone cannot express

what I owe them for their encouragement and whose patient care enabled me to complete this daunting yet well-worth journey Special thanks to Adrian who read and corrected every single draft of this thesis, for putting up with me all, and for cracking me up, time after time,

always knowing when it's most needed Because of you, I feel lucky everyday

Publications Articles

Khong WX, Chow VTK and Alonso S (2010) Exploring the versatility of the autotransporter BrkA for the presentation of enterovirus 71 vaccine candidates at

the surface of attenuated Bordetella pertussis Procedia in Vaccinology 2:66-72

Khong WX, Yan B, Yeo H, Tan EL, Lee JJ, Ng JK, Chow VT, and Alonso S (2012) A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection J Virol 86:

2121-31

Khong WX, Foo DGW, Trasti SL, Tan EL, and Alonso S (2011) Sustained high levels

of IL-6 contribute to the pathogenesis of enterovirus 71 in a neonate mouse model J

Virol 85: 3067-76

Lin XF, Jia Q, Khong WX, Yan B, Premanand B, Alonso S, Chow VT, and Kwang J

(2012) Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection

PLoS One 7:e29751

Review

Khong WX, Yeo H and Alonso S (2012) Enterovirus 71: Pathogenesis, Control and Models of Disease Future Virology Accepted

Table of Contents

ACKNOWLEDGEMENTS II PUBLICATIONS III LIST OF FIGURES IX LIST OF TABLES XII SUMMARY XIII LIST OF ABBREVIATIONS XVI

CHAPTER 1 LITERATURE REVIEW 1

1.1 V IROLOGY 1

1.1.1 Classification 1

1.1.2 Genomic and organization of EV71 3

1.1.3 Virus entry and spread in humans 6

1.1.4 Life cycle and replication 7

1.2 E PIDEMIOLOGY 11

1.2.1 Clinical epidemiology 11

1.2.2 Molecular epidemiology 13

1.3 C LINICAL FEATURES 18

1.3.1 Mucocutaneous and respiratory 18

1.3.2 Neurological and systemic manifestations 19

1.3.3 Pathological observations 22

1.4 P ATHOGENESIS 26

1.4.1 Viral determinants of virulence 26

1.4.2 Host genetic factors 28

1.4.3 Immunopathogenesis 29

1.4.3.1 Cytokine and chemokine-induced bystander damage 31

1.4.3.2 Lymphocyte depletion 33

1.4.3.3 Virus spread using immune target cell 33

1.4.3.4 Antibody-dependent enhancement 36

1.5 C ONTROL OF VIRAL INFECTIONS 37

1.5.1 Virus surveillance and social distancing 37

1.5.2 EV71 Vaccine development 40

1.5.3 Treatment against EV71 45

1.6 A NIMAL MODELS 53

1.6.1 Non-human primate animal model 53

1.6.2 Mouse models 54

1.7 S PECIFIC AIMS 57

CHAPTER 2 MATERIALS AND METHODS 59

2.1 M OLECULAR BIOLOGY 59

2.1.1 Detection of specific IgM and IgG antibodies 59

2.1.2 Cytokine quantification by ELISA 60

2.2 V IRUS WORK 60

2.2.1 Virus strains 60

2.2.2 Virus propagation 62

2.2.3 Purification and concentration of virus 62

2.2.4 Virus quantification 63

2.2.4.1 Virus quantification by 50% tissue culture infective dose (TCID 50 ) assay 63

2.2.4.2 Virus quantification by real-time PCR 64

2.2.4.3 Virus quantitation by plaque assay 65

2.3 C ELL BIOLOGY 66

2.3.1 The rhabdomyosarcoma cell line 66

2.3.1.1 Maintenance and storage 66

2.3.1.2 Plaque reduction neutralization test (PRNT) 67

2.3.2 Primary cells 68

2.3.2.1 Isolation and differentiation of mouse bone-marrow derived dendritic cells (BMDCs)

68

2.3.2.2 Isolation of murine splenocytes 68

2.3.2.3 Isolation of cells from lymph nodes 69

2.3.2.4 Isolation of T-lymphocytes 70

2.3.3 BMDC infection 70

2.3.4 Quantification of cell viability 71

2.3.4.1 XTT assay 71

2.3.4.2 PI staining 72

2.3.5 Allogeneic mixed lymphocyte reaction 72

2.3.6 Measurement of cell proliferation via 3 H-thymidine incorporation 73

2.3.7 Flow cytometric analysis 73

2.3.7.1 Surface marker expression 73

2.3.7.2 Carboxyfluorescein succinimidyl ester (CFSE) staining 74

2.4 A NIMAL WORK 75

2.4.1 Ethics statement 75

2.4.2 Neonatal mice 76

2.4.2.1 EV71 infection of neonatal mice 76

2.4.2.2 Anti-IL-6 monoclonal antibody treatment 76

2.4.2.3 Isolation of intestinal RNA for viral quantification 76

2.4.3 AG129 mice 77

2.4.3.1 EV71 infection of AG129 mice 77

2.4.3.2 Passive transfer of antibody 77

2.4.3.3 Ribavirin treatment 78

2.4.3.4 Quantification of blood and tissue viral loads 78

2.4.4 Histology 79

2.4.5 Adoptive transfer of BMDC 80

2.5 S TATISTICS 80

CHAPTER 3: ROLE OF INTERLEUKIN-6 IN THE IMMUNOPATHOGENESIS OF EV71 INFECTION 82 3.1 I NTRODUCTION 82

3.2 R ESULTS 84

3.2.1 Systemic and local levels of IL-6 are elevated in EV71-infected mice 84

3.2.2 Suppression of serum IL-6 levels in EV71-infected mice by antibodies 85

3.2.3 Anti-IL-6 treatment protects mice from lethal EV71 infection 88

3.2.4 Anti-IL-6 antibody treatment prevents tissue damage in EV71-infected mouse neonates 91

3.2.5 Anti-IL-6 antibody treatment did not affect the viral load 95

3.2.6 Anti-IL-6 antibody treatment increased serum IL-10 production 97

3.2.7 Anti-IL-6 treatment at the time of infection is detrimental to the mice 99

3.3 D ISCUSSION 105

CHAPTER 4: EV71 INFECTION OF BONE-MARROW DERIVED DENDRITIC CELLS (BMDCS) 111

4.1 I NTRODUCTION 111

4.2 R ESULTS 113

4.2.1 BMDCs are permissive to EV71 infection 113

4.2.2 EV71 infection increases BMDC viability 115

4.2.3 Cytokine profiles in BMDCs infected with EV71 118

4.2.4 Differential phenotypic modulation of BMDCs infected with live EV71 and heat-inactivated EV71 120

4.2.5 EV71-infected BMDCs show defects in the activation of T H 1 cells in vitro 122

4.2.6 EV71-infected BMDCs show defects in the activation of T H 1 cells in vivo 125

4.2.7 EV71 infection increases BMDCs mobility 128

4.3 D ISCUSSION 131

CHAPTER 5 DEVELOPMENT OF A NOVEL MOUSE MODEL OF EV71 INFECTION 136

5.1 I NTRODUCTION 136

5.2 R ESULTS 140

5.2.1 Two-week-old or younger AG129 mice develop fatal EV71 infection 140

5.2.2 AG129 mice are susceptible to EV71 infection via ip and oral route in a dose-dependent manner 142

5.2.3 EV71 strain 41 displays neurotropism in AG129 mice 144

5.2.4 Histopathological examination of EV71-infected mice 148

5.2.5 Pro-inflammatory cytokines are up-regulated in EV71-infected mice 151

5.2.6 Adaptive immune response in EV71-infected AG129 mice 153

5.2.7 Model validation 155

5.3 D ISCUSSION 159

CHAPTER 6 INVESTIGATIONS ON EV71 VIRULENCE DETERMINANTS IN THE AG129 MOUSE MODEL 163

6.1 I NTRODUCTION 163

6.2 R ESULTS 166

6.2.1 Comparison of clinical outcomes following infection in AG129 mice 166

6.2.2 Fatality was associated with tissue damages in CNS of AG129 mice 169

6.2.3 Fatal strains displayed neurotropism in AG129 mice 171

6.2.4 Pro-inflammatory cytokines were up-regulated in mice infected with fatal-causing strains

174

6.2.5 Adaptive immune response in EV71-infected AG129 mice 176

6.2.6 Fatal-causing strains induced greater cytotoxicity in vitro 180

6.2.7 Comparative genomic analysis of EV71 strains 184

6.3 D ISCUSSION 186

CHAPTER 7 CONCLUSION AND FUTURE WORK 192

7.1 R OLE OF I NTERLEUKIN -6 IN THE IMMUNOPATHOGENESIS OF EV71 INFECTION 192

7.2 R OLE OF DC IN EV71 INFECTION 196

7.3 D EVELOPMENT OF A NOVEL MOUSE MODEL FOR EV71 INFECTION 199

7.4 I NVESTIGATIONS ON EV71 VIRULENT DETERMINANTS IN THE AG129 MOUSE MODEL 202

CHAPTER 8 REFERENCES 206 APPENDIX I: REAGENTS FOR GROWTH MEDIA I

APPENDIX II: MISCELLANEOUS BUFFERS II APPENDIX III: TCID 50 ASSAY IV APPENDIX IV: PUBLICATIONS V

List of Figures CHAPTER 1

Figure 1.1 Enterovirus 71 (EV71) structure and genome structure of the virion 5

Figure 1.3 Distribution of EV71 isolates identified globally from 1970 to 2000 16 Figure 1.4 Distribution of inflammation in brain sections of EV71 patients 23 Figure 1.5 The postulated pathology of EV71-associated acute pulmonary oedema.25

CHAPTER 3

Figure 3.1 Systemic IL-6 levels in EV71-infected mouse neonates.80

Figure 3.2 IL-6 productions in the brain, muscle, intestines, spleen, and lungs from

Figure 3.3 Survival rate and clinical score of EV71-infected mouse neonates either

untreated or treated with anti-IL-6 antibodies post-infection 90 Figure 3.4 Histological examination of the muscles, intestines, and spleen from EV71-

infected mice either untreated or treated with anti-IL-6 antibodies

Figure 3.5 Spleen cell composition in EV71-infected mice either untreated or treated

Figure 3.6 Viral load in the intestines of EV71-infected mice either untreated or

treated with anti-IL-6 antibodies post-infection 96 Figure 3.7 Serum IL-6 and IL-10 levels in EV71-infected mice either untreated or

treated with anti-IL-6 antibodies post-infection 98 Figure 3.8 Survival rate and clinical score of EV71-infected neonatal mice either

untreated or co-treated with anti-IL-6 antibodies 101 Figure 3.9 Histological examination of the limb muscle, intestines, and spleen from

EV71-infected mice either untreated or co-treated with anti-IL-6 antibodies

102

Figure 3.10 Spleen cell composition in EV71-infected mice either either untreated or

Figure 3.11 Viral load and systemic IL-6 levels in EV71-infected mice either untreated

CHAPTER 4

Figure 4.1 Virus production upon infection of bone marrow-derived dendritic cells

Figure 4.2 BMDCs increase viability upon stimulation 117

Figure 4.3 Differential cytokine profiles by BMDCs stimulated with live and

Figure 4.4 EV71 infection impairs responsiveness of BMDCs to TLR ligands 121 Figure 4.5 In vitro proliferative response of lymphocytes against EV71-infected

Figure 4.6 T cells from mice receiving EV71-infected BMDCs showed diminished

Figure 4.7 EV71 infection enhanced BMDC migration by increased expression of

CHAPTER 5

Figure 5.1 Age-dependent mortality of AG129 mice intraperitoneally infected with

Figure 5.2 Survival rate of AG129 mice infected with a dose range of EV71 143

Figure 5.3 Virus titers in organs from AG129 infected with EV71 via the ip and oral

Figure 5.4 Viral RNA in organs from AG129 infected with EV71 via the oral route

147 Figure 5.5 Histological examination of EV71-infected mice 149 Figure 5.6 Detection of EV71 particles in the brain by immunohistochemistry 150

Figure 5.7 Systemic cytokine profile in EV71-infected AG129 152 Figure 5.8 Adaptive immune response in EV71-infected AG129 154 Figure 5.9 Passive protection of EV71-infected AG129 mice 157 Figure 5.10 Effect of ribavirin treatment on EV71-infected AG129 mice 158

CHAPTER 6

Fiure 6.1 Strain-specific clinical outcomes in AG129 mice 170 Figure 6.2 Representative histological analyses of EV71-infected mice 175 Figure 6.3 Virus titers in organs from AG129 ip infected with MS, C2, S10 and S41

178 Figure 6.4 Systemic cytokine levels in EV71-infected mice 180 Figure 6.5 Adaptive immune response in EV71-infected AG129 184

Figure 6.7 In vitro analysis of EV71 strain virulence 183

List of Tables CHAPTER 1

Table 1.2 Enterovirus 71 genotypic subgroups reported to be circulating in the

Table 1.3 Neurological syndromes associated with EV71 infection 21 Table 1.4 Anti-EV71 activity of selected compounds 49 Table 1.5 Summary of established animal models for EV71 infection 56

CHAPTER 2

Table 2.1 All EV71 virus strains used in this study 60 Table 2.2 List of antibodies used for flow cytometry analysis 74

CHAPTER 3

Table 3.1 Systemic IL-6 levels in EV71-infected mice either untreated or treated

with anti-IL-6 neutralizing antibodies post-infection 87 Table 3.2 IL-10/IL-6 ratios in EV71-infected mice either untreated or treated with

CHAPTER 6

Table 6.2 Amino acid substitutions in fatal and non-fatal causing EV71 clinical

Summary

Enterovirus 71 (EV71) is responsible for Hand, Foot and Mouth Disease (HFMD) and has been consistently associated with the most severe complications including death While most research efforts have been devoted to understand the neuropathogenesis of EV71, the immunopathogenesis aspect of the viral infection has remained elusive The aim of this thesis was thus to address some of the salient questions in EV71 immunopathogenesis in order to fill the important gaps in our understanding of the virulence associated with this virus

A number of observations in patients have reported elevated levels of pro- inflammatory cytokines and suggested their involvement in the pathogenesis Here, we show in the neonate mouse model for EV71 infection that sustained high levels of interleukin-6 (IL-6) induced upon viral infection are detrimental to the host, leading to severe tissue damage, and eventually death of the animals Consistently, administration of anti-IL-6 neutralizing antibodies after the onset of the clinical symptoms successfully improved survival rate and clinical score of the infected animals As there is still neither vaccine nor treatment available against EV71, anti-IL-6 antibody treatment may represent a possible therapeutic approach to prevent from the most severe complications of the disease

Furthermore, we have investigated the potential cellular source of production of IL-6 and

we have shown that mouse bone-marrow derived dendritic cells (BMDCs) release high levels of IL-6 upon productive infection with EV71 Further investigation revealed that EV71-infected BMDCs are impaired in their ability to migrate to the draining lymph

nodes and activate nạve T-cells, supporting a possible immune evasion mechanism triggered by EV71 to circumvent host’s immune surveillance against the virus

To gain further insight into the mechanisms involved in EV71 immunopathogenesis, we have embarked on the development of a novel mouse model of EV71 infection We report here that interferon (IFN)-α/β and γ-receptors knock-out mice (AG129) are susceptible to EV71 infection through both the intraperitoneal and oral route The infected mice displayed progressive limb paralysis prior to death Dissemination of the virus was dependent on the route of inoculation, but eventually resulted in virus accumulation in the central nervous system from both animal groups, indicating a clear neurotropism of the virus Histopathological examination revealed massive damage in the limb muscles, brainstem and anterior horn areas However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion

induced-We then carried out a comparative phenotypic analysis of EV71 isolates in the AG129 mouse model Our results indicated that morbidity and mortality in mice were highly

correlated with the virus capability to spread to the CNS in vivo and the cytotoxicity of the virus in vitro They also support that muscle damage observed in the infected animals

is not due to a direct cytopathic effect of the virus but correlate with the ability of the virus to induce brain damage A full genome comparison of these EV71 isolates could potentially lead to the identification of genetic determinants underlying the neurovirulence of EV71

Overall, our work has contributed to a better understanding of the mechanisms involved

in EV71 pathogenesis with the development of a novel mouse model that also represents

a valuable platform for vaccine and drug testing

List of Abbreviations

2Apro 2A protease

3Cpro 3C protease

3’UTR 3’untranslated region

5’UTR 5’untranslated region

ALN Axillary lymph node

ANS Autonomic nervous system

AFP Acute flaccid paralysis

APC Antigen-presenting cells

ARDS Acute respiratory distress syndrome

BALT Bronchus-associated lymphoid tissue

BE Brainstem encephalitis

BLN Bronchial lymph node

BMDC Mouse bone-marrow derived dendritic cell

BSA Bovine serum albumin

CCL C-C chemokine ligand

CCR C-C chemokine receptor

CD Cluster of differentiation

CFSE Carboxyfluorescein succinimidyl ester

CNS Central nervous system

conA Concanavalin A

CO2 Carbon dioxide

CPE Cytopathic effect

CSF Cerebral spinal fluid

CstF-64 Cleavage stimulation factor-64

CTLA4 Cytotoxic T-lymphocyte antigen 4

CV Coxsackievirus

DC Dendritic cell

DC-SIGN Dendritic cell-specific intercellular adhesion molecule-2-grabbing

non-intergrin DMEM Dulbecco’s modified Eagle’s medium

DMSO Dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid

eIF4G Eukaryotic initiation factor 4G

ELISA Enzyme-linked immunosorbent assay

EMCV Encephalomyocarditis virus

HEV Human enterovirus

HFMD Hand, Foot and Mouth disease

HIV Human immunodeficiency virus

HI EV71 heat-inactivated EV71

HLA Human leukocyte antigen

IP-10 IFN-γ-induced protein 10

IPV Formaldehyde-inactivated polio vaccine

IRES Internal ribosome entry site

IVIG Intravenous immunoglobin

mAb Monoclonal antibody

MCP-1 Monocyte chemo-attractant protein 1

MHC Major histocompatibility complex

MIP-2 Macrophage inflammatory protein 2

MLN Mesenteric lymph node

MLR Mixed lymphocyte reactions

MOI Multiplicity of infection

MRI Magnetic resonance imaging

NK Natural killer

OPD o-Phenylenediamine dihydrochloride

OPV Oral poliovirus vaccine

ORF Open reading frame

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PEG Polyethylene glycol 8000

PFU Plaque forming unit

P.I Propidium iodide

PI Post-infection

PLN Popliteal lymph node

PRNT Plaque reduction neutralization test

PSGL-1 P-selectin glycoprotein ligand-1

RBC Red blood cell

RIG-I Retinoic acid inducible gene I

RPMI Roswell Park Memorial Institute medium

RT-PCR Reverse transcription-PCR

SCARB2 Scavenger receptor B2

S10 Strain 10

S41 Strain 41

TNF Tumor necrosis factor

TCID50 50% of Tissue Culture Infective Dose

TLR Toll-like receptor

WBC White blood cell

WHO World health organization

Chapter 1 Literature Review 1.1 Virology

1.1.1 Classification

Taxonomically, the major etiological agent of the Hand, Foot and Mouth disease (HFMD), Enterovirus 71 (EV71) belongs to human enterovirus A species classified under

the Enterovirus genus in the Picornaviridae family Traditionally, the human

enteroviruses (HEVs) were classified into four subgroups based on their pathogenicity in

human, namely Echoviruses, Coxsackie A and B viruses, Polioviruses and other Enteroviruses (Nasri et al, 2007) However, this system was later revamped due to its

lack of specificity Instead, serologically distinct HEVs isolated since 1974 were named numerically in subsequence, beginning with HEV68 The original classification of HEV has been gradually substituted by a taxonomic scheme based on molecular and biological properties of the viruses, enabling the revised classification to recognize more than 100 subtypes and separate them into four species (Table 1.1) In this system, members of an HEV species “share greater than 70% aa (amino acid) identity in P1, share greater than 70% aa identity in the nonstructural proteins 2C+3CD, share a limited range of host cell receptors, share a limited natural host range, have a genome base composition (G+C) which varies by no more than 2.5%, share a significant degree of compatibility in

2005)

Table 1.1 Human enterovirus species and serotype

Enterovirus species A

Enterovirus species B

Enterovirus species C

Enterovirus species D

Numbers represent the designated serotype number of each human enteroviruses

Adapted from Bible et al., 2007 with permission

1.1.2 Genomic and organization of EV71

EV71 is a small, non-enveloped virus with a positive-stranded RNA genome size of about 7.4kb (Brown & Pallansch, 1995) The virus genome is packaged within the viral capsid and

consists of a 5’untranslated region (5’UTR), a single open reading frame (ORF) encoding a

polyprotein of 2194 amino acids, a short 3′ untranslated region (3’UTR) and a poly-A tail of

variable length (Fig 1.1) The 5’UTR contains an internal ribosome entry site (IRES), which

is a critical determinant for the translation of viral RNA and for its neurovirulence (Evans et

al, 1985) Instead of a cap structure, the 5’ terminus of the viral RNA at this region is

modified by the presence of a covalently bound small protein VPg (3B protein) The 3′UTR region contains a pseudo-knot like structure and is important for the replication of EV71

The polyprotein is subdivided into three regions, namely P1, P2 and P3 (Fig 1.1) The P1 region encodes four viral structural (VP1 to VP4), while the other two regions encode seven non-structural proteins (2A to 2C and 3A to 3D) (Brown & Pallansch, 1995) Once synthesized, the nascent polyprotein is believed to be co- and post-translationally cleaved by viral proteinases 2A (2Apro) to produce P1 protein, the latter is further cleaved by 3CD (a fusion of 3C and the viral polymerase 3D) to yield VP1, VP3 and VP0 (precursor of VP2 and

VP4) (Nicklin et al, 1987; Basavappa et al, 1994) Typically, the virus capsid comprises 60

identical subunits (protomers), each of which contains each of the four structural viral proteins (VP1-VP4) that is symmetrically arranged on an icosahedral lattice (Fig 1.1) Among them, VP1, VP2 and VP3 are the main structural components of the virion, whereas VP4 is completely internalized and is not, therefore, exposed to the host antibody response

(Hogle et al, 1985) The capsid proteins play the roles of not only receptor binding on the

surface from susceptible host cells but also contain the antigenic determinants of the virus

Two viral proteases, 2A protease (2Apro) and 3C protease (3Cpro) are encoded by the structural protein encoding region In addition to proteolytic processing of the viral polyprotein, the proteases have been suggested to play multiple roles in virus replication During an EV71 infection, 2Apro is involved in the cleavage of eukaryotic initiation factor 4G

non-(eIF4G), which is important for host protein synthesis (Kuo et al, 2002) The protein 3Cpro

was shown to suppress the host innate immune response by inhibiting retinoic acid inducible gene I (RIG-I)-mediated Type I interferon (IFN) response, thereby facilitating virus

replication (Lei et al, 2010) Furthermore, transient expression of the two proteases were also found able to induce cell apoptosis (Li et al, 2002; Kuo et al, 2002) Protein 2C is one of the

most highly conserved proteins among the picornaviruses due to its critical role in forming the viral replication complex by binding and rearranging mammalian cytoplasmic

membranes (Tang et al, 2007) Protein 3D codes for viral RNA-dependent RNA polymerase

which forms a replication complex with the viral factors to initiate RNA chain elongation (Brown & Pallansch, 1995)

VP1 VP2

Figure 1.1 EV71 structure and genome structure of the virion The single ORF is

flanked by UTRs at the 5' and 3' ends The ORF is divided into three regions: the P1 region

of the genome encodes all the structural proteins while the P2 and P3 regions encode seven non-structural proteins (2A-2C and 3A-3D) A variable length poly-A tail is found at the 3'

UTR UTR= untranslated region VPg= virus encoded protein Reproduced from Solomon et al., 2010 with permission

1.1.3 Virus entry and spread in humans

Humans are the only known natural hosts of EV71 The replication cycle of EV71 is thought

to be similar to most other enteroviruses including polioviruses (PV) (De Jesus, 2007) The virus is transmitted predominantly via the fecal-oral route, but reports have shown that in some occasions, EV71 can also spread through contact with virus-contaminated oral, vesicular and respiratory fluid as well as fomites (Pallansch MA, 2001)

Once in the alimentary tract, EV71 is thought to replicate initially at the sites of virus

multiplication in the regional lymph nodes (deep cervical and mesenteric lymph nodes) (Pallansch MA, 2001) Subsequently, virus begins to appear in the throat and in the feces—

et al, 2001) At this time, it is possible that virus may spread to the central nervous system (CNS) Clinical observations and experimental studies suggest that CNS invasion may occur through a disrupted blood-brain barrier or through retrograde axonal spread along cranial or

spread also occurs via a low titre viremia to the susceptible cells in reticuloendothelial system (liver, spleen, bone marrow and lymph nodes) as well as heart, lung, pancreas, skin, mucous membranes Despite much speculation, the exact dissemination route of the virus from its initial site of infection to other tissues and organs especially the CNS is still unclear and awaits to be further investigated

1.1.4 Life cycle and replication

The replication cycle of EV71 begins in the human host when the virion infects a susceptible host cell Studies have shown that human enterocytes (Coca-2), human T cell line (Jurkat), a macrophage cell line (THP-1), human dendritic cells (DCs) and human peripheral blood

mononuclear cells may become infected by EV71 in vitro (Chen & Yeh, 2009; Lin et al,

2009) These epithelial cells and immunocytes could be the initial site of replication of EV71

as they are present in abundance in the Peyer’s patches and regional lymph nodes In addition, human endothelial, rhabdomyosarcoma and neural cells are productively infected

by EV71 in vitro, although their actual contribution in supporting EV71 dissemination in vivo remained unclear (Liang et al, 2004; Chang et al, 2004; Pallansch MA, 2001)

Current data indicated that the virus cell entry into susceptible host cells involves several processes including viral surface attachment, receptor binding and finally uptake through endocytic pathways (Fig 1.2) At least five types of human cellular receptors specific to EV71 have been recently identified A sialomucin membrane protein found mainly on the surface of cells of hematopoietic origin, human P-selectin glycoprotein ligand-1 (PSGL-1,

CD162) has been shown to be functional receptor for EV71 infection (Nishimura et al,

2009) Transient expression of PSGL-1 renders the normally unsusceptible mouse L929 cells

to support EV71 entry, replication and subsequently exhibit typical cytopathic effects Further study revealed that tyrosine sulfation of PSGL-1 is essential for EV71 binding as mutation of the tyrosine sulfation sites significantly impairs the EV71 interaction with PSGL-

1, and inhibits PSGL-1-mediated viral replication in Jurkat T cells (Nishimura et al, 2010) However, a number of cell types that do not express PSGL-1 can also be infected by EV71 This suggests that the virus might use more than one receptor to infect a host cell, much akin

to other enteroviruses, Immature human dendritic cells (DCs) can be infected by EV71 via a

C-type lectin receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion Grabbing Non-integrin, CD209), which is found exclusively on dendritic cells Antibody blockade of DC-SIGN, could inhibit viral entrance, thereby reducing the infectious ability of EV71 significantly (Lin et al, 2009b) A ubiquitously expressed cellular receptor, scavenger receptor B2 (SCARB2, lysosomal integral membrane protein III, CD36b like-2) has been implicated as a functional receptor for EV71 as cells expressing SCARB2 allow EV71 propagation while the infection was prevented upon receptor blockade with a specific antibody against SCARB2 (Yamayoshi et al, 2009) Further analysis revealed that amino acids 142-204 of human SCARB2 are critical for the virus binding activity (Yamayoshi & Koike, 2011) Finally, annexin II and a sialic acid from a human intestinal epithelial cell line have both been suggested as the putative cellular receptors of EV71 in separate studies (Yang

molecule-3-et al, 2011; 2009)

Upon binding to a specific receptor, pores are formed in the cell membrane through which the virion RNA is released into the host cell cytoplasm Being positive-sense, the virus genome is translated directly into a large polypeptide that is promptly cleaved by the viral proteases 2Apro and 3Cpro into 11 mature structural and non-structural proteins Meanwhile, the host’s cap-dependent protein translation is effectively shut down via the cleavage of host factor eIF4G by 2Apro (Kuo et al, 2002) By remodeling intracellular membrane, viral proteins induce many membranous structures such as autophagosome-like vesicles to provide sites for its replication (Jackson et al, 2005) The positive viral RNA strand is replicated by the viral RNA polymerase 3D, to produce negative strand RNA, which is subsequently used

as template for the synthesis of more positive viral RNA strands

The newly synthesized viral RNAs are finally packaged into progeny virions and released

through the lytic cellular exit route Once infected, cells typically display cytopathic effects

(CPE) that comprise a series of cellular changes (Schlegel et al, 1996) The cell nucleus

gradually alters in morphology until it acquires a characteristic crescent shape This is followed by migration of the chromatin, in which chromosomal DNA is found in increasingly smaller regions of the nucleus that are often associated with the nuclear membrane (pkynosis) Ribosomes are aggregated in the cytoplasm and clusters of membranous vesicles form in great numbers Eventually, the cells get rounded and lysed

Figure 1.2 Intracellular life cycle of EV71

1.2 Epidemiology

1.2.1 Clinical epidemiology

EV71 was first isolated from the stool of a child aged 9 months with encephalitis in California, USA in year 1969, although an earlier isolate has since been identified (Schmidt

et al, 1974; van der Sanden et al, 2009) In the 1970s, two large EV71 epidemics occurred in

Europe The first, in Bulgaria, caused 44 deaths and 451 children to present non-specific

febrile illness or neurological disease (Chumakov et al, 1979) However, the epidemic was

initially attributed to PVs because of epidemiological, clinical and pathological

characteristics (Shindarov et al, 1979) Three years later, the second major outbreak was

reported in Hungary, with 1550 cases (826 aseptic meningitis, 724 encephalitis) and 47

deaths (Nagy et al, 1982) Link between EV71 and HFMD was only established in 1973 during small-scale epidemics in Sweden (Blomberg et al, 1974) and Japan (Hagiwara et al,

1978) Subsequently EV71 only caused small sporadic outbreaks in Hong Kong and

Australia (Gilbert et al, 1988; Samuda et al, 1987)

It was in the last two decades, that there was a surge in the scale of epidemics and neuropathogenicity of EV71-associated HFMD particularly in the Asia-Pacific region In the outbreak that occurred in Sarawak, Malaysia in 1997, a total of 2,618 HFMD cases were

caused by EV71 and 34 fatalities were reported between May and July (Chan et al, 2000) It

was in this outbreak that pulmonary oedema (PE), a new clinical manifestation that has led to

cardiopulmonary arrest in many children was observed (Chan et al, 2000) Around the same

time, 4 fatalities were reported in Peninsular Malaysia while several cases of severe

neurological disease were reported in Japan (Komatsu et al, 1999; Cardosa et al, 1999) The

largest EV71 epidemic to date occurred in Taiwan in 1998, with 1.5 million people estimated

to be infected and more than 400 children were hospitalized, of whom 78 died from severe

neurological complications and cardiopulmonary collapse (Ho et al, 1999) Since then,

several smaller scale cyclical epidemics that occur every 2-3 years have been recorded in

many areas including Western Australia, Korea, Japan, Vietnam and Singapore (Bible et al,

2007) Amongst, three major outbreaks were reported in Singapore in 2000, 2006 and 2008,

with approximately 5,800 of HFMD cases recorded and a total of 5 fatalities (Ang et al, 2009; Wu et al, 2010) Phylogenetic analysis revealed that 75% of the cases reported in

Singapore’s outbreaks were caused by EV71 The latest large Asian-Pacific epidemic occurred in China in 2008, where 490,000 infections and 126 fatalities In the epicenter,

Anhui Province alone, more than 6,000 HFMD cases and 22 deaths were reported (Zhang et

al, 2010) Figure 1.3 depicts a summary of the global reports of EV71 infection since 1970,

which clearly illustrates three separate waves of EV71 activity since its identification

Other than the geographical changes, there was also a clear change in clinical presentation among the patients throughout the years Aseptic meningitis was the most frequent neurological involvement before the 1990s, when EV71 epidemics occurred predominantly

in regions outside Asia (Blomberg et al, 1974) In contrast, brainstem encephalitis (BE),

especially affecting the medulla, associated with cardiopulmonary dysfunction has become a notable feature and the primary cause of death in EV71 epidemics in Asia, in particular

during outbreaks in Malaysia in 1997 and Taiwan in 1998 (Cardosa et al, 1999; Huang et al, 1999) (Prager et al, 2003; Zhang et al, 2010) However, the association of the circulating strains during specific endemic and their roles in the pathogenesis of severe neurological disease remains to be elucidated

1.2.2 Molecular epidemiology

Most phylogenetic analyses of EV71 rely on the sequence of capsid protein VP1 due to its high degree of diversity and lack of involvement in recombination Over the decades, 3 distinct EV71 genotypes (A, B and C) have been identified, and each group displays at least

15% divergence from the others (Brown et al, 1999) Group A consists of one member, the prototype BrCr strain, which was first identified in California in 1970 (Schmidt et al, 1974)

There was no record of this genotype outside the USA until 2008, when isolates were reported in Anhui province of Central China, although the surveillance data from Chinese Centre for Disease Control and Prevention did not seem to indicate any group A viruses (Yu

et al, 2010)

Group B can be further divided into five subgroups, B1-B5 The B1 and B2 strains were

predominantly circulating in the United States in mid-1980s (Brown et al, 1999) Thereafter,

the newly appeared subgroups B3 and B4 had been responsible for nearly all EV71 epidemics in the rest of the world, and were identified as the predominant strains in

Malaysia, Singapore and Western Australia (Chang et al, 2008; McMinn et al, 2001; 2001; Cardosa et al, 2003) Subgroup B5 was first isolated in Japan and Sarawak in 2003 from epidemics in Brunei, Sarawak and Taiwan in 2006 (Shimizu et al, 1999; Podin et al, 2006; Huang et al, 2009)

Viruses from genotype C were identified in the mid-1980s (Brown et al, 1999) Since then,

low level circulation of subgroup C1 viruses were recorded sporadically except for the major

community outbreak in Sydney (Sanders et al, 2006) Subgroup C2 viruses caused outbreaks

in Taiwan (1998), Australia (1999), and it was also found in Japan in 1997–99 and 2001–02

(McMinn et al, 2001; 2001; Cardosa et al, 2003) The first isolation of Subgroup C3 was in Japan (1994), and in Korea in 2000 (Jee et al, 2003; Iwai et al, 2009) Subgroup C4 has been

the predominant circulating subtype in mainland China since 2000, and has been reported in Japan, Vietnam and Taiwan in recent years (Lin et al, 2006; Tu et al, 2007; Zhang et al,

2010) Subgroup C5 has been reported in southern Vietnam and Taiwan (Tu et al, 2007; Huang et al, 2008) The EV71 genotypic subgroups reported to be circulating in the Asia-Pacific region between 1970 and 2010 are summarized in Table 1.2

Epidemiological studies showed that each genogroup could either circulate predominantly or co-circulates with other genotype within the same epidemic region during the outbreaks For instance, the co-circulation of four distinct genogroups (B3, B4, C1, C2) in Malaysia between 1997–2000 has been well documented In particular B3 and B4 were identified as the major causes of the EV71 epidemic in Malaysia during 1997, while C1 and B4 were

responsible for the year 2000 epidemic (Herrero et al, 2003)

With an estimated variation rate of 1.35 x 10-2 substitution per nucleotide, it is clear that EV71 has high mutability and that the continuous evolution in its genetic make up is likely to impact on its epidemiology and pathological potential (Drake, 1999) As a single-stranded RNA virus, EV71 lacks the proof-reading activity of DNA polymerases, resulting in an average of one mutation per new genome copy Furthermore, genomic recombination is

frequently used amongst enteroviruses as a mechanism to produce variants (Santti et al,

1999), while neutralizing epitopes on non-polio enterovirus capsid proteins can be altered, presumably as a response to selection pressures (Halim & Ramsingh, 2000) Taken collectively, this suggests that recombination and mutation may benefit the spread of EV71

in the human population For instance, older subgroups of EV71 have been circulating and causing low levels of disease for many years, whereas some of the EV71 strains that belong

to newly described subgroups such as B5 are more likely to be responsible for explosive

outbreaks due to low herd immunity (Huang et al, 2009; van der Sanden et al, 2010) A

thorough understanding of the relationship between the genetic changes and their consequences on host-pathogen interactions is therefore essential for successful control and understanding of the virus

Sweden Japan Bulgaria USA Japan Hungary

France

Australia

USA

Hong Kong

Taiwan USA

Brazil

Malaysia Singapore Taiwan Canada

Australia Korea Japan Singapore

Figure 1.3 Distribution of EV71 isolates identified globally from 1970 to 2000

Reproduced from Bible et al., 2007 with permission

Table 1.2 EV71 genotypic subgroups reported to be circulating in the Asia-Pacific region between 1970 and 2010

1973 1980 1986 1990 1993 1994 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Malaysia

C1, C2, B3*, B4

B4, C2, C4

C2

B4, C1

C1, C2

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)

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