Investigations on the immunopathology of enterovirus 71 1

INVESTIGATIONS ON THE IMMUNOPATHOLOGY OF
ENTEROVIRUS 71










KHONG WEI XIN

(B. Sc. (Hons.), NUS










A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


ii

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.



iii
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.











iv
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 VIROLOGY 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 EPIDEMIOLOGY 11
1.2.1 Clinical epidemiology 11
1.2.2 Molecular epidemiology 13
1.3 CLINICAL 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 PATHOGENESIS 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 CONTROL 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 ANIMAL MODELS 53



v

1.6.1 Non-human primate animal model 53
1.6.2 Mouse models 54
1.7 SPECIFIC AIMS 57

CHAPTER 2 MATERIALS AND METHODS 59

2.1 MOLECULAR BIOLOGY 59
2.1.1 Detection of specific IgM and IgG antibodies 59
2.1.2 Cytokine quantification by ELISA 60
2.2 VIRUS 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 CELL 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 ANIMAL WORK 75
2.4.1 Ethics statement 75
2.4.2 Neonatal mice 76



vi
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 STATISTICS 80

CHAPTER 3: ROLE OF INTERLEUKIN-6 IN THE IMMUNOPATHOGENESIS OF EV71 INFECTION . 82

3.1 INTRODUCTION 82
3.2 RESULTS 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 DISCUSSION 105

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

4.1 INTRODUCTION 111
4.2 RESULTS 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 DISCUSSION 131




vii
CHAPTER 5 DEVELOPMENT OF A NOVEL MOUSE MODEL OF EV71 INFECTION 136
5.1 INTRODUCTION 136
5.2 RESULTS 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 DISCUSSION 159

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

6.1 INTRODUCTION 163
6.2 RESULTS 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 DISCUSSION 186

CHAPTER 7 CONCLUSION AND FUTURE WORK 192

7.1 ROLE OF INTERLEUKIN-6 IN THE IMMUNOPATHOGENESIS OF EV71 INFECTION 192
7.2 ROLE OF DC IN EV71 INFECTION 196
7.3 DEVELOPMENT OF A NOVEL MOUSE MODEL FOR EV71 INFECTION 199
7.4 INVESTIGATIONS ON EV71 VIRULENT DETERMINANTS IN THE AG129 MOUSE MODEL 202

CHAPTER 8 REFERENCES 206

APPENDIX I: REAGENTS FOR GROWTH MEDIA I



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

ix
List of Figures

CHAPTER 1
Figure 1.1 Enterovirus 71 (EV71) structure and genome structure of the virion. 5

Figure 1.2 Intracellular life cycle of EV71. 10

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
EV71-infected mouse neonates. 85

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 post-
infection. 93

Figure 3.5 Spleen cell composition in EV71-infected mice either untreated or treated
with anti-IL-6 antibodies post-infection. 94

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




x
Figure 3.10 Spleen cell composition in EV71-infected mice either either untreated or
co-treated with anti-IL-6 antibodies. 103

Figure 3.11 Viral load and systemic IL-6 levels in EV71-infected mice either untreated
or co-treated with anti-IL-6 antibodies. 104


CHAPTER 4
Figure 4.1 Virus production upon infection of bone marrow-derived dendritic cells

with EV71. 114

Figure 4.2 BMDCs increase viability upon stimulation. 117

Figure 4.3 Differential cytokine profiles by BMDCs stimulated with live and heat-
inactivated EV71. 119

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
BMDCs. 124

Figure 4.6 T cells from mice receiving EV71-infected BMDCs showed diminished
response to re-stimulation with EV71. 127

Figure 4.7 EV71 infection enhanced BMDC migration by increased expression of
CCR7. 130


CHAPTER 5
Figure 5.1 Age-dependent mortality of AG129 mice intraperitoneally infected with
EV71. 141

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
route. 146

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




xi
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 6.6 In vitro plaque formation assay. 185
Figure 6.7 In vitro analysis of EV71 strain virulence. 183

xii
List of Tables

CHAPTER 1

Table 1.1 Human enterovirus species and serotype. 2

Table 1.2 Enterovirus 71 genotypic subgroups reported to be circulating in the Asia-
Pacific region between 1970 and 2010. 17

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
anti-IL-6 antibodies post-infection. 98



CHAPTER 6

Table 6.1 EV71 strains used in this study. 166

Table 6.2 Amino acid substitutions in fatal and non-fatal causing EV71 clinical
isolates. 185



xiii
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



xiv
nodes and activate naïve 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 induced-
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.

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.




xv
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.



xvi
List of Abbreviations


2A
pro
2A protease
3C
pro

3C protease
3’UTR 3’untranslated region
5’UTR 5’untranslated region
x g Gravitational acceleration
°C Degree Celsius
AA Amino acid
ADE Antibody-dependent enhancement
Ag Antigen
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
BW Body weight
CCL C-C chemokine ligand
CCR C-C chemokine receptor
CD Cluster of differentiation



xvii
CFSE Carboxyfluorescein succinimidyl ester
CNS Central nervous system
conA Concanavalin A
CO

2
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
DNA Deoxyribonucleic acid
ECV Echovirus
EDTA Ethylenediaminetetraacetic acid
eIF4G Eukaryotic initiation factor 4G
ELISA Enzyme-linked immunosorbent assay
EMCV Encephalomyocarditis virus
EV71 Enterovirus 71
FBS Fetal bovine serum
FcγR Fcγ receptors
g Gram
h Hour



xviii
HEV Human enterovirus
HFMD Hand, Foot and Mouth disease
HIV Human immunodeficiency virus

HI EV71 heat-inactivated EV71
HLA Human leukocyte antigen
HRP Horseradish peroxidase
IFN Interferon
Ig Immunoglobulin
IHC Immunohistochemistry
IL Interleukin
ip. Intraperitoneal
IP-10 IFN-γ-induced protein 10
IPV Formaldehyde-inactivated polio vaccine
IRES Internal ribosome entry site
IVIG Intravenous immunoglobin
KO Knockout
LN Lymph node
LPS Lipopolysaccharide
mAb Monoclonal antibody
MCP-1 Monocyte chemo-attractant protein 1
MFI Mean fluorescence intensity
MHC Major histocompatibility complex
MIP-2 Macrophage inflammatory protein 2
MLN Mesenteric lymph node
MLR Mixed lymphocyte reactions



xix
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
PE Pulmonary oedema
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
PV Poliovirus
RBC Red blood cell
RD Rhabdomyosarcoma
RIG-I Retinoic acid inducible gene I
RNA Ribonucleic acid
RPMI Roswell Park Memorial Institute medium
RT-PCR Reverse transcription-PCR
SCARB2 Scavenger receptor B2
SI Stimulation index



xx
S10 Strain 10
S41 Strain 41
TNF Tumor necrosis factor
TCID
50

50% of Tissue Culture Infective Dose
TLR Toll-like receptor
WBC White blood cell
WHO World health organization

CHAPTER 1 LITERATURE REVIEW


1
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
proteolytic processing, replication, encapsidation, and genetic recombination” (Fauquet,
2005)

CHAPTER 1 LITERATURE REVIEW


2














Table 1.1 Human enterovirus species and serotype.

Enterovirus
species A
Enterovirus
species B
Enterovirus
species C
Enterovirus
species D

Polioviruses

1-3


Coxsackie A
viruses
2-8, 10, 12,
14, 16
9
1, 11, 13, 15,
17-22, 24

Coxsackie B
viruses


1-6


Echoviruses

1-9, 11-21,
24-27, 29-33



Enteroviruses
71, 76, 89-92

69, 73-75,
77-88, 93,
97, 98, 100,
101, 106,
107

95, 96, 99,
102, 104,
105, 109,
116
68, 70, 94

Numbers represent the designated serotype number of each human enteroviruses.
Adapted from Bible et. al., 2007 with permission.
CHAPTER 1 LITERATURE REVIEW


3
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 (2A
pro
) 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.
CHAPTER 1 LITERATURE REVIEW


4

Two viral proteases, 2A protease (2A
pro
) and 3C protease (3C
pro
) are encoded by the non-
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, 2A
pro
is involved in the cleavage of eukaryotic initiation factor 4G
(eIF4G), which is important for host protein synthesis (Kuo et al, 2002). The protein 3C

pro

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).