Prophylactic and therapeutic potential of synthetic peptides against enterovirus 71

2.5.3.1 Protection afforded by maternal-transferred 2.5.3.2 Passive protection afforded by mice immune sera 86 EPITOPES FROM THE VP1 CAPSID PROTEIN OF ENTEROVIRUS 71 USING SYNTHETIC PEPT

DAMIAN FOO GUANG WEI

NATIONAL UNIVERSITY OF SINGAPORE

2008

DAMIAN FOO GUANG WEI

Associate Professor Poh Chit Laa for the opportunity to pursue my postgraduate

studies under her supervision and to undertake this project Thank you for your invaluable guidance and support throughout the course of this study The knowledge and experiences gained in your laboratory were truly invaluable

Assistant Professor Sylvie Alonso for her unwavering guidance, sound advice and

most importantly, for her friendship Thank you for your constant encouragement, support and the opportunity to continue my postgraduate studies under your close supervision I am indebted to you for providing me with the motivation to develop a passion towards science and sharing your research experiences with me

Associate Professor Vincent Chow Tak Kwong for his co-supervision, advice and

guidance throughout the course of this study

Associate Professor Lu Jinhua, Assistant Professor Kevin Tan Shyong Wei and Assistant Professor Theresa Tan May Chin for being my PhD qualifying examiners

and the opportunity for me to continue with my postgraduate studies

Mr Ramachandran and Mrs Phoon Meng Chee for their help during the animal

study and their technical advices in tissue culture work and in vitro

invaluable advices and concern Thank you for the joy and laughter we have shared

Chew Ling ♥ for her patience and understanding throughout my postgraduate studies Thank you so much for your endless encouragement, support and help especially with the bacterial work Also, thank you for accompanying me in lab during my late night stints Your love and sincerity has indeed changed me into a better person

My parents for their unconditional love, concern and understanding in every possible

ways Thank you for your constant support throughout my studies You have given

me everything a child can possibly dream of Without you, I won’t be who I am today

God for his eternal guidance, love and being there in my times of need You have

given me spiritual courage and strength to face new challenges and overcome all difficulties Continue to guide me and strengthen my faith in you Amen

Acknowledgements ii

Table of contents iii

List of Tables xi

List of Figures xii

Abbreviations xiv Summary xx

CHAPTER 1 LITERATURE REVIEW 1.1 Picornaviruses 1

1.2 Genome and organization of enteroviruses 4

1.2.1 The enteroviral capsid proteins 6

1.2.2 Infection cycle 8

1.3 Enterovirus 71 (EV71) infection 10

1.3.1 Epidemiological studies 10

1.3.2 Phylogenetic studies 12

1.3.2.1 VP1-based classification 12

1.3.2.2 VP1- and VP4-based classification 13

1.3.2.3 Relationship between subgenogroups and outbreak occurrence 14

1.3.3 Clinical features of diseases caused by enterovirus 71 (EV71) 19

1.3.3.1 Hand, foot and mouth disease (HFMD) 19

1.3.3.2 Other EV71-associated diseases 22

1.3.4 Immunopathogenesis of EV71 infection 23

1.4.2 Immunofluorescence assay 26 1.4.3 Enzyme-Linked Immunosorbent Assay (ELISA) 27

1.4.4.1 Reverse Transcription Polymerase Chain Reaction

1.5.2.1 Intravenous immunoglobulin (IVIG) 37

1.8 Animal models for Enterovirus 71 (EV71) 50

2.1.1.2 Culture and storage of bacterial cells 55 2.1.1.3 Preparation of chemically competent

2.2.1.1 Regeneration and culture of Rhabdomyosarcoma

2.2.2.1 Isolation of peripheral blood mononuclear cells

2.3.3.3 Conventional RT-PCR amplification 69 2.3.3.4 Hybridization probe-based

2.3.4.1.1 Preparation of plasmids by the modified

alkaline lysis method of Birnbolm and

2.3.4.1.2 Preparation of plasmids by the modified boiling method of Holmes and Quigley

2.3.4.1.3 Plasmid purification using the Wizard™

SV Miniprep DNA Purification Kit

2.3.4.2 Restriction endonuclease digestion of DNA 74 2.3.4.3 Agarose gel electrophoresis of DNA 75

2.3.4.4 Purification of DNA using the GFX™ Purification kit

2.4.1.1 Sodium dodecyl sulphate - polyacrylamide gel

2.4.2.1 Electrophoretic transfer of proteins 79 2.4.2.2 Immunogenic development of Western blots 79 2.4.3 Expression and analysis of recombinant GST-tagged

2.4.3.1 Growth and induction of bacteria 80

2.4.3.3 Purification of recombinant GST-tagged

2.4.3.3.1 MicroSpin™ GST Purification kit

(GE Healthcare Life Sciences, UK) 81 2.4.3.3.2 GSTrap™ Fast Flow column kit

(GE Healthcare Life Sciences, UK) 81

2.4.4 Enzyme-linked Immunosorbent Assay (ELISA) 82

2.4.4.1 Detection of specific mouse or

2.5.3.1 Protection afforded by maternal-transferred

2.5.3.2 Passive protection afforded by mice immune sera 86

EPITOPES FROM THE VP1 CAPSID PROTEIN OF ENTEROVIRUS 71 USING SYNTHETIC PEPTIDES

3.2.1 Identification of EV71-neutralizing antisera from mice

immunized with synthetic peptides (preliminary study) 91 3.2.2 EV71-neutralizing antisera from mice immunized with SP55,

SP70 or heat-inactivated homologous EV71 whole virion 91 3.2.3 Immunoreactivity of antisera from mice immunized with

3.2.6 In vitro protection afforded by antisera from mice immunized

with SP55, SP70 or heat-inactivated homologous EV71 strain 41 against heterologous EV71 strains 105

3.2.9 In vivo passive protection against lethal EV71 challenge

3.2.9.1 Homologous EV71 strain 41 challenge 113 3.2.9.2 Heterologous EV71 strains challenge 114 3.2.10 Histological examination in EV71-infected

3.2.11 Detection of EV71 by real-time RT-PCR

3.2.12 Cytokine profiles in suckling Balb/c mice protected

against lethal homologous EV71 strain 41 challenge 122

LINEAR EPITOPE OF ENTEROVIRUS 71 USING SYNTHETIC PEPTIDES FOR DETECTING HUMAN ANTI-EV71 IgG ANTIBODIES IN WESTERN BLOT

4.2.3 Detecting human anti-EV71 IgG antibodies using purified

recombinant GST-VP1 and GST-SP32 fusion proteins in

4.2.4 Specificity of the purified recombinant GST-SP32

fusion protein as a capture antigen in Western blot 143

5.2.5 Cytokine profile upon antigenic stimulation 158

PUBLICATIONS

potentially be caused by more than one enterovirus 3 Table 1.2 Summary of main HFMD outbreaks from 1997 to present 11 Table 2.1 Bacterial strains and plasmids used in this study 57

Table 2.3 Nucleotide sequences of EV71-specific primers and

Table 3.1 Immunospecificity of SP12-, SP55- and SP70-immune sera 99 Table 3.2 Total and IgG sub-type responses in SP12-, SP55- and

Table 3.3 Neutralizing antibody titers elicited by SP55, SP70 and

heat-inactivated homologous EV71 whole virion in mice against

Table 3.4 Survival rates of suckling Balb/c mice upon challenged with the

Table 5.1 Sequences and locations of predicted promiscuous regions 152 Table 5.2 EV71 exposure of volunteers and predicted peptide binding

Table 5.3 Antigen-specific cytokinea secretion by stimulated CD4+ T cells 161

Figure 1.2 Diagrammatic representation of the EV71 icosahedra virus capsid 7

Figure 1.4 Classification of 113 EV71 strains into genogroups based

on the VP1 gene (nucleotide position 2442 to 3332) 16 Figure 1.5 Phylogenetic tree showing classification of 25 EV71 field isolates into

subgenogroups based on alignment of the complete VP1 sequence

Figure 3.1 Infection of RD cells with a viral dose of 103 TCID50

Figure 3.2 In vitro microneutralization assay using a representative serum sample

from mice (n=5) immunized with the heat-inactivated homologous

Figure 3.3 In vitro microneutralization assay using a representative serum sample

from mice (n=5) immunized with the synthetic peptide SP70 95 Figure 3.4 In vitro microneutralization assay using a representative serum sample

from mice (n=5) immunized with the synthetic peptide SP55 96 Figure 3.5 Western blot analysis using the synthetic peptide-antisera as

Figure 3.6 Kyte and Doolittle hydrophobicity profiles of the VP1 capsid protein

Figure 3.7 Alignment of amino acid sequences represented by the synthetic

peptides SP55 and SP70 against heterologous EV71 strains from different subgenogroups based on the VP1 amino acid sequences 104 Figure 3.8 Viral infection of suckling Balb/c mice with the homologous EV71

strain 41 at a lethal dose (103 TCID50 per mouse) 109

Figure 3.11 In vivo passive protection of suckling Balb/c mice upon challenged

Figure 3.12 In vivo passive protection study conferred by different anti-EV71

Figure 3.13 Detection of EV71 infection in small intestines of suckling Balb/c mice

upon challenged with the homologous EV71 strain 41 at a lethal dose

Figure 3.14 Detection of EV71 by real-time RT-PCR hybridization probe assay

Figure 3.15 Cytokine profile in suckling Balb/c mice upon EV71 challenge 123 Figure 3.16 Immunoreactivity of VP1 capsid protein against human sera

Figure 3.17 Immunoreactivity of VP1 capsid protein against mice immune sera

Figure 4.1 Expression of purified recombinant GST-VP1 and GST-SP32

Figure 4.2 Western blot analysis of antigen reactivity with pooled EV71-positive

Figure 4.3 Alignment of amino acid sequences represented by the synthetic

peptide SP32 against heterologous EV71 strains from different subgenogroups based on the VP1 amino acid sequences 144 Figure 5.1 An output of ProPred analysis of the VP1 amino acid sequence in

Figure 5.2 Proliferation of CD4+ T cells upon stimulation with peptides or

Figure 5.3 Proliferation of SP2-stimulated CD4+ T cells in the presence of

Cytoxicity

HLA Human Leukocyte Antigen

Virus

IPTG

Isopropyl-ß-D-thiogalactopyranosid

MCS Multiple Cloning Site

PE Polyethylene

WHO World Health Organization

Enterovirus (EV71) is the agent of Hand, foot and mouth disease (HFMD), a common and mild illness amongst infants and young children However, in recent years, this pathogen has posed a serious threat; large scale outbreaks of HFMD have been reported in the Asia-Pacific region with an increasing number of cases of neurological complications which resulted in high fatality rates

In this study, characterization of the linear neutralizing epitopes on the VP1 capsid protein of the Enterovirus 71 strain 41 (5865/SIN/00009) (belonging to subgenogroup B4 and isolated from a fatal case in Singapore) was undertaken Antisera were raised in adult Balb/c mice against 95 overlapping diphtheria toxoid-conjugated synthetic peptides of 15 amino acids in length spanning the entire VP1 capsid protein Two synthetic peptides, designated SP55 (VP1 amino acid residues

162 to 177) and SP70 (VP1 amino acid residues 208 to 222) were capable of eliciting neutralizing antibodies against EV71

Based on in vitro microneutralization assay, the synthetic peptide SP70 was

able to elicit a higher EV71-neutralizing response with a neutralizing antibody titer of 1:32 in mice when compared to the synthetic peptide SP55, eliciting an EV71-neutralizing antibody titer of 1:8 The anti-SP70 antiserum was found to be almost as efficient as the immune serum raised against the heat-inactivated homologous EV71 whole virion with a neutralizing antibody titer of 1:64 In addition, the total IgG response specific to EV71 whole virion measured in the anti-SP55 or anti-SP70 antiserum was found to be as high as that measured in the immune serum raised

neutralizing antibodies elicited are likely belonging to the IgG1 subtype

The amino acid sequences represented by SP55 and SP70 lie towards the terminal part of the VP1 capsid protein of EV71 strain 41 The hydrophobicity profiles showed that these regions are located within the major hydrophilic regions of VP1 and hence they are expected to be exposed at the surface of the protein Alignment with databases showed that the amino acid residues represented by SP70 are highly conserved amongst the VP1 sequences of 25 representative EV71 strains

C-from different subgenogroups In vitro microneutralization assay has shown that the

immune serum raised against SP70 was able to neutralize heterologous EV71 strains with similar efficiencies to that obtained with the homologous EV71 strain 41, thereby suggesting that SP70 might represent an interesting and promising peptide-based vaccine candidate for EV71

In addition, when passively administered to one-day-old suckling Balb/c mice challenged with a lethal dose of 103 TCID50 virus/mouse, the anti-SP70 antibodies

were able to confer 80% in vivo protection comparable to antiserum raised against the

homologous heat-inactivated EV71 whole virion The level of protection conferred by the anti-SP70 antiserum against heterologous EV71 strains was almost similar to that obtained against the homologous strain, supporting that the VP1 amino acid sequences represented by SP70 contain a highly conserved neutralizing linear epitope

neutralizing anti-SP70 antibodies plays a major role in the inhibition of EV71

replication in vivo which significantly reduced the viral titer The cytokine profiles for

EV71-challenged mice showed elevated IL-6 and IFN-γ levels in unprotected mice whereas significant lower levels were observed for mice which were protected by passively-transferred immune sera This observation suggests a correlation between pro-inflammatory cytokines and the severity of EV71 infection

The use of synthetic peptide(s) as capture antigen(s) in immunoassays represents an interesting approach for the serodiagnostic of EV71 infection as it would avoid the need for propagating infectious viruses Antigenic sites on VP1 protein of EV71 strain 41 (5865/SIN/00009) were mapped by Pepscan analysis using the 95 overlapping synthetic peptides spanning the entire VP1 amino acid sequence against EV71-neutralizing sera from pediatric patients A major IgG-specific immunodominant linear epitope (VP1 amino acid residues 91 to 111), defined by the core amino acid sequence ‘LEGTTNPNG’, was identified

Therefore, a 15 amino acid-based synthetic peptide SP32 (DLPLEGTTNPNGYAN) which contains the core sequence of the immunodominant

VP1 linear epitope was over-expressed in Escherichia coli as a soluble recombinant

GST-SP32 fusion protein When used as a capture antigen in Western blot, the recombinant GST-SP32 fusion protein significantly reacted with human anti-EV71 IgG antibodies with high specificity when compared to both the recombinant GST-VP1 fusion protein and EV71 whole virion as capture antigen In addition,

enteroviruses Altogether, these data indicate that recombinant GST-SP32 fusion protein which harbored the immunodominant VP1 linear epitope of EV71 could be potentially used as a capture antigen in Western blot for detecting human anti-EV71 IgG antibodies

The identification of human CD4+ T-cell epitopes within a protein vaccine candidate is of great interest as it provides a better understanding of the mechanisms involved in protective immunity and may therefore help in the design of effective vaccines and diagnostic tools The entire amino acid sequence representing the VP1 capsid protein of EV71 strain 41 was submitted to analysis using a virtual matrix-based prediction program (ProPred) for the identification of promiscuous HLA-DR ligands Three regions spanning amino acids 66 to 77, 145 to 159 and 247 to 261 of VP1 were predicted to bind more than 25 different HLA-DR alleles

The corresponding peptides (SP1 to SP3) were then tested for their abilities to induce proliferation of CD4+ T cells isolated from peripheral blood mononuclear cells

of five human volunteers screened positive for previous EV71 exposure and one EV71-negative volunteer Upon stimulation with either peptide, CD4+ T cell proliferative responses were observed for all EV71-positive volunteers, indicating the presence of EV71-specific memory CD4+ T cells The amplitude of the proliferative responses was peptide- and HLA-DR-dependent, and correlated well with the ProPred predicted binding efficiencies

differentiation into Th1-type subset Among the three peptides, SP2 induced the highest proliferative response and cytokine production In addition, the SP2-induced proliferative response could be inhibited with anti-major histocompatibility complex (MHC) class II antibody, indicating that SP2 may represent a MHC class II-restricted CD4+ T-cell epitope Hence, this study demonstrates that the ProPred algorithm can accurately predict the presence of human CD4+ T-cell epitopes within the VP1 capsid protein of EV71, and therefore represents a useful tool for the design of subunit vaccines against EV71 The identification of CD4+ T-cell epitopes also provides a better understanding in protective immunity and may help in diagnostic tools against EV71

CHAPTER 1 LITERATURE REVIEW

1.1 Picornaviruses

Picornaviruses are one of the oldest known viruses At present, the Picornaviridae family is divided into five main genera which consist of rhinoviruses, enteroviruses, aphthoviruses, cardioviruses and heptaviruses (REACH) The enteroviruses mostly inhabit the alimentary tract and include polioviruses, coxsackieviruses, echoviruses, human enteroviruses 68 to 71 and human hepatitis A virus (Melnick, 1996)

Not until 1908 was poliovirus identified by two Austrian physicians, Karl Landsteiner and E Popper Following their discovery, polio became a reportable disease entity, and the state of Massachusetts began counting polio cases in 1909 (Bradshaw, 1989) The acid-sensitive rhinoviruses and aphthoviruses are known to cause common cold and the foot-and-mouth disease in cattle, respectively The acid stability of enteroviruses enabled them to be well-ingested and reach the intestinal

tracts of both animals and humans (Levy et al., 1994) The human enteroviruses were

originally classified on the basis of human disease manifestations, replication and

pathogenesis in newborn mice, and propagation using in vitro cell culture method

However, based mostly on molecular properties, they have recently been re-classified into five different species which includes human enteroviruses A through D and

polioviruses (King et al., 2000)

Most enterovirus infections are either mild or asymptomatic However, infections in neonates are frequently life-threatening Study has shown that enteroviruses can also cause common chronic diseases such as dilated

cardiomyopathies, insulin dependent diabetes mellitus and chronic fatigue syndrome

(Muir et al., 1998) Several enteroviruses have been shown to be associated with gastrointestinal disorders, meningitis/encephalitis and respiratory illness (Levy et al.,

1994) Table 1.1 summarizes the various clinical symptoms associated with enteroviruses in infections

Table 1.1 Clinical manifestations of enteroviruses Each symptom may potentially be caused by more than one enterovirus (Melnick, 1996)

Clinical Manifestations Enterovirus Serotypes

B2 to B5; Echovirus 4, 6, 9, 11, 30; Enterovirus 70, 71

A7, A9, A10, B1 to B6; Echovirus 1 to 11,

13 to 23, 25, 27, 28, 30, 31; Enterovirus 70,

71 Hand, foot and mouth disease

(HFMD)

Coxsackievirus A5, A10, A16, Enterovirus 71

Herpangina Coxsackievirus A2 to A6, A8, A10

Acute hemorrhagic conjunctivitis Coxsackievirus A24, Enterovirus 70

71 Pericarditis, myocarditis Coxsackievirus B1 to B5

1.2 Genomic and organization of enteroviruses

Enteroviruses possess a single positive-stranded RNA genome of approximately 7,500 nucleotides The complete genomic sequences of several Enterovirus 71 (EV71) strains have been determined, including the prototype BrCr strain (Accession no U22521), the neurovirulent MS/7423/87 strain (Accession no U22522) (Brown and Pallansch, 1995), the fatal 5865/sin/000009 strain (Accession

no 316321) and the non-fatal 5666/sin/002209 strain (Accession no 352027) isolated from two patients during the Hand, foot and mouth disease (HFMD) outbreak in

Singapore in the year 2000 (Singh et al., 2002)

The enteroviral genome comprises a 5’ untranslated region (5’UTR), a long open reading frame (ORF) encoding a polyprotein of 2,194 amino acids, a short 3’ untranslated region (UTR) and a poly-adenylated tail The 5’UTR contains determinants for translation of the viral RNA by internal ribosome entry site (IRES)

mechanism for the amplification of viral RNA and for its neurovirulence (Evans et

al., 1985), and its end is modified by the presence of a covalently bound protein VPg

The polyprotein is sub-divided into three regions, namely P1, P2 and P3 The P1 region encodes four structural proteins (VP1 to VP4), the P2 and P3 regions encode seven non-structural proteins (2A to 2C and 3A to 3D, respectively) (Figure 1.1) The polyprotein is co- and post-translationally cleaved to give rise to four structural proteins, namely VP1, VP2, VP3 and VP4 The 3’UTR region contains a pseudo-knot like structure that is crucial for negative-stranded RNA synthesis (Brown and Pallansch, 1995)

Figure 1.1 Genome structure of EV71 The single open reading frame (ORF) is

flanked by UTRs at the 5' and 3' ends A variable length poly-A tail is found at the 3' UTR The ORF is divided into three regions: the P1 region encodes four structural proteins VP1 to VP4, the P2 and P3 regions encode seven non-structural proteins 2A

to 2C and 3A to 3D, respectively

207bp

762bp 726bp

1.2.1 The enteroviral capsid proteins

The single large open reading frame (ORF) encodes a polyprotein of approximately 250 kDa in size which is proteolytically processed by virus-encoded proteinases to yield structural and non-structural proteins This is a full-length molecule on which RNAs are translated, while others are packaged as viral genomes

by the newly synthesized coat proteins (Harper, 1998; Johnson and Sarnow, 1995) The viral capsid is icosahedra (T=1) in symmetry and is composed of sixty identical units (protomers) Each promoter is comprised of four structural proteins: VP1 to VP4 Among them, VP1, VP2 and VP3 are the main structural components of the

viron, whereas VP4 is relatively unstructured and wholly internal (Hogle et al., 1985)

The VP1 capsid protein contains all three major neutralization sites that have been identified on the surface of poliovirus However, VP2 and VP3 capsid proteins

contain two and one neutralization site, respectively (Van der Marel et al., 1983; Van Wezel et al., 1983) Since X-ray crystallographic structures have been obtained for

several picornaviruses, a consensus structural model for the EV71 coat protein was also developed to facilitate diagnostic immunoassays and vaccine development

(Ranganathan et al., 2002) The unique surface features of EV71 were characterized

by homology modeling and comparing its structural model with experimental structures of other enteroviruses such as bovine enterovirus, rhinovirus and poliovirus Although the antigenic determinants for EV71 have not been fully characterized but surface topography analysis and protein structure modeling have shown that the 3D structure of VP1 protein forms peaks separated by canyons and the immunogenic sites were surface-exposed and thus constituted a large immunogenic site for EV71 (Figure 1.2) Among all enteroviruses, a study has indicated that the VP1 region appears to be

the most immunodominant amongst the other capsid proteins (Oberste et al., 1999)

Figure 1.2 Diagrammatic representation of the EV71 icosahedra virus capsid

The capsid protein surface is assigned colors by depth, the deepest being dark grey and the outermost being white The antigenic regions of VP1 are denoted by light

green, red, green, yellow and salmon (Ranganathan et al., 2002) (Permission granted

by publisher, Adis)

1.2.2 Infection cycle

Typically, in an early event of any infection by enteroviruses, the initial uncoating of viral genome is the primary objective to gain entry into the host cell To begin, endocytosis of viruses generally involves receptors, which is mediated by clathrin-coated pits and vesicles (Racaniello, 1995) Upon entry into the host cell, the virus genome is translated to produce the RNA polymerase and other enzymes As shown in Figure 1.3, the entire replicative cycle of enteroviruses occur in the cytoplasm of infected cells During viral replication, one of the products made is the viral RNA-dependent RNA polymerase which copies the genomic RNA to produce a negative-sense strand This forms the template for positive-stranded genomic RNA synthesis which occurs via a multi-stranded replicative intermediate complex, some of which are translated whereas others are believed to be packaged as viral RNA into

preformed capsids (Ansardi et al., 1996; Whitton et al., 2005) Once infected, cells

typically displayed cytopathic effects (CPE) that comprise a series of cellular changes (Schlegel and Kirkegaard, 1995) The cell nucleus gradually alters in morphology until it acquires a characteristic crescent shape late in infection 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 1.3)

Figure 1.3 Life cycle of Picornaviruses (Whitton et al., 2005) (Permission granted

by Nature publishing group)

1.3 Enterovirus 71 (EV71) infection

1.3.1 Epidemiological studies

EV71 was first identified in the United States from the stool of an infected

infant in the year 1969 (Schmidt et al., 1974) Australia was the first country in which

EV71 was isolated outside of the United States during an epidemic of aseptic

meningitis between 1972 and 1973 (Kennett et al., 1974) In the year 1975, the

neurovirulence of EV71 was manifested during a large EV71 outbreak in Bulgaria

which resulted in 44 fatalities (Chumakov et al., 1979) However, an association of

EV71 with the Hand, foot and mouth disease (HFMD) was not made until the year

1973, during the small epidemics in both Sweden and Japan (Blomberg et al., 1974; Hagiwara et al., 1978)

In the last 10 years, there was an increase of epidemics and neuropathogenicity of HFMD caused by EV71, particularly in the Asia-Pacific region Before 1997, outbreaks caused by EV71 were of smaller scale and most were not associated with acute neurological disease Since 1997, several large epidemics and high-level endemic circulations of EV71 strains have been reported A summary of the main outbreaks from 1997 to present is presented in Table 1.2 Ever since the major HFMD outbreaks within the Asia-Pacific region in 2000, small scale outbreaks were also reported in countries such as Malaysia, Taiwan, China, Hong Kong, Brunei, India, Singapore, USA and Germany from year 2000 to 2006 (http://www.promedmail.org) Recently, in 2006, a large scale of HFMD outbreak occurred in Sarawak, Malaysia and 13 fatalities were reported At the same time, approximately 3,000 of non-fatal HFMD cases were recorded in Singapore and 75%

of the cases were caused by EV71 (Ministry of Health, Singapore)

Table 1.2 Summary of main HFMD outbreaks from 1997 to present

Year Location Estimation of

Total Cases

Agent Involved

Fatal Cases

1.3.2 Phylogenetic studies

The comparison of nucleotide and deduced amino acid sequences have identified four major phylogenetic groups within the enterovirus genus: coxsackievirus A16 (CA16)-like viruses (cluster A), a coxsackievirus B (CB)-like group containing all CB and echoviruses, as well as CA9 and enterovirus 69 (EV69) (cluster B), poliovirus-like viruses (cluster C), and EV68 and EV70 (cluster D) (Pöyry

et al., 1996; Pringle, 1999) EV71 was also found to be very closely linked to CA16

where they share a nucleotide sequence homology of approximately 77% and 89% homology based on deduced amino acid sequences and hence falls within cluster A

1.3.2.1 VP1-based classification

The understanding of the molecular epidemiology and evolution of EV71 took

a major step forward when Brown et al (1999) reported the phylogenetic analysis of

EV71 To investigate the genetic variability of various EV71 strains and their associations with outbreaks, the complete cDNA sequence (891bp) encoding the VP1 capsid protein of EV71 strains isolated from various countries over a 30-year period was analyzed and the monophylogenetic serotype was further divided into three

distinct genogroups A, B and C (Brown et al., 1999) Genogroup A contains a single

member, the prototype EV71 strain BrCr-CA-70 whereas genogroup B consists of 65 strains isolated from 1972 to 1997 in the United States, Australia, Columbia and East Malaysia (Sarawak) Genogroup C is made up of 47 strains isolated from 1986 to

1988 from the United States, Australia, the Republic of China, Canada and mainland

Malaysia (Brown et al., 1999) (Figure 1.4) Within each genogroup, EV71 strains

were classified into subgenogroups (B1 to B5 and C1 to C4) mostly based on their VP1 or VP4 gene sequences Within similar subgenogroup, EV71 strains share more

than 92% nucleotide sequence identity whereas the nucleotide sequence identity between the three genogroups ranges from 78% to 83%

1.3.2.2 VP1- and VP4-based classification

Phylogenetic relationships of EV71 strains isolated from major outbreaks in the Asia-Pacific region were also established based on other parts of the EV71

genome such as the 5’UTR (Abubakar et al., 1999; Wang et al., 2000) and the VP4 (Shimizu et al., 1999; Chu et al., 2001; Cardosa et al., 2003; Lin et al., 2006) Chu et

al (2001) examined a partial VP4 region (207 bp) of 23 EV71 Taiwanese strains from

the 1998 epidemic and 21 other strains deposited in the GenBank, (the prototype BrCr strain, the neurovirulent 7423/MS/87 strain, three strains isolated during the 1986

outbreak in Taiwan, and 16 strains isolated from Japan) Cardosa et al (2003)

analyzed the partial VP1 and VP4 regions of 128 EV71 strains isolated from the year

1970 to 2002 from the United States, Japan, Taiwan, Malaysia, Singapore, China, Bulgaria, Hungary and the United Kingdom Analysis of either the VP1 or VP4 gene sequences provided similar phylogenetic classification of the EV71 strains However, higher bootstrap values were observed in the VP1 dendrograms, hence providing

greater confidence when elucidating new genogroups (Cardosa et al., 2003) Similar

phylogenetic classification between the VP1 and VP4 regions was further shown in two recent separate studies which carried out genetic analysis of a 414 bp region within the VP1 sequence and a 207 bp region within the VP4 sequence on the EV71

strains isolated in Taiwan from the year 1988 to 2005 (Lin et al., 2006; Kung et al.,

2007) Both studies demonstrated the predominance of the subgenogroup B1 before the year 1998 whilst the subgenogroup C2 was the major etiologic group in the 1998 outbreak The subgenogroup B4, which was a minor etiologic group during the 1998

outbreak, became the major sub-genogroup isolated from 1999 to 2003 During the year 2004, the subgenogroup C4 emerged to be the predominant sub-genogroup in Taiwan

1.3.2.3 Relationship between subgenogroups and outbreak occurrence

There seems to be no correlation between the severity of disease and the genetic lineage of the EV71 strains since viruses from all three genogroups are

capable of causing disease (Brown et al., 1999) McMinn et al (2001a) compared the

complete VP1 gene sequence of 66 EV71 strains isolated from Malaysia, Singapore, Taiwan and Western Australia between 1997 and 2001 and established two sub-lineages within the genogroup B which circulated in Southeast Asia between 1997 and 2001 EV71 strains in the subgenogroup B3 were the predominant strains isolated from the epidemics in Sarawak and peninsular Malaysia in 1997 and in Western Australia in 1999 (Figure 1.5) Partial genetic analysis (500 bp) of the VP1 capsid protein from 18 EV71 strains involved in the 1998 outbreak in Taiwan has shown that

majority of the viruses belonged to subgenogroup C2 (Shih et al., 2000b) Based on

the phylogenetic analysis of the VP1 region of 45 EV71 strains which were isolated

over a 6-years period in Yamagata, Mizuta et al (2005) found that outbreaks in

Yamagata were mainly caused by EV71 strains belonging to the subgenogroups B4, C2 and C4 However, there were a few isolated EV71 strains that could not be classified into any of the three subgenogroups and they were classified under a new subgenogroup known as B5 (Figure 1.6) More EV71 strains were classified into the

subgenogroup B5 when Ooi et al (2007) analyzed the VP1 regions of EV71 isolated

from 277 patients in Malaysia from 2000 to 2004 It was shown that 168 patients were infected with EV71 strains from the subgenogroup B4 whereas 68 patients were

infected with strains from the subgenogroup C1 and 41 patients were infected with the newly emerged EV71 strain belonging to the subgenogroup B5

At present, a total of 10 subgenogroups have been identified It was established that there is a great diversity of EV71 strains circulating in the Asia-Pacific region and other parts of the world However, no significant differences in genome sequence were found between EV71 strains isolated from fatal and non-fatal cases In the year 2000, EV71 strains belonging to the subgenogroup B4 were responsible for the HFMD outbreak in Singapore Using a comparative sequence

analysis, Singh et al (2002) showed that the fatal Singapore strain 5865/sin/000009

which belongs to the subgenogroup B4 had 99% nucleotide and 100% amino acid homology with the non-fatal Singapore strain 5666/sin/002209 from similar subgenogroup However, both strains displayed significant differences when compared to other EV71 strains including the prototype BrCr strain (genogroup A) and the neurovirulent strain MS/7423/87 (subgenogroup B2) Therefore, there is no particular subgenogroup that is associated with severe neurological complications

(Singh et al., 2002; Cardosa et al., 2003) Due to its potential in causing severe

neurological diseases, further studies on EV71 are necessary to understand the factors responsible for neurovirulence and epidemic potential