The generation of native human monoclonal antibodies with neutralising activity for dengue virus 3

Since alteration of the recognition motif for integrin did not abolish virus-cell binding, it has been proposed that receptor-mediated endocytosis of DV involves two or more receptors: a

Chapter 1 - Introduction

1.1 Dengue virus

1.1.1 Classification

Dengue viruses (DV) are members of the Flavivirus genus of the Flaviviridae

family which comprises of over 70 members separated into groups using molecular phylogenetic analyses and analyses of serological relatedness Viruses

in the Flavivirus genus can cause serious diseases in humans and animals, and most of them are anthropod-borne (arboviruses) and thus are transmitted to vertebrate hosts by mosquitoes or ticks Several members of the Flavivirus genus, such as Yellow Fever virus (YFV), West Nile virus (WNV), Japanese Encephalitis virus (JEV) and in particular Dengue virus (DV) are highly pathogenic to humans and constitute major international health problems (Mackenzie, Gubler et al 2004; Gould and Solomon 2008) Within the genus, the viruses can be further subdivided into antigenic complexes according to serological criteria, or into clusters, clades and species on the basis of molecular phylogenetics (1.1.6) and is summarized in Table 1.1 (Kuno, Chang et al 1998)

Table.1.1 Flavivirus Classification The above dendrogram shows the

relationships between selected flaviviruses (2001) Lippincott Williams and Wilkins, Philadelphia In Fields Virology

Based on the gene sequence of a non-structural protein, NS5, the flaviviridae are

clustered into three distinct groups which correlate with the mode of transmission (mosquito-borne, tick-borne, unknown vector respectively)(Kuno, Chang et al 1998) DV was determined to be evolved from a common ancestor 1,000 years ago and that human transmission started between 125 and 325 years ago (Twiddy, Holmes et al 2003; Mackenzie, Gubler et al 2004) It is still undetermined whether the virus originated from Africa or from Asia but dengue transmission

was first maintained in a sylvatic lifecycle within the Aedes species of mosquitoes

(Twiddy, Holmes et al 2003) Sporadic outbreaks of dengue fever first occurred

in predomestic regions due to transmission by Aedes albopictus and Aedes

aegypti The adaptation of Aedes species, mainly the Aedes aegypti, to urban and

densely inhabited areas is proposed to have created optimal conditions for human

transmission resulting in the emergence of dengue epidemics (Gubler 2002; Gubler 2004; Mackenzie, Gubler et al 2004)

1.1.2 Epidemiology

At present, close to 2.5 billion people living in more than 100 dengue endemic countries in the tropical/sub-tropical belt are considered to be at risk of dengue infection (Pinheiro and Corber 1997) Approximately 50 million people are infected each year with DV with over 500 000 people requiring hospitalization All four DV serotypes are infectious to humans Severe disease was first detected

in Southeast Asia and the Western Pacific region Over the years, the geographic distribution has increased to include South Asia, South and Central America, the Caribbean, and Africa This is mainly due to the increased accessibility to infected areas with the aid of modern transport Over 3 billion people live in endemic areas and therefore at risk of developing the disease, thus making dengue an emerging disease and global threat The outbreaks in Hawaii, along the Texas-Mexico border and Puerto Rico make the widespread appearance of dengue a possibility

in the United States (FIG.1.1)

FIG.1.1 Global prevalence of DF and DHF as shown by WHO World map

showing the prevalence of DV in 2005

http://www.who.int/csr/disease/dengue/impact/en/index.html

All four DV serotypes are prevalent in Singapore In 2006, DV1 remained the predominant serotype after the major 2004-2005 outbreak and more than 75 % of the samples are DV1 positive In early January 2007, the predominant circulating serotype switched from DV1 to DV2 The proportion of DV2 positive samples rose from 57.9 % to 91 % at mid-2007 The rise of DV3 cases were detected in

2008 Early attempts to step up prevention of the spread of the serotype that had been uncommon in Singapore have been implemented to prevent another outbreak The fatality rates are low in Singapore with 0.32 % in 2006 and 0.27 %

in 2007 and rates remained unchanged the years after (Ooi, Goh et al 2006)

1.1.3 Structure of dengue virions

Members of the Flaviviridae family are characterized by having enveloped

virions of small size containing three structural proteins, the envelope (E), core (C) and precursor membrane (prM) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) The E, C and prM proteins have

495, 120 and 165 amino acids respectively (Mukhopadhyay, Kuhn et al 2005)

prM is processed to the mature M protein late in secretion in the trans Golgi

compartment by furin (Stadler, Allison et al 1997) Multiple copies of the C protein (11kDa) encapsulate the viral RNA genome to form the viral nucleocapsid (Chang, Luh et al 2001; Jones, Ma et al 2003; Ma, Jones et al 2004) The E protein has three distinct structural domains termed domain I, II and III respectively (Rey, Heinz et al 1995; Modis, Ogata et al 2003; Modis, Ogata et al 2005; Nybakken, Nelson et al 2006) Domain I (DI) is structurally positioned between Domain II (DII), the homodimerizaton domain, and the immunoglobulin-like domain III (DIII) (FIG 1.2) The mature dengue virion has a diameter of about 500Å and consist of a viral genome of around 10.8kb packed by the dimeric capsid proteins The resulting nucleocapsid is enclosed by a host-derived lipid bilayer containing 180 copies of the E and M protein that form an icosahedral symmetry (T=3) (Kuhn, Zhang et al 2002) This mean that the three E monomers present in each icosahedral asymmetric unit exist in three chemically distinct environments and may therefore play a distinct role in different stages of the infection Based on the shape of the monomer and the location of the antibody

epitopes, Rey et al postulated that the E protein has a „flat‟ topology along the surface of the virus lipid bilayer (Rey, Heinz et al 1995)

FIG.1.2 Ribbon drawing of E protein Dengue E protein dimer with three

defined domains within each monomer: Domain I in red, Domain II in yellow with fusion loop in green, and Domain III in blue (Modis, Ogata et al 2003)

1.1.4 Organization of the Flavivirus genome

The viral genome consists of a single stranded, positive-sense RNA and approximately 10.8kb in length (Chambers, Hahn et al 1990) (FIG.1.3) The genome has one open reading frame encoding a single polyprotein The 5‟ end of the RNA contains a type I cap and is followed by the conserved dinucleotide sequence AG The type I cap is generated when the first nucleotide in the transcript correspond to this position Genomic RNA of mosquito-borne flaviviruses appears to lack a 3‟ poly-(A) tract and instead terminate with the conserved dinucleotide CU The flaviviral genome contains an open reading frame of over 10000 bases, flanked by 5‟ and 3‟ untranslated regions (UTR) containing conserved RNA elements No other conserved open reading frame

(ORF) has been identified in either the genomic sense RNA or its compliment (Chambers, Hahn et al 1990) The amino terminus of the genome encodes the prM, C and E proteins that constitute the virus particle Seven NS proteins are essential for viral replication and are encoded by the remainder of the genome The C protein is involved with packaging of the viral genome and forming the nucleocapsid (NC) prM and E are glycoproteins, each of which contains two transmembrane helices Before it is cleaved during particle maturation to yield the

pr peptide and the M protein (75 amino acids), the prM protein may function as a chaperone for folding of the E protein The E protein contains a cellular receptor binding site and a fusion peptide (Stadler, Allison et al 1997; Allison, Schalich et

al 2001; Lorenz, Allison et al 2002) Since flaviviruses only encode 10 proteins, the host cell protein synthesis, nucleic acid synthesis, membrane trafficking machinery and functions are exploited in order to complete the viral infectious cycle

FIG.1.3 Schematic representation of the polyprotein processing for flaviviruses The top region depicts the structural and non-structural ORF and the

5‟ and 3‟ UTR The bottom region depicts co-translational cleavage by signalase and NS3 protease separating structural and non-structural proteins occurs at the C-terminus of E and the roles of virus proteins Figure adapted from (Fernandez-Garcia, Mazzon et al 2009)

1.1.5 Replication strategy

1.1.5.1 Receptor interaction

Infection with one of the arthropod-borne flaviviruses begins when the vector takes a blood meal and the virus is introduced into the host Despite a small number of reports suggesting other entry mechanisms of dengue virus such as entry via direct fusion with the plasma membrane (Hase, Summers et al 1989;

Lim and Ng 1999), the receptor-mediated endocytosis is generally accepted as the principle mode of entry Autopsy studies have indicated the virus infects dendritic cells (DCs), monocytes/macrophages, B cells (Tassaneetrithep, Burgess et al 2003), T cells, endothelial cells, hepatocytes and neuronal cells (Clyde, Kyle et al

2006) Further evidence for this broad cellular tropism in vivo has included the

detection of DV in Langerhans cells (skin-resident DCs) (Scott, Nisalak et al 1980; King, Nisalak et al 1999; Wu, Grouard-Vogel et al 2000; Neves-Souza, Azeredo et al 2005) after inoculation with an experimental dengue vaccine and in monocytes and B cells in peripheral blood from naturally infected patients (Scott, Nisalak et al 1980; King, Nisalak et al 1999; Wu, Grouard-Vogel et al 2000; Neves-Souza, Azeredo et al 2005) Several groups have attempted to characterize the cellular receptors of DV infection A number of different mammalian cell receptors have been proposed Evidence of heparin sulfate (Chen, Maguire et al 1997), heat shock protein 70 (HSP70) and HSP90 (Reyes-Del Valle, Chavez-Salinas et al 2005), GRP78/BiP (Jindadamrongwech, Thepparit et al 2004), CD14 (Chen, Wang et al 1999), 37 kDa/67 kDa high affinity laminin receptor (Thepparit and Smith 2004), mannose receptor (MR) (Miller, de Wet et al 2008), DC-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209) (Tassaneetrithep, Burgess et al 2003) and liver/lymph node-specific ICAM-3-grabbing nonintegrin (Tassaneetrithep, Burgess et al 2003) have been provided A growing body of evidence suggests that DC-SIGN provides a critical bridge between viral replication in insect vectors and infection

of the vertebrate host though it should be noted that much of this datails based on

human cell lines or animal models (Navarro-Sanchez, Altmeyer et al 2003; Tassaneetrithep, Burgess et al 2003; Lozach, Burleigh et al 2005)

DC-SIGN is a tetrameric C-type lectin that is unique for pathogen capture and antigen presentation (Cambi, Gijzen et al 2003) This receptor is constitutively expressed on DCs, including Langerhans cells, the proposed cells present at the anatomical site of initial infection following the bite of a DV-infected mosquito (van Kooyk and Geijtenbeek 2003) The four DV serotypes of DV strains utilize DC-SIGN to enter into and infect productively immature DCs (Navarro-Sanchez, Altmeyer et al 2003; Tassaneetrithep, Burgess et al 2003; Martina, Koraka et al 2008) The carbohydrate recognition domain (CRD) of DC-SIGN interacts with the N-glycosylated carbohydrate moieties of the E protein DC-SIGN possesses the remarkable capacity to distinguish between high-mannose glycans typical of insect-derived glycoproteins and the complex glycosylation of host-derived proteins (van Kooyk and Geijtenbeek 2003; Lozach, Burleigh et al 2005) implies that DV have evolved an elegant strategy to initiate infection of human cells by taking advantage of the ligand specificity of this pattern recognition receptor

A comparative study of two dengue strains showing differences in infectivity revealed an unique amino acid in the E-protein leading to the loss of N-linked glycosylation sites and therefore a decrease in infectivity (Ishak, Takegami et al 2001) DC-SIGN-mediated infection may be an important component of DC maturation (Lozach, Burleigh et al 2005), which is a crucial for allowing DCs to leave the skin and migrate to the lymph nodes, where they present processed antigens to T cells and initiate adaptive immune responses (Mellman and

Steinman 2001) In addition, Sakuntabhai and colleagues (2005) provided evidence of DC-SIGN playing a critical role in the initial viral dissemination and pathogenesis by identifying a single nucleotide polymorphism in the DC-SIGN promoter that is strongly associated with protection against DF, thus supporting this concept of pathogenesis (Despres, Sakuntabhai et al 2005) Since alteration

of the recognition motif for integrin did not abolish virus-cell binding, it has been proposed that receptor-mediated endocytosis of DV involves two or more receptors: a ubiquitous, lower-affinity receptor such as DC-SIGN that captures the virus at the cell surface, increasing the local concentration, and a less-common high affinity receptor that mediates internalization of the virion (van der Most, Corver et al 1999) A recent report has suggested that another DV binding receptor, identified as C-type lectin domain family 5, member A (CLEC5A, MDL-1), induces DAP12 phosphorylation and proinflammatory cytokine production However, in contrast to DC-SIGN, CLEC5A does not promote viral entry Over the last decade, several candidate receptors and/or attachment factors have been identified, which suggests that DV is capable of utilizing multiple targets for viral entry

1.1.5.2 Viral entry and the E protein

DV upon engagement with target cellular receptors that traffic into the endocytic pathway, where the acidic environment triggers major conformational changes in

E protein that induce fusion of the viral and host cell membranes (Modis, Ogata et

al 2004) These conformational changes cause dimers seen in the mature virus to undergo large rotations in the transition to the fusogenic form During these

rearrangements, the virus expands radially by at least 10% to avoid steric clashes (Kuhn, Zhang et al 2002) (FIG.1.4) The E glycoprotein suggests a common class

II fusion mechanism for flaviviruses based on the insertion of a β barrel-type structure into the host cell membrane Class I and II fusion proteins were first differentiated by Lescar et al based on a description of a variety of properties related to the activation and position of the fusogenic peptide (Lescar, Roussel et

al 2001; Jahn, Lang et al 2003) Flaviviruses such as DV possess a hydrophobic sequence of about a dozen amino acid residues known to be essential for fusion These sequences are components of helices and β barrel-type structures The E glycoprotein has two conserved helices in the „stem‟ region (residues 398-420 and 426-448 in DV2), between the ectodomain and the transmembrane region (Allison, Stiasny et al 1999; Zhang, Chipman et al 2003) The helices and β-barrels hide all main chain amino and carbonyl polar groups by forming hydrogen bonding networks The exteriors of these structures are hydrophobic to help accommodate them among the aliphatic chains that constitute a membrane The fusion peptides of flaviviruses are located in homologous E protein (Rey, Heinz et

al 1995) Although the hydrophobic fusion peptides are not in equivalent positions, they are in neighboring loops of the β structure at the distal end of domain II A flexible hinge region between domains I and II has been shown to be required for structural changes which is necessary for the exposure of the fusion peptide (Modis, Ogata et al 2003; Modis, Ogata et al 2004) Rey and colleagues suggested that it is in this region that the largest structural changes occurs when

the pH is lowered, as it is required for the fusion process to be initiated (FIG.1.5) (Rey, Heinz et al 1995)

FIG.1.4 Proposed rearrangement of E dimer in Flaviviruses upon exposure

to low pH (A) T=3 fusogenic structure The radial expansion of the particle in

(B) upon low pH induced conformational change Arrows in (B) indicate the direction of E rotation (Kuhn, Zhang et al 2002)

FIG.1.5 Schematic diagram of E glycoprotein in neutral and the proposed acidic pH conformation E glycoproteins are shown as yellow cylinders with the fusion peptide as

green curve Fusion peptide is exposed upon conformational change in the presence of acidic pH The membrane is shown in grey (Kuhn, Zhang et al 2002)

1.1.5.3 Translation of the DV genome

Upon viral entry, DV, that is encoded by positive-sense genome RNA utilizes the same template for both translation and genome replication However, the two processes cannot occur simultaneously After input strand translation, the virus switches to production of viral RNA, which involves a negative-strand template for further generation of the positive strands Since the positive strand serves as both viral genome and mRNA, it is produced in excess of the negative strand The mechanism by which this asymmetric synthesis occurs has yet to be elucidated Cells infected with DV detected at later stages of infection are found to harbor vesicle packets within the cytoplasm of the cells Mackenzie et al showed that several viral NS proteins as well as double-stranded RNA (an intermediate of transcription), are localized within the vesicle packets(Mackenzie 2005) The inner side of the vesicles is likely the site of viral RNA replication Electron tomography by Welsch et al revealed that pores are present in invaginations of

ER membranes to retain an open connection to the surrounding cytoplasm of the infected cell (Welsch, Miller et al 2009)

Non-structural proteins of DV are essential for the transcription and translation processes necessary for viral propagation Of the viral NS proteins, the most extensively characterized are the NS3 protein and the cofactor NS2B, and NS5 NS3 protein does not contain long stretches of hydrophobic amino acids but becomes membrane-associated via the interaction with NS2B protein, which together constitutes the functional viral protease, NS2B-3 (Falgout, Pethel et al 1991) The N-terminal 180 amino acids of NS3 protein contain the catalytic

domain of the viral protease, NS2B-3, as defined by the sequence alignment with known serine proteases of the trypsin superfamily (Bazan and Fletterick 1989), by deletion analyses (Wengler, Czaya et al 1991) and by site-directed mutagenesis

of residues in the putative catalytic triad or the substrate-binding pocket (Chambers, Hahn et al 1990; Wengler, Czaya et al 1991) The residues of the catalytic triad, His-Asp-Ser, are conserved among flaviviruses (Gorbalenya, Donchenko et al 1989) NS3 protein is required for viral RNA synthesis through its nucleoside triphosphatase and helicase functions (Li, Clum et al 1999), as well

as 5‟ triphosphatase activity, which is prior to the capping of the RNA (Bartelma and Padmanabhan 2002; Benarroch, Selisko et al 2004) In addition, NS3 protein has been shown to interact with human nuclear receptor binding protein, which modulates intracellular trafficking between the ER and the Golgi compartment, to impact its cellular distribution and to induce some of the membranous invaginations seen during infection (Chua, Ng et al 2004) NS3 epitopes are commonly found in the repertoire of the DV-specific cytotoxic T cells Mutations

of the NS3 protein helicase domain have been shown to abolish viral replication (Matusan, Pryor et al 2001) These data are consistent with the proposed role of NS3 protein in the function of the flaviviral replication complex The flavivirus NS5 is essential for the capping pathway NS5 protein is a viral RNA-dependent RNA polymerase, as well as a methyltransferase Medin and colleagues have provided evidence to show that NS5 protein also induces transcription and translation of interleukin-8 (IL-8), a neutrophil chemoattractant via activation of CAAT/enhancer binding protein The role of IL-8 is discussed further in 1.1.8.2

Although functions of NS1 (40-50 kDa) protein have yet to be fully elucidated, several lines of evidence have suggested that NS1 protein is involved in replication of viral RNA NS1 protein is expressed in three forms The first identified form resides in the ER and colocalizes with the viral replication complex The second form is a membrane-anchored form, and the third form is secreted (Lindenbach and Rice 2003) NS1 protein is glycosylated at two sites, N130 and N207, and glycosylation of both residues is required for viral replication in mosquito cells and for neurovirulence in mice (Crabtree, Kinney et

al 2005) Secreted NS1 protein is another dominant target of humoral immunity and may play a significant role in the pathogenesis of disease (1.1.8.2)

The remaining NS proteins such as NS2A, NS4A and NS4B have not been thoroughly characterized They are implicated in localizing viral proteins and viral RNA to sites of RNA synthesis, virion assembly and were recently discovered to block IFN signaling by reducing STAT activation (Munoz-Jordan, Sanchez-Burgos et al 2003; Ho, Hung et al 2005; Ashour, Laurent-Rolle et al 2009) While three proteins in combination are most effective, NS4B protein alone is a potent inhibitor of beta interferon (IFN-β) and gamma interferon (IFN-γ) signaling NS2A protein has an important role in the incorporation of genomic RNA into the budding virion (Kummerer and Rice 2002) as studies have shown that purified NS2A protein binds RNA This led to the hypothesis postulating NS2A protein may shuttle genomic substrates out of the membrane-bound replication complexes to sites of packaging (Mackenzie, Khromykh et al 1998; Liu, Chen et al 2003) Like NS3 protein, NS4A protein also played a role in the

induction of invaginations of the ER membrane (Roosendaal, Westaway et al 2006; Miller, Kastner et al 2007) Further studies are necessary to decipher the molecular mechanisms by which NS4A induces these membrane changes, and to investigate what other additional roles do the NS proteins participate in

1.1.5.4 Virus assembly and propagation

Structural analysis of newly assembled immature virions revealed that a single virion contains 180 prM/E heterodimers that project vertically outward from the viral surface as 60 trimeric spikes It is markedly different from mature particles which are spikeless and smooth (Mukhopadhyay, Kim et al 2003; Zhang, Chipman et al 2003) CryoEM has shown the hinge angle between domains I and

II of each of the three symmetry-independent E proteins to differ approximately

by 5-15° from the crystal structures and about 30° from the cryoEM structure of the mature particle (Zhang, Corver et al 2003) The immature particles formed in the ER mature as they travel through the secretory pathway The slightly acidic

pH (~5.8 - 6.0) of the trans Golgi network triggers dissociation of the prM/E

heterodimers, which leads to the formation of 90 dimers that lie flat on the surface

of the particle, with prM capping the fusion peptide of the E protein This global structural reorganization of the glycoproteins enables the cellular endoprotease furin to cleave prM Furin cleavage occurs at a Arg-X-(Arg/Lys)-Arg (where X is any amino acid) recognition sequence and leads to the generation of membrane-associated M and a pr peptide A recent study has shown that the pr peptide

remains associated with the virion until the virus is released to the extracellular milieu to infect neighboring cells (Yu, Zhang et al 2008) (FIG.1.6)

FIG.1.6 Life cycle of DV The picture depicts the various cleavage processes

within the cell contributing to major conformational changes during the life cycle (van der Schaar, Rust et al 2008) DV binds and enters cells via receptors by receptor mediated endocytosis Endosomal acidification results in an irreversible trimerization of the viral E protein, exposing the fusion domain After uncoating, the viral RNA is translated at ER-derived membranes, where it is processed into three structural and seven NS proteins After the viral replication complex is synthesized, viral RNA translation switches off and RNA synthesis begins Subsequently, successive rounds of translation are followed by assembly in the

ER The virion is maturated in the Golgi compartment and exits via the host secretory pathway

1.1.6 Phylogeny of DV

The four serotypes of dengue are phylogenetically different to an extent as observed between different flaviviruses which in turn, highlights their independent zoonotic transfer and their separate evolutionary development (Twiddy, Woelk et al 2002; Klungthong, Putnak et al 2008) Serotype 1 and 4 share about 70 % homology and Serotype 1 and 3 share about 50 % homology Serotype 2 however, does not seem to be closely related to any of the other serotypes (Blok 1985) Rico-Hesse (2003) first performed a study to determine intra-serotype variation using the junction between the E/NS1 genes of Dengue virus serotype 1 (DV1) and Dengue virus serotype 2 (DV2) At present the entire sequence of E-protein (1485 bp) is used and recent phylogenetic studies revealed that three to five genotypes could be found within a serotype (Rico-Hesse 2003) DV1 can be subdivided into five genotypes representing geographical locations, namely Americas/Africa, South Pacific, Asia Thailand, and different host characteristics such as human or sylvatic (Rico-Hesse 2003) Using 177 DV1 E protein gene sequences, three genotypes with geographical distribution was maintained Genotype 1 consists of viruses from Africa/Asia and Asia/Oceania Genotype 2 from Asia and Oceania and finally Genotype 3 composed of three smaller clades of Asia, Africa and Latin America

The phylogeny of DV2 is the best studied due to an abundance of sequence data Five possible genotypes were detected They are Cosmopolitan, Asian 2, Asian 1, American/Asian and American Studies have shown the different genotypic

strains have little epidemiological impact on human populations It was shown that the introduction of South-East Asian genotype into the Americas correlated with the occurance of DHF/DSS (Rico-Hesse 2003) and later experiments underline the early replicative advantage of the South-East Asian genotype (Armstrong and Rico-Hesse 2003; Cologna and Rico-Hesse 2003; Cologna, Armstrong et al 2005)

Dengue virus serotype 3 (DV3) consists of four or five distinct genotypes depending on the analysis performed (Messer, Gubler et al 2003; Rico-Hesse 2003; Klungthong, Putnak et al 2008) Genotype 1 includes viruses from South-East Asia and the Pacific, whereas Genotype 2 is only represented by viruses from Thailand Genotype 3 was isolated in the Indian Subcontinent as well as Africa and Genotype 4 comprised of viruses from Puerta Rico and Tahiti Genotype 3 showed interesting patterns in terms of evolutionary and clinical aspects Further clustering of this genotype into smaller clades [Clade A (pre-DHF emergence) and Clade B (post-DHF emergence)] revealed correlations with DHF (Messer, Gubler et al 2003)

Not much data was available for Dengue virus serotype 4 (DV4) which is the first

to separate from the phylogenetic tree (Rico-Hesse 2003) Considering DV4 to be the most divergent of all four serotypes, it was suggested that strain migration of DV4 in the Americas was influenced by different patterns in host immunity (Carrington, Foster et al 2005) Upon introduction, DV4 introduced from Asia was first isolated in 1981 and showed a tremendous increase in genetic diversity reflecting rapid transmission throughout the region (Mota, Ramos-Castaneda et al

2002) Gradually, genetic diversity was retained reflecting the low mutation rate and reports of DV4 activity declined which is underlined by decreased transmission and rare occurrence in dengue outbreaks (Carrington, Foster et al 2005) There are currently the Malaysia, South-East Asia and Indonesia genotypes but further studies are essential for the elucidation of DV4 evolution

Circulation of multiple serotypes in highly endemic regions leads to serotype switch and intraserotypic clade replacement are commonly observed phenomena that play a crucial role in shaping transmission dynamics The presence of multiple serotypes may alleviate the frequency of DHF/DSS causing dengue to be hyperendemic in certain regions of the world (Ocampo and Wesson 2004) It was observed that intraserotypic clade replacement increased the overall abundance of

a first serotype but simultaneously decreased the population of the second serotype due to mutations affecting viral fitness In turn, the later decline of the prevalence of the first serotype was associated with the increase in prevalence of the second serotype (Zhang, Mammen et al 2005) Finally, it was suggested that existing clades belonging to the first serotype were antigenically more distinct to the second serotype than the clades that did not survive the period of the second serotype This may suggest an active role of cross protection in serotype switch but does not completely explain the phenomenon (Zhang, Mammen et al 2005) and in contrast, other studies proposed stochastic explanations for clade replacement as well as serotype switch (Myat Thu, Lowry et al 2005) Therefore, further studies are required to determine the complex interactions of selective and

stochastic forces that shape DV evolution and determine genotypic diversity, which better elucidate the phenomenon of ADE

In addition to the four dengue serotypes, DV exists as quasispecies due to the fact that viral RNA-dependent RNA polymerases are of notoriously low fidelity and tend to incorporate mutations into the progeny RNA strand, coupled with the lack

of proofreading from the second strand, results in the creation of quasispecies Recently, studies focusing on the C, E and NS2B genes have indicated that DV exhibits substantial sequence diversity in humans (Wang, Lin et al 2002; Wang, Sung et al 2002) and mosquitoes (Lin, Hsieh et al 2004) To address the question

of the degree of association of intrahost sequence diversity or particular sequence signatures to DV pathogenesis, a large scale full length sequencing of viral RNAs from human DV infections is necessary

1.1.7 Pathogenesis of DV

Most primary dengue infections result in clinically silent infections, at least in young children Clinical disease usually takes the form of an acute febrile illness termed dengue fever (DF), from which nearly all patients recover Other severe forms of the disease, such as Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) occur with significantly lower frequency DHF is characterized by high fever, hemorrhagic phenomena and plasma leakage and may be life threatening DHF is classified into four grades, the more severe of which (grades III and IV) are associated with DSS DSS manifests itself with

extreme fevers, headache, sore throat, cough, nausea and vomiting The shock occurs after two to six days of symptoms, followed by collapse, weak pulse, and blueness around the mouth (FIG.1.7) These forms may have case fatality rates of 1% or higher especially in infants and young children admitted in hospitals (McBride and Bielefeldt-Ohmann 2000) Although DF is a debilitating disease, it

is DHF and DSS which cause most concern There are hundreds of thousands of cases of DHF each year and without appropriate treatment, mortality can be over

20 % About 95 % of cases occur in children under 15 years of age and at least 5% of these infants, although higher adult morbidity has been reported in some recent outbreaks (Guzman and Kouri 2002) The pathogenesis of DHF is not well characterized, but key epidemiological studies indicate that it often occurs when a dengue-immune person becomes secondarily infected with a virus of a different serotype Halstead and colleagues (1982) reported that DHF and DSS were 15-80 times more likely to manifest in secondary infections than primary infections Furthermore, this was positively associated with pre-existing dengue-virus-specific, non-neutralizing antibodies, thus implicating the immune response in the pathogenesis of severe forms of dengue (Halstead 1982) This has been shown in

primate models having higher viral loads both in vitro and in vivo when observed

in subjects challenged with secondary infection of a different serotype (Halstead and O'Rourke 1977; Halstead 1979)

FIG.1.7 Course of dengue infection and timing of diagnosis Onset of fever

and viremia happens 4 to 6 days after initial infection from a mosquito bite Production of antibodies can occur as early as 6 days Non-neutralizing antibodies may contribute to dengue hemorrhagic shock (Halstead 2007)

Cologna and colleagues have also indicated that there may be differences in the phathogenecity of dengue genotype and that there is a selection for virulent DV

in humans and mosquitoes (Cologna, Armstrong et al 2005) Some dengue genotypes are more capable of producing DHF epidemics in a population base of variable immune status The origin and spread of DHF in the Western Hemisphere can be linked to viruses of the Southeast Asian genotype, whereas the American genotype viruses have been isolated solely from patients presenting DF This suggests biological differences among viral genotypes Subsequent studies demonstrated that Thai DV2 strains (Asian genotype) replicated at higher titers

than American genotype DV2 strains in human monocyte-derived macrophages and DCs (Pryor, Carr et al 2001; Cologna and Rico-Hesse 2003)

A number of studies have identified candidate gene variants that may predispose

or protect an individual towards developing DHF/DSS based on observations of Afro-caribbean individuals having less frequently developed severe dengue than caucasians These include specific human leukocyte antigens (HLAs) alleles and non-HLA gene polymorphisms Out of the non-HLA polymorphic alleles, the FcγRII, vitamin D receptor (Loke, Bethell et al 2002), tumor necrosis factor alpha (TNF-α) (Fernandez-Mestre, Gendzekhadze et al 2004), CTLA-4 (Chen, Wang et al 2009) and transforming growth factor (TGF-β) (Chen, Wang et al 2009) affect the severity of the disease Several HLA class I alleles (A*01, A*0207, A*24, B*07, B*46, B*51) and class II alleles (DQ*1, DR*1, DR*4) were found to be associated with severe diseases susceptibility whereas individuals expressing HLA B*13, B*14 and B*29 were found to be protected (Chiewsilp, Scott et al 1981; Loke, Bethell et al 2001; LaFleur, Granados et al 2002; Stephens, Klaythong et al 2002; Zivna, Green et al 2002; Polizel, Bueno et

al 2004) All these studies suggested that degree of dengue disease severity may

be affected by pre-existing antibodies, sequence variation, level of virus or viral proteins in the bloodstreams of patients and host genetic factors Defining the link between disease risk and HLA type, race (Halstead, Streit et al 2001; Guzman, Kouri et al 2002), or receptor (Sakuntabhai, Turbpaiboon et al 2005) and cytokine (Fernandez-Mestre, Gendzekhadze et al 2004) polymorphisms may

serve as a prognostic tool to identify individuals at elevated risk of severe disease and also a better understanding of DV pathogenesis

al 2005) Secreted IFN binds to IFN receptors present on the same cells as well as

on neighboring cells and activates the JAK/STAT pathways leading to expression

of more than 100 effector proteins (Aebi, Fah et al 1989) IFN-mediated responses induce an antiviral state and initiate a variety of processes including metabolic control to limit virus infection Moreover, these responses promote the adaptive immune response through stimulation of DC maturation and by direct activation of B and T cells (Erickson and Gale 2008) However, as mentioned above in 1.1.5.3, NS2A, NS4A and NS4B proteins are thought to block IFN signaling by reducing STAT activation (FIG.1.8) Interestingly, the ability of DV

to suppress type I IFN response has been shown to be strain-dependent, as within each serotype, both non-suppressive and suppressive strains that block STAT1/STAT2 pathways are found (Umareddy, Tang et al 2008) In Taiwan, adults with DF and DHF who survived infection exhibited significantly higher levels of circulating IFN-γ than controls, but non-survivors did not have significantly higher levels (Chen, Lei et al 2006) One of the main producers of IFN-γ are natural killer (NK) cells which can recognize and kill virus-infected cells (Lanier 2008) In response to flavivirus infection such as DV, early activation of NK cells has been observed but this is transient (Shresta, Kyle et al 2004) Flaviviruses appear to increase MHC-I expression on infected cells Moderate enhancement of MHC-I expression results in significant inhibition of

NK cytolytic activity and flavivirus-induced aggregation of MHC-I molecules on the surface of target cells appears to result in greatly enhanced engagement by NK inhibitory receptors (Hershkovitz, Zilka et al 2008) It is suggested that cell surface expression of a cellular or viral factor that interacts with NK activating receptors due to DV infection increased MHC-I expression and counteracts the activation signals to NK cells MHC-I upregulation can also be a consequence of IFN-induced signaling The innate immune system, the IFN system and NK cells emphasize the importance of innate immune subversion of DV

FIG.1.8 Type I Interferon signal transduction pathway and putative inhibition by flavivirus Virus infection induces secretion of type I interferons,

which bind to cell surface IFN-α receptors on infected and nearby cells Binding

of IFN receptors leads to activation of JAK1 and TYK2 kinases via tyrosine phosphorylation STAT2 and later, STAT1 are phosphorylated and form heterodimers, and bind to IRF9 to form ISGF3 ISGF3 translocate to the nucleus and initiate transcription of interferon-stimulated genes by binding to interferon-stimulated response elements (ISREs), leading to transcriptional upregulation of hundreds of cellular genes and induction of an antiviral state Proposed sites of inhibition by flaviviruses are shown in the boxes (Fernandez-Garcia, Mazzon et

al 2009)

1.1.8.2 Adaptive immunity to DV

Over many years, laboratories across the globe have attempted to dissect the contributions by components of the adaptive immune system in DV immunopathology The hallmark of pathogenesis of DHF/DSS is the loss of endothelial integrity, which is assumed to be the result of an abnormal immune

response against the virus The levels of cytokines and immune mediators such as TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-18, MCP-1, IFN-α and IFN-γ are significantly increased in patients with DHF/DSS (Hober, Poli et al 1993; Green, Vaughn et al 1999; Azeredo, Zagne et al 2001; Chakravarti and Kumaria 2006; Dong, Moran et al 2007; Basu and Chaturvedi 2008; Bozza, Cruz

et al 2008; Dejnirattisai, Duangchinda et al 2008) (FIG.1.9) Cytokines are thought to play a direct role in the immunopathogenesis of DV, due specifically to their proinflammatory effects on vascular endothelial cells The role of cytokines

in increased vascular permeability has been substantiated in murine model systems Cytokines can induce the secretion and production of other cytokines This complex interactive network of induction results in further increases in the levels of cytokines and other chemical mediators Cytokines often exhibit synergistic effects such as TNF-α, IL-1 and IFN-γ together induce a larger increase in permeability compared to when each cytokine is acting alone In addition, IFN-γ is able to upregulate the expression of Fcγ receptors on phagocytes and appears to be important in protection and viral clearance A study with participants receiving a monovalent DV vaccine showed a higher ratio of TNF-α- to IFN-γ- producing cells when stimulated with antigens from a heterologous serotype than from a homologous serotype (Mangada and Rothman 2005) suggesting that TNF-α induces vascular permeability and IFN-γ plays a protective role TNF-α is strongly implicated in the pathogenesis of DV (Braga, Moura et al 2001; Chakravarti and Kumaria 2006) Antibody blocking TNF-α

abrogates the increase of the marker of activation in endothelial cells, ICAM-1, in

vitro in dengue-infected cells (Cardier, Marino et al 2005) Furthermore, several

studies have shown increased levels of TNF-α in infected mouse models and TNF-α antibodies attenuate the disease (Atrasheuskaya, Petzelbauer et al 2003; Shresta, Sharar et al 2006) Other cytokines such as IL-6, IL-8 and IL-10 also play distinct roles in the manifestation of DHF and DSS Comparing fatal DHF patients to surviving DHF patients, one study found high levels of IL-6 and IL-10

anti-in fatal cases In addition, IL-10 level was elevated anti-in DHF versus DF patients (Perez, Garcia et al 2004; Chen, Liu et al 2005; Chen, Lei et al 2006) As mentioned, IL-8 is a chemoattractant, modulated by NS5 protein of DV that attracts neutrophils and may activate monocytes IL-8 mRNA levels are higher in DHF patients than DF patients in peripheral blood mononuclear cells in the days before defervescence IL-8 was released in endothelial cell monolayers infected with DV in addition to cytoskeletal rearrangements and increased permeability Treatment with IL-8 leads to partial ablation of vascular permeability (Talavera, Castillo et al 2004) In addition, the presence of antibodies to IL-6 prevents the increase of inflammatory mediators such as tissue plasminogen activator from endothelial cells and abrogates vascular permeability (Huang, Lei et al 2003) Lastly, complement anaphylatoxin C5a and terminal complement complex SC5b-

9 were present in pleural fluids of patients with DHF and DSS and may contribute

to vascular permeability present in these patients (Avirutnan, Mehlhop et al 2008)

The cytokine response in dengue infection is closely linked to T-cell activation (Avirutnan, Mehlhop et al 2008) Chaturvedi and colleagues (2009) observed a

release of IFN-γ, TNF-α, IL-2 and IL-6 initially and then IL-4, IL-10 and IL-5 later in PBMCs during the course of DV infection (Chaturvedi 2009) This suggests a change from a Th1 to Th2 response in DV infection In addition, Pacsa

et al provided supporting evidence of this Th1 to Th2 switch by observing a decline of IL-12 in patient‟s serum (Pacsa, Agarwal et al 2000) The “original antigenic sin” model has been proposed that postulates that an inappropriate immune response occurs in secondary infection due to clonal expansion of cross-reactive memory T cells specific for the previous rather than a current infection, resulting in delayed viral clearance and/or increased cytokine secretion (Mongkolsapaya, Dejnirattisai et al 2003) It is suggested that, presentation of viral peptides on the cell surface of antigen-presenting cells, in association with HLA molecules, induce proliferation of CD4+ T cells to produce cytokines

Recent studies have provided clues on the functions of the CD8+ T cells in dengue One group reported that CD8+ T cells isolated from Thai patients were highly activated and underwent apoptosis (Mongkolsapaya, Dejnirattisai et al 2003) This may be necessary for elevated T cell numbers to return to baseline levels upon viral clearance On the contrary, another study has argued that activation of memory CD8+ T cells during heterologous secondary DV infection results in massive production of cytokines and immune modulators characteristic

of DHF and DSS (Mathew and Rothman 2008) A good animal model is lacking for the better understanding of CD8+ T cells in dengue Following massive activation of memory T cells, the macrophages are activated to produce free

radicals, reactive oxygen species and nitrites which in turn encourage apoptosis in the target cells

Plasma leakage syndrome is the primary mechanism underlying disease severity While endothelium itself appears to be susceptible to dengue infection (Huang, Lei et al 2003; Warke, Xhaja et al 2003), extensive cell death or damage does not appear to be responsible for the increase in permeability Indeed, the endothelial permeability is transient and appears as a result of soluble mediators released by the endothelium or by immune cells Treatment of endothelial cells with culture supernatants from dengue-infected macrophages resulted in an increase in cell permeability in the absence of infection (Carr, Hocking et al 2003) It is likely that the process of ADE, leading to increase viral replication, which eventually leads to T cell activation and cytokine production, does not happen sequentially but is rather a complex network of self-reinforcing pathological events towards DHF and DSS As mentioned in Section 1.1.7, recent studies identified certain DV epitopes of HLA types may be associated with immune enhancement (Zivna, Green et al 2002; Simmons, Dong et al 2005; Mongkolsapaya, Duangchinda et al 2006) or protection (Chiewsilp, Scott et al 1981; Loke, Bethell et al 2001; LaFleur, Granados et al 2002; Stephens, Klaythong et al 2002; Zivna, Green et al 2002; Polizel, Bueno et al 2004) The rapid induction of cross-reactive memory T cells with HLA bearing mainly NS3 protein-specific DV epitopes during a secondary infection would be consistent with the increased incidence of DHF and DSS The magnitude of T cell responses has been correlated with disease severity Studies in humans, both controls and

infected patients, have indicated that both serotype-specific and cross-reactive T cells play a role in T cell response during infection Moreover, some T cell clones cross-react with other flaviviruses such as Yellow fever and Japanese encephalitis (Scott, Eckels et al 1983)

FIG.1.9 Immunopathogenesis of DV infection IFN-γ is produced by NK and

CD8+ T cells and activates macrophages and CD4+ T cells Infected phagocytes and endothelial cells produce TNF-α and NO which contributed to increase vascular permeability Albumin in the plasma escapes from the bloodstream together with DV and soluble NS1 Changes in vascular permeability in DV infections have classically been measured by monitoring levels of albumin in the plasma (Clyde, Kyle et al 2006)

Human humoral immunity develops approximately 6-8 days after a bite from a DV-infected mosquito (FIG.1.7) The antibody produced by plasma cells is mainly directed against E and prM glycoproteins present on the surface of the virus There are a number of different mechanisms postulated to prevent virus from entering the cell One way is for antibodies to alter the spatial distances

between the glycans on the E proteins, thereby inhibiting the interaction of the virus with DC-SIGN (Pokidysheva, Zhang et al 2006) Another route may involve binding to DIII of the E glycoproteins to prevent binding of the virus to its primary entry receptor (Hung, Hsieh et al 2004) Several studies have shown that the neutralizing epitopes of DV are clustered at the tip of Domain II (DII) of the E protein, (which is the location of the fusion peptide), the hinge region between Domain I (DI) and DII of E protein and between DI and Domain III (DIII) of E protein (Hung, Hsieh et al 2004; Gromowski and Barrett 2007; Lok, Kostyuchenko et al 2008) These are sites that participate in structural rearrangements of the viral surface glycoproteins that occur at low pH as the immature virus morph to maturity and at the initial stages of infection where infectious mature virus fuses with the endosomal membrane inside the host cell (Section 1.1.5) DIII of the E protein was thought to be the main target for viral neutralization until recently, one group provided evidence on the presence of other neutralizing epitopes on E protein besides those on DIII, as neutralizing effects were retained despite depletion of DIII antibodies of patient‟s serum NS1 protein antibodies can also be generated due to the surface expression of NS1 protein on the surface of infected cells and its secretion from infected tissues (FIG.1.9) Anti-NS1 antibodies have shown to be protective in DV infection in mice and activate complement-mediated lysis of DV-infected cells (Schlesinger, Brandriss et al 1987; Henchal, Henchal et al 1988; Costa, Freire et al 2006; Kurosu, Chaichana et al 2007) Antibodies play a dual role in DV infection as

they can enhance and neutralize DV infectivity in vitro and in vivo

1.1.9 Antibody dependent enhancement (ADE)

While infection with one dengue serotype typically confers protective immunity

to that serotype, an individual may still be infected at a later date by a different dengue serotype In a study in Bangkok in the 1960‟s, it was observed that over

85 % of DHF patients had high levels of antibodies against other dengue serotypes, presumed to be a consequence of prior infection (Halstead, Nimmannitya et al 1970) This leads Halstead and colleagues to formulate the hypothesis of antibody dependent enhancement (ADE) Primary infections give rise to heterotypic, non-neutralizing but enhancing antibodies which, in a subsequent infection (by another serotype) enhance the entry of virus into Fc-γ receptor-positive cells such as monocytes, DCs and macrophages that are permissive for DV infection Studies with E protein-specific antibodies suggest that when virion opsonization occurs at a level of antibody occupancy that does not exceed the threshold required for virus neutralization, these antibodes may enhance the efficiency of virus attachment to the cell surface and facilitate entry

of virions via Fc receptor-mediated endocytosis (Halstead and O'Rourke 1977; Pierson, Xu et al 2007)

The antibody-virus complexes bind via the Fc portion of the antibody to FcγRI and FcγRII bearing cells, which engulf the complex and allow the virus to infect the cell This results in greater number of infected cells and with a resulting increase in virus burden and eventually the release of mediators of vascular permeability, leading to enhancement of disease (Halstead and O'Rourke 1977) High viremia titers during dengue fever, peaking 2 days before defervescence,

have been shown to correlate with progression to DHF at defervescence (Vaughn, Green et al 2000; Libraty, Endy et al 2002) A study utilizing THP-1 cells infected with DV-immune complexes showed a downregulation of production of IL-12, IFN-, TNF-, and NO, and upregulation of IL-6 and IL-10 indicating that the Fc receptor-mediated entry may also suppresses the antiviral immune response, thereby promoting virus particle production (Chareonsirisuthigul, Kalayanarooj et al 2007)

However, there is a 2 % - 4 % probability for a dengue patient who is experiencing a secondary infection to progress to more severe forms of the disease, suggesting that perhaps other factors may contribute to dengue pathogenesis In addition, several groups observed that DHF manifestations were present in patients with primary infections For example, in the Bangkok study, approximately 10 % of children with DHF/DSS, all four years old or more, were primary infections and two of these were fatal (Scott, Nimmannitya et al 1976) Furthermore, a retrospective study of a Greek cohort experiencing DV1 outbreak

in 1927-1928 confirmed that fatal cases had occurred in many patients with primary infections (Rosen 1986) These studies provide evidence that questions the relative contribution that ADE to DV pathogenesis Perhaps the confirmation

of this hypothesis is hampered by the absence of a reliable animal model Recently, Zellweger and colleagues demonstrated in mice that DV-specific antibodies can sufficiently increase severity of disease such that progression from

a mostly non-lethal illness to a fatal disease resembling human DHF/DSS is observed, though this data should not be over-interpreted given the restricted IgG

subclass response in these mice Thay also reported an increase in infection in liver sinusoidal endothelial cells of the mice which resulted in increased systemic levels of virus This does suggest that a subprotective humoral response may have pathological consequences (Zellweger, Prestwood et al 2010)

The extent of ADE in dengue pathogenesis may be dependent on the affinity of IgG subclass antibodies binding to the Fcγ-receptor as well as the physical properties of these IgG subclasses In the 1960's extensive studies, performed with specific polyclonal rabbit antisera against homogeneous human IgG myeloma proteins, revealed the existence of four distinct subgroups of human IgG, which were designated IgG1, IgG2, IgG3 and IgG4, respectively (Grey and Kunkel 1964) The four subclasses show more than 95 % homology in the amino acid sequences of the constant domains of the y-heavy chains The four IgG subclasses show their most conspicuous differences in the amino acid composition and structure of the 'hinge region', which is the part of the molecule containing disulfide bonds between the y-heavy chains This region, between the Fab (Fragment antigen binding) arms and the two carboxy-terminal domains CH2 and

CH3) of both heavy chains, determines the flexibility of the molecule (Burton, Gregory et al 1986) Hinge-dependent Fab-Fab and Fab-Fc flexibility may be important in triggering effector functions such as complement activation and Fcγ-receptor binding The length and flexibility of the hinge region varies among the IgG subclasses The flexibility of the hinge region decreases in the order IgG3>IgG1>IgG4>IgG2 This probably relates to the higher activity of IgG3 in

triggering effector functions, when compared to the other subclasses (Grey and Abel 1967)

Although all receptors can bind IgG immune complexes, individual receptors display significantly different affinities for IgG subclasses (Nimmerjahn and Ravetch 2005) It was demonstrated that IgG3 bind 3-fold better than IgG1, approximately 100-fold better than IgG4 and IgG2 in FcγRI and FcγRII IgG3 binds approximately as well as IgG1 to FcγRIII It implies that IgG3 antibodies can efficiently activate Fcγ-receptor expressing NK cells, monocytes and macrophages In summary, the affinity of IgG subclasses binding to activating Fcγ-receptors are as follows IgG3 > IgG1 > IgG4 = IgG2 (Bruhns, Iannascoli et

Fcγ-al 2009) Such considerations on antibody affinity to Fcγ-receptors may prove important in the design of antibody-based therapeutics and active vaccination protocols

Perhaps ADE is neither sufficient nor entirely necessary for severe disease As mentioned above in Section 1.1.7, the genetic background, virulence of the infecting viral strain, immune status of the patient, combined with levels of underlying disease and even nutritional status (Thisyakorn and Nimmannitya 1993) may be risk factors for dengue Resolution of this controversy may provide new insights for the development of vaccines and better management of DV-infected patients

1.2.2 Epidemiology

More than 90 % of people are infected with EBV by the time they enter adulthood (Henle, Henle et al 1979) Most of these were infected during early childhood and the virus persists throughout their lifetime In the United States, as many as 95% of adults between 35 and 40 years of age have been infected Infants become susceptible to EBV as soon as maternal antibody protection disappears Many children become infected with EBV, and these infections are asymptomatic or are indistinguishable from the other mild illnesses of childhood In Singapore, approximately 99 % of the population are EBV infected, due to high urban

population density and cultural practices such as communal sharing of food in ethnic communities

1.2.3 Structure of EBV virions and organization of the virus genome

EBV has a toroid-shaped protein core with DNA enclosed within, a nucleocapsid,

a protein tegument and an outer envelope The outer envelope has an abundance

of structural glycoproteins (Gp) Gp350/220 The BAMHI restriction fragment of the B95.8 laboratory strain genome has been completely sequenced (Baer, Bankier et al 1984) The EBV genome is a double stranded DNA molecule of 172kb and has reiterated 0.5kb terminal repeats and a reiterated 3kb internal direct repeat The EBV genes are named after the BAMHI restriction fragment containing the RNA start site and their leftward or rightward transcriptional orientation depending on the two promoters Cp and Wp A specific set of EBV proteins obtained from LCLs play an important function in the engagement of the lytic or latent program of the virus The proteins are six EBV nuclear antigens (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C) and EBNA leader protein (EBNA-LP)) and three latent membrane proteins (LMP1, LMP2A and LMP2B) (Robertson, Ooka et al 1996) EBER1 and EBER2, which are small polyadenylated (hence non-coding) RNAs were found in abundance of 105-107copies per cell in LCLs and were proposed to play a role in the splicing of other viral transcripts including primary EBNA and LMP transcripts (Robertson, Ooka