Chapter - 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 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 immunoglobulinlike 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 (FernandezGarcia, 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, ChavezSalinas 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 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209) (Tassaneetrithep, Burgess et al. 2003) and liver/lymph nodespecific 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 10 Kaufman, B. M., P. L. Summers, et al. 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Component Volume per reaction (ul) 1ug Total RNA 2uM gene specific primer (GSP) 10mM DNTP Mix DEPC-Treated Water Final Volume 10 Component Volume per reaction (ul) 10X RT Buffer 25mM MgCl2 0.1M DTT RNase Out (40U/ml) SuperScript III RT (200U/ul) Final Volume 10 193 2.5 Reaction mix for cDNA synthesis of Human Immunoglobulin Genes Component Volume per reaction (ul) 10XPCR Mix 50mM MgCl2 1.5 10mM DNTP Mix 10uM sense primer 10uM antisense primer Taq Pol (5U/ul) 0.4 cDNA Water 38.1 Final Volume 50.0 2.6 Reaction mix for attachment of linker to heavy and light chains Component Volume per reaction (ul) -ve 10XPCR Mix 50mM MgCl2 1.5 1.5 10mM DNTP Mix 10uM sense primer 10uM antisense primer 0.4 0.4 100ng DNA H chain - 100ng DNA L chain - Water 38.1 40.1 Final Volume 50.0 50.0 Taq Pol (5U/ul) 194 2.18.2 Reaction Mix for Scfv digestion Reaction mix for Not digestion was as follows: Vol (ul) Not I (10 000U/ml) NEB 2.0 10X BSA 10.0 10X Buffer 10.0 DNA (1µg) H2O Top up to 100ul Total Volume 100.0ul Reaction mix for Sfi1 digestion was as follows: Vol (ul) Sfi I (20 000U/ml) NEB 1.0 10X BSA 10.0 10X Buffer 10.0 DNA 50.0 H2O Top up to 100ul Total Volume 100.0ul Reaction mix for ligation was as follows: T4 DNA Ligase (400 000U/ml) NEB 10X T4 DNA Ligase Buffer DNA (insert) pCANTAB 5E (vector) H2O Total Volume Sample 1.0 -ve 1.0 DO1 (+ve) 1.0 2.0 20ng 150ng Top up to 20ul 20.0ul 2.0 150ng Top up to 20ul 20.0ul 2.0 20ng 150ng Top up to 20ul 20.0ul 195 2.19.1 Reaction mix for heavy and light chain amplification Reaction components 1x reaction mix 4x Master mix 5x iProof HF buffer 10μL 40μL 10mM dNTP mix μL 4μL Upstream Primer [10μM] 2.5μL 10μL Downstream Primer [10μM] 2.5μL 10μL MgCl2 [50mM] 1μL 4μL Miniprep DNA template [2ng/μl] 5μL 20μL Nuclease-free water 27.5μL 110μL iProof DNA Polymerase [2U/μL] 0.5μL 2μL Total volume 50μL 200μL 2.19.5.1 Reaction mix in one Protein LoBind tube for double-digestion of Variable Heavy Chain PCR product Reaction components Volume Purified PCR product [10μg] variable 10x NEBuffer 10μl BSA (100x) 1μl ApaLI [10U/μl] 4μl (40U) NsiI [10U/μl] 6μl (60U) Nuclease-free water Total volume Top up to 100μl 100μl 196 Reaction components Volume Purified PCR product [10μg] variable 10x NEBuffer 10μl BSA (100x) 1μl MfeI [10U/μl] 6μl (60U) XhoI [20U/μl] 3μl (60U) Top up to 100μl Nuclease-free water 100μl Total volume 2.19.5 Reaction mix in one Protein LoBind tube for double-digestion of Variable Light Chain + Constant Lambda Chain PCR product was used. Reaction components Volume Purified PCR product [10μg] variable 10x NEBuffer 10μl BSA (100x) 1μl ApaLI [10U/μl] 5μl (50U) AscI [10U/μl] 5μl (50U) Nuclease-free water Total volume Top up to 100μl 100μl 2.19.6 Reaction mix in one Protein LoBind tube for double-digestion of the vector to allow cloning in the Variable Light Chain fragment 197 Reaction components Volume pDSO IgG1 framework vector [20μg] variable 10x NEBuffer 20μl BSA (100x) 2μl ApaLI [10U/μl] 8μl (80U) PstI [20U/μl] 8μl (160U) Nuclease-free water Total volume Top up to 200μl 200μl 2.19.6 Reaction mix in one Protein LoBind tube for double-digestion of the vector to allow cloning in the Variable Heavy Chain fragment. Reaction components Volume pDSO IgG1 framework vector [20μg] variable 10x NEBuffer 20μl BSA (100x) 2μl MfeI [10U/μl] 10μl (100U) XhoI [20U/μl] 5μl (100U) Nuclease-free water Total volume Top up to 200μl 100μl 2.19.7 Reaction mix for the prevention of self-religation of plasmids. reaction components were placed in a microtube for double-digestion of plasmid Reaction components Volume Double-digested pDSO IgG1 framework vector [20μg] 200μl 10x Antartic Phosphatase reaction buffer 24μl Antartic Phosphatase [5U/μl] Total volume 16μl (80U) 240μl 198 2.19.10 Reaction mix for double-digestion of plasmid constructs containing the Variable Light Chain fragment. Reaction components Volume Master Mix (35 reactions) Miniprep DNA 5.0μl - 10x NEBuffer 1.0μl 35μl BSA (100x) 0.1μl 3.5μl ApaLI [10U/μl] 0.4μl (4U) 14μl PstI [20U/μl] 0.6μl (6U) 21μl Nuclease-free water 2.9μl 101.5μl Total volume 10.0μl Use 5μl/reaction 2.19.10 Reaction mix for double-digestion of plasmid constructs containing the Variable Heavy Chain fragment. Reaction components Volume Master Mix (35 reactions) Miniprep DNA 5.0μl - 10x NEBuffer 1.0μl 35μl BSA (100x) 0.1μl 3.5μl PstI [20U/μl] 0.5μl (4U) 17.5μl XhoI [20U/μl] 0.5μl (6U) 17.5μl Nuclease-free water 2.9μl 101.5μl Total volume 10.0μl Use 5μl/reaction 199 [...]... alter the spatial distances 33 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. .. into the Americas correlated with the occurance of DHF/DSS (Rico-Hesse 20 03) and later experiments underline the early replicative advantage of the South-East Asian genotype (Armstrong and Rico-Hesse 20 03; Cologna and Rico-Hesse 20 03; 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 20 03; ... et al 20 03; Zhang, Chipman et al 20 03) 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 20 03) The immature particles formed in the ER mature as they travel through the secretory pathway The slightly... 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. .. 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... 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... the generation of membraneassociated M and a pr peptide A recent study has shown that the pr peptide 17 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. .. (Falgout, Pethel et al 1991) The N-terminal 180 amino acids of NS3 protein contain the catalytic 14 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... 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... 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 . 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. 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. and about 30 ° from the cryoEM structure of the mature particle (Zhang, Corver et al. 20 03) . The immature particles formed in the ER mature as they travel through the secretory pathway. The slightly