siRNA BASED INHIBITION OF DENGUE VIRUS AND
PREDICTION OF POSSIBLE VACCINAL TARGETS
UmmarRaheel
2010-NUST-TfrPhD-V&I-44
Atta-ur-Rahman School of Applied Biosciences National University of Sciences & Technology Islamabad Pakistan 2015 siRNA BASED INHIBITION OF DENGUE VIRUS AND
PREDICTION OF POSSIBLE VACCINAL TARGETS
UmmarRaheel
A thesis submitted in partial fulfillment of the requirement for the
degree of Doctor of Philosophy in
Virology and Immunology
Supervision
Supervisor
Dr.Najam Us SaharSadafZaidi
Atta-ur-Rahman School of Applied Biosciences National University of Sciences & Technology IslamabadPakistan 2015
Dedicated to
My Almighty Allah and my Mother…
For providing me strength and patience
Acknowledgements
ACKNOWLEDGMENTS
All praises for almighty Allah the creator of the universe. Who created human mind and give us the power to conceive ideas and make decisions. Who guided us to follow the right pathway and without his guidance we were blind and ignorant.
I am extremely thankful to my supervisor Dr.SadafZaidi for her never ending help, guidance and patience throughout my studies. I am highly thankful to National
University of Sciences and technology and Higher Education Commission for providing me funding for my research. I am thankful to Dr. Muhammad Tahir, Dr.HajraSadia and
Dr.RoquyyaGul (UOL) my thesis committee members, for their support and guidance. I am also thankful to Dr. Peter John and Dr.AttyaBhatti for their support during my study.
I owe immense acknowledgement to Prof AravindaDesilva, Department of
Microbiology and Immunology, University of Chapel Hill North Carolina, USA for accepting me in his lab as visiting research scholar. I did most of the research work in his lab. He allowed me to utilize all available facilities for my research work in addition with constant guidance and support.
During my PhD research work, I met many amazing people who were my research colleagues and great friends and I knew I could rely on them whenever I needed.
My research work would not have been possible without the assistance of Dr Muhammad
Imran, Dr Mushin Jamal, NasirRaiz, Muhammad Fahim, IrshadulHaq, HashaamAkhatar and every member of AttaUr Rahman School of Applied Biosciences.
v
Acknowledgements
I can never forget the role of my mother who inspired me and prayed for my success when I was down and she did not let me fall. I thank Allah every day for blessing me with a great mother. I must acknowledge my Fiancée for her support during the last two years; it was her constant encouragement that made me withstand difficulties and disappointments. I will never forget the role of my family in reaching this milestone.
Finally I would again thank Dr.SadafZaidi for being a great person who was not only my supervisor in research but also a source of inspiration.
UmmarRaheel
vi
Table of Contents
TABLE OF CONTENTS
Title Page #
Acknowledgements v
List of Abbreviations vii
List of Tables xii
List of Figures xiii
Abstract xvii
Chapter -1
Introduction 01
1.1 Dengue virus 01
1.1.1 Epidemiology 02
1.2 DENV infection in Pakistan 04
1.3 DENV genome 05
1.4 Replication and life cycle of DENV 06
1.5 Pathogenesis 08
1.6 In vitro culturing 09
1.7 Animal models 10
1.8 RNA interference 11
1.9 Insilico analysis 12
1.10 Aim and objectives 13
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Table of Contents
Chapter -2
Review of Literature 23
2.1 Genome organization of Flavivirus 24
2.2 DENV proteins 25
2.3 Dengue virus 31
2.4 DENV variations 33
2.5 Pathogenesis 34
2.5.1 High virulence 34
2.5.2 Host innate immunity 35
2.5.3 Antibody dependent autoimmune responses in DF 35
2.5.4 Cellular autoimmunity in DHF/DSS 36
2.5.5 Antibody Associated with Enhancement of DF 36
2.6 Viral targets for DENV inhibitors 38
2.6.1 Structural proteins’ based inhibition of DENV 38
2.6.2 Nucleoside based inhibition of DENV 39
2.6.3 Methyltransferases inhibition of DENV 39
2.6.4 Protease (NS2B/NS3) inhibition of DENV 41
2.6.5 Host enzymes based inhibition of DENV 42
2.6.6 Kinases based inhibition of DENV 43
2.6.7 Glucosidase based inhibition of DENV 43
2.6.8 Cytokines and receptors based inhibition of DENV 44
2.6.9 Computational design of peptide inhibitors against DENV 45
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Table of Contents
2.7 Inhibition of DENV by siRNA 45
2.8 Vaccine development for DENV 47
2.8.1Chimeric vaccine 48
2.8.2 DNA vaccine 48
2.8.3 Live attenuated vaccines 49
2.8.4 Subunit vaccine 50
2.9 Epitope variation a challenge for DENV vaccine design 51
2.9.1DENV T Cell antigenic diversity analysis 52
2.9.2 T cell epitope mapping of DENV 52
2.9.3 Insilico analysis of DENV T cell epitopes 53
2.9.4 B cell epitope mapping antigenic diversity analysis 53
2.9.45 Insilico analysis of DENV B cell epitopes 54
Chapter -3
Materials and Methods 57
3.1 SerotypessiRNA designing/ bioinformatics 57
3.2 Mammalian cells and DENV 57
3.3 siRNA transfections of cell cultures 58
3.4 Focus assay 58
3.5 Immunofluorescence Assay (IFA) 59
3.6 Western blot analysis 60
3.7 Realtime PCR analysis of DENV RNA 60
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3.8.1 RNA extraction for qPCR 60
3.8.2 Optimizing of qPCR 61
3.9 Animal model of DENV2 61
3.9.1 Intracerebral injection 61
3.9.2 RNA Extraction from different tissues 62
3.9.3 Primer Designing for DENV RNA detection 63
3.9.4 Complementary DNA (cDNA) synthesis 64
3.9.5 Polymerase chain reaction 64
3.9.6 Purification of PCR products 64
3.9.7 Ligation 65
3.9.9 Transformation 65
3.10 Screening and selection of clones 66
3.10.1 Colony PCR 66
3.10.2 Plasmid DNA isolation and restriction digestion 67
3.11 Sequencing 68
3.12 Prediction of vaccine targets 68
3.12.1 DENV2 sequence comparison and phylogenetic analysis 68
3.12.2 Predication of epitopes on DENV2 Pakistani Isolate 68
3.13 Insilicomodeling of DENV2 structural proteins from Pakistani isolate 69
Chapter-4
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Results 70
4.1 Nucleotide analysis of un-translated terminal repeats 70
4.1.1 Alignment results for 3'UTR of DENV2 70
4.1.2 Alignment results for 3'UTR of DENV3 73
4.1.3 Alignment results for 5'UTR of DENV2 77
4.1.4Alignment results for 5'UTR of DENV3 80
4.1.5Alignment results for structural region of DENV2 genome 90
4.1.6 Alignment results for structural region of DENV3 genome 99
4.2 siRNAdesigning and bioinformatics 89
4.3 Focus assay 102
4.4Immunofloroscence assay 107
4.5 Western blotting 120
4.9 Cloning, sequencing of DENV2 structural genes 131
4.9 DENV2 structural genes phylogenetic analysis 141
4.10 Full length epitopic sites prediction of Pakistani DENV2 structural proteins 145
4.11 Full length B cell epitopic sites prediction of Pakistani DENV2 capsid protein 146
4.12 Full length epitopic sites prediction of Pakistani DENV2 prM protein 153
4.13 Full Length epitopic Sites Prediction of Pakistani DENV2 E protein 160
4.14 DENV2 structural proteins homology modeling 167
Chapter -5
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Table of Contents
Discussion 174
Chapter -6
References 185
xii
List of Tables
LIST OF TABLES
Table 2.1DENV RNA elements and there functions 30
Table 2.2 DENV proteins and there functions 31
Table 3.1 Primers for detection of DENV2 structural genes 63
Table 4.1 Designed siRNAs and their location in DENV2 genome 100
Table 4.2 Designed siRNAs and their location in DENV3 genome 101
Table 4.3 Focus assay triplet experiment for DENV2 load estimation 103
Table 4.4 Focus assay triplet experiment for DENV3 load estimation. 105
xii
List of Figures
LIST OF FIGURES
Figure # Title Page #
1.1 Epidemiology of DENV 03
1.2 Replication cycle of DENV 06
1.3 Genome organization of DENV 09
1.4 DENV RNA elements and proteins 11
1.5 RNA interference pathways 30
4.1 Sequence analysis of DENV2 3'UTR 72
4.2 Sequence analysis of DENV3 3'UTR 76
4.3 Sequence analysis of DENV2 5'UTR 79
4.4 Sequence analysis of DENV3 5'UTR 81
4.5 Sequence analysis of DENV2 structural region 89
4.6 Sequence analysis of DENV3 structural region 98
4.7 Focus assay of siRNADENV2 transfected Vero-81 cells 104
4.8 Focus assay of siRNADENV3 transfected Vero-81 cells 106
4.9 IFA images of DENV2UTR5'siRNA1 treated Vero-81 cells 108
xiii
List of Figures
4.10 IFA images of DENV2UTR5'siRNA2 treated Vero-81 cells 109
4.11 IFA images of DENV2SsiRNA1 treated Vero-81 cells. 110
4.12 IFA images of DENV2SsiRNA2 treated Vero-81 cells. 111
4.13 IFA images of DENV2UTR3'siRNA1 treated Vero-81 cells 112
4.14 IFA images of DENV2UTR3'siRNA2 treated Vero-81 cells 113
4.15 IFA images of DENV3UTR5'siRNA1 treated Vero-81 cells 114
4.16 IFA images of DENV3UTR5'siRNA2 treated Vero-81 cells 115
4.17 IFA images of DENV3SsiRNA1 treated Vero-81 cells. 116
4.18 IFA images of DENV3SsiRNA2 treated Vero-81 cells. 117
4.19 IFA images of DENV3UTR3'siRNA1 treated Vero-81 cells 118
4.20 IFA images of DENV3UTR3'siRNA2 treated Vero-81 cells 119
4.21 Western blot analysis of mature and immature DENV2 121
4.22 Western blot analysis of siRNA transfected cells post- 122
DENV2 infection.
4.23 Western blot analysis of siRNA transfected cells post- 123
DENV3 infection.
4.24 Q-PCR for analysis of DENV2 RNA levels. 125
xiv
List of Figures
4.25 Q-PCR for checking DENV3 RNA levels. 126
4.26 Dissection of DENV2 mice model 127
4.27 Amplification of DENV2 C from different organs of mice. 129
4.28 Amplification of DENV2 prM from different organs of 130 mice.
4.29 Amplification of DENV2 C gene from local DENV2 isolate. 132
4.30 Colony PCR of DENV2 C gene. 133
4.31 DENV2 C clone confirmation by restriction digestion. 134
4.32 Amplification of DENV2 prM gene from local DENV2 135 isolate.
4.33 Colony PCR of DENV2 prM gene. 136
4.34 DENV2 prM clone confirmation by restriction digestion. 137
4.35 Amplification of DENV2 E gene from local DENV2 isolate 138
4.36 Colony PCR of DENV2 E gene. 139
4.37 DENV2 E clone confirmation by restriction digestion 140
4.38 Phylogenetic tree of DENV2 C gene 142
4.39 Phylogenetic tree of DENV2 prM gene. 143
4.40 Phylogenetic tree of DENV2 E gene. 144
xv
List of Figures
4.41 Bepipred linear epitope prediction of DENV2 C protein. 147
4.42 Chou and Fasman Beta-turn prediction of DENV2 C protein. 148
4.43 Emini surface accessibility prediction of DENV2 C protein. 149
4.44 Karplus and Schulz flexibility prediction of DENV2 C 150 protein.
4.45 Kolaskar and tongaonkar antigenicity of DENV2 C protein. 151
4.46 Parker hydrophilicity prediction of DENV2 C protein 152 DENV2 infection. 4.47 Bepipred linear epitope prediction DENV2 of prM. 154
4.48 Chou and Fasman Beta-turn prediction DENV2 of prM. 155
4.49 Emini surface accessibility prediction DENV2 of prM. 156
4.50 Karplus and Schulz flexibility prediction DENV2 of prM 157
4.51 Kolaskar and tongaonkar antigenicity prediction DENV2 of 158
prM.
4.52 Parker hydrophilicity prediction DENV2 of prM. 159
4.53 Bepipred linear epitope prediction of E protein. 161
4.54 Chou and Fasman Beta-turn prediction of E protein. 162
4.55 Emini surface accessibility prediction of E protein. 163
4.56 Karplus and Schulz flexibility prediction of E protein. 164
xvi
List of Figures
4.57 Kolaskar and tongaonkar antigenicity prediction of E 165
protein.
4.58 Parker hydrophilicity prediction of E protein 166
4.59 Phyre2 web server generated model of DENV2 C protein 168
represented by a colored 3D structure
4.60 Phyre2 web server generated model of DENV2 C protein 169
represented by a colored 3D structure
4.61 Phyre2 web server generated model of DENV2 prM protein 170
represented by a colored 3D structure
4.62 Phyre2 web server generated model of DENV2 prM protein 171
represented by a colored 3D structurewith epitopes
4.63 Phyre2 web server generated model of DENV2 E protein 172
represented by a colored 3D structure
4.64 Phyre2 web server generated model of DENV2 E protein 173
represented by a colored 3D structurewith epitopes
xvii
Abstract
ABSTRACT
Dengue virus (DENV) is an insect borne virus classified in the family
Flaviviridae. DENV is the most widespread arthropod borne virus across the globe and according to latest epidemiological reports dengue fever (DF) is one of the major cause of concern for global health.Amongst all four DENV serotypes predominately circulating serotypes in Pakistan are DENV2 and DENV3. RNA interference (siRNA and miRNA) is at the forefront of molecular approaches for inhibitory studies. The present study was aimed at inhibition of both these serotypes via siRNA interference; moreover structural genes were cloned for insilico analysisto facilitate strategies for the development of vaccines against DENV.
Both DENV2 and DENV3 were targeted by synthetic siRNAsdesigned against conserve regions in the genome. Six siRNAs were designed against both DENV2 and
DENV3 from three vital regions of genome 5'untranslated region (5'UTR), 3'UTR and structural genes. Transfections were performed by Lipofectamine and level of inhibition was observed by performing several viral titer estimation assays. Results showed thatDENV3UTR3'siRNA2 targeting 3' UTR was able to inhibit DENV3 replication.The replication cycle of DENV has revealed that in human Dendritic cells (DCs),Furin enzyme converts the immature DENV pre-membrane protein (prM) to mature M protein assisted by change in cellular pH.Therefore, in the later part of study fully mature
DENV2 were allowed to infect and later targeted by RNAi. Results from this study showed that siRNA (DENV2SsiRNA2) targeting the M region of genome was able to
xvi
Abstract
drastically knock down DENV2 in Vero-81 cells. Substantial inhibition was also seen by siRNA (DENV2UTR3'siRNA2) targeting 3' UTR region ofDENV2 genome.
Structural genes were cloned from locally isolated DENV2 serotype. All three genes (Capsid, prM and Envelope) were subjected to insilico analysis involving construction of phylogenetic trees and prediction of epitopic domains. It was observed that both Pakistani and Indian DENV2 share a strong sequence similarity. Inhibition of both DENV serotypes via RNAi highlights the effectiveness of molecular approaches for anti-dengue drug development. Moreover, findings of insilico study of Pakistani DENV2 serotype will assist in the development of regionally targeted vaccine for densely populated South Asian region.
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Chapter 1 Introduction
Chapter 1
INTRODUCTION
1.1 DENGUE VIRUS
Dengue Virus (DENV) is a positive sense RNA virus which belongs to family
Flaviviridae, DENV has four serotypes (DENV1-4), is transmitted by mosquitoes
Aedesaegyptiand Aedesalbopictus(Rigau-Pérez et al., 1998). Amongst arthropods borne viruses DENV has highest rates in terms of annual cases and deaths, owing largely to growing population and travel(Lai et al., 1991). DENV has RNA genome which contains
5' cap on one side and 3' poly A tail (Gibbons and Vaughn, 2002). One of the reason for a lack of vaccine against DENV is antigenic difference amongst four serotype(Avirutnan et al., 2006).
DENV genome is positive sense single stranded RNA virus having a size of approximately 10.7 kb. The 5'end of genome known as untranslated terminal region
(5'UTR) is a100 nucleotide long stretch whereas genome ends at 3'UTR which has more than 400 nucleotides. Genome analysis revealed that there is a 5' cap at 5'UTR on the other hand 3'UTR is naked and lacks a polyadenylation at 3' end (Alvarez et al., 2005b).
UTRs are responsible for genome cycling, organization and replication for production of progeny DENVs. Several host cellular as well as viral factors interact with these UTRs for DENV replication (Alvarez et al., 2005a; Alvarez et al., 2008; Cui et al., 1998; De
Nova-Ocampo et al., 2002; Garcı́a-Montalvo et al., 2004). The region of genome towards the 5' end encodes structural proteins capsid (C), membrane (M) and envelope (E) at the
1
Chapter 1 Introduction
same time the rest of the portion of genome encodes nonstructural proteins (NS) (Rice et al., 1985).
1.1.1 EPIDEMIOLOGY
Worldwide DENV is prevalent in tropical and sub-tropical regions, urban populations of countries in Southeast Asia, South Asia and South America are at high risk(Lai et al., 1991). Dengue fever (DF) can be a self-limited or in some cases it can progress to sever conditions like dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Both DHF and DSS are characterize by high fever, enhanced vascular permeability, thrombocytopenia, these complications can cause death in the absence of proper management (Avirutnan et al., 2006; Gibbons and Vaughn, 2002;Huang et al.,
2003).
According to WHO, DF was the most prevalent insect borne viral illness in 2012, due to the rising number of annual cases (Murray et al., 2013). Economic losses relating with DENV were mentioned in recent reports showing that cost associated with DF. In
South Asian and American countries was roughly US$3 billion (Shepard et al., 2013).
Although precise number of DF cases are still not known however, DENV has be isolated from 125 countries worldwide which is the indication of the magnitude of this global crisis (Ferreira, 2012).
In 1970s rigorous measures were taken for controlling mosquito populations for curbing Malaria and DF, even with these efforts sporadic break were recorded (Guzman et al., 1984a; Guzman et al., 1984b; Halstead, 1980). According to World Health
Organization (WHO) number of reported cases between 1950s to 1990s were approximately 2.5 million with more than 42000 deaths(Kautner et al., 1997). In the mid
2
Chapter 1 Introduction
1990s DF was confirmed in patients from 24 American countries, with many countries facing worst epidemics. In recent times first reported epidemic in Indian Sub-continent was reported in 1996. Since 2006 several DF epidemics were seen in South Asia, according to WHO's estimates in Pakistan most number of cases were reported in the outbreak of 2011 as shown in Figure 1.1.
Figure 1.1: Epidemiology of DENV: Geographical distribution of DF in the world and countries which are located in the risk zones.The global incidence of dengue fever in the year 2013 according to WHO estimates. The prevalence of dengue cases is depicted by brown color. (WHO report, 2013)
3
Chapter 1 Introduction
1.2 DENV INFECTION IN PAKISTAN
Dengue fever is endemic in Pakistani population and highest number of cases are reported during or post monsoon seasons (Jahan, 2011). During mid 1980s DF was reported for the first time in children which were having persistent high fever (Akram et al., 1998). The Baluchistan province was hit by a deadly DF outbreak in the year 1995 resulting in 57 deaths (Paul et al., 1998). With each passing year number of DF cases were rising and in 2003 a major outbreak resulted in approximately 1000 cases and 7 confirmed deaths in the Haripur region located in KPK province (Tang et al., 2008). One year later in 2004, more than 2500 confirmed DF cases were reported in the Punjab region and molecular analysis revealed that DENV2 was predominant serotype in both of these outbreaks.
One of the worst year for DF in Pakistan was 2006 in which 5400 cases were reported and 55 people lost their lives, however a large number of cases were not reported owing to the inadequate medical facilities. For this outbreak two serotypes which were held responsible were DENV2 and DENV3 respectively (Khan et al., 2007).
Lahore was severely affected by three circulating serotypes (DENV 2, 3 and 4) and resulted in 1800 DF cases in 2008. This co-circulation led to tremendous increase in the rate of DHF and resulted in several deaths. Country wide outbreak in the year 2010 resulted in more than 5000 confirm cases and several deaths, both DENV1 and DENV2 were present in the outbreak (Mahmood et al., 2012).
The largest recorded outbreak of DF occurred in 2011, with more than 20,000 countrywide confirmed cases and a staggering 300 deaths. The current trend show that
4
Chapter 1 Introduction
Pakistani population is facing a severe threat from DF and an outbreak can strike at any time. There is an urgent need for development of anti-dengue drug and vaccine in addition with measures for controlling vector population (Jahan, 2011).
1.3 DENV GENOME
The whole DENV poly-protein produced by the open reading frame (ORF) is in the following sequence 5'- C-prM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4BNS5-3' as shown in Figure 1.3. The 3'end of DENV genome encodes non structural proteins, the proper roles of NS proteins is still not clear, however NS1 to N4A/B in association with
C protein are in the encapsidation of DENV RNA. Larger structural proteins M and E form a lipid bilayer structure which ultimately becomes envelope of new DENV. One of the important step in DENV maturation is cleavage of prMto M which occurs in the
Trans Golgi network. Furin is a pH dependent enzyme, when the pH of Golgi network drops this activates the cellular Furin resulting in cleavage of prM(Mackenzie and
Westaway, 2001).
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Chapter 1 Introduction
Figure 1.3: Genome organization of DENV: Genomeorganization of DENV starting with 5'UTR along with three major structural genes (C, prM and E) while ending at the
3'UTR region in between there are seven nonstructural genes (NS1, NS2A, NS2B, NS3,
NS4A, NS4B and N55). Both structural as well as non structural genes play a vital role in replication of DENV(Guzman et al., 2010).
1.4 Replication and life cycle of DENV
In humans major targets for DENV replication are immune cells; during the early phase of DENV infection virus enters macrophages, monocytes and dendritc cells (DC).
After entry and replication DENV spreads to several organs including Liver, kidney and spleen (Upanan et al., 2008). While in mosquitoes initial sight of replication of DENV is the midgut region, later on DENV spreads to several organ(Mercado-Curiel et al., 2008;
Molina-Cruz et al., 2005; Salazar et al., 2007).
Adsorption studies revealed that DENV enters into the host cell via clathrin mediated endocytosis (Heinz et al., 2004). Next step involves the release of Nucleocapsid
(NC) in the cytoplasm, naked genome is the replicated and translated for production of progeny viral particles utilizing host machinery. Translation of DENV RNA results in formation of endoplasmic reticulum (ER) bound poly-protein which is later cleaved to form structural and non structural proteins(Cahour et al., 1992). After the cleavage of structural proteins the interaction between these proteins and genome results in encapsidation of DENV RNA, DENV C-RNA complex formation occurs in the membrane of ER, nevertheless this whole interaction is still unclear (Ma et al., 2004).
The process of virus assembly and release is still not clear but the process of virus
6
Chapter 1 Introduction
maturation depends on the activity of cellular enzyme furin, pre-membrane (prM) is cleaved to membrane (M) by furin which is the final step of virus maturation before release form the host cell(Stadler et al., 1997) as shown in Figure 1.2.
Figure 1.2: Replication cycle of DENV: Complete Following initial attachment the particle engages in further interactions and finally enters cells by receptor-mediated endocytosis. The viral RNA genome is released into the cytoplasm and translated at the rough ER, giving rise to a polyprotein that is cleaved into mature proteins. RNA replication occurs when negative-sense copy (–RNA) that serves as a template for the production of excess amounts of positive-sense progeny RNAs (+RNA). Assembly of
7
Chapter 1 Introduction
DENV particles initiates in close proximity to the ER, where core protein and viral RNA accumulate. After acquiring viral envelope virus buds out of cell(Alcaraz-Estrada et al.,
2010).
1.5 PATHOGENESIS
Pathogenesis of DENV starts at the site of mosquito bite, DENV enters into the
DC and macrophages where initial replication takes place (Marovich et al., 2001; Wu et al., 2000). Subsequently DENV migrate to the lymph nodes for further multiplication and spreading, from these lymph nodes DENV enter into the blood stream and spread throughout the body. DENV virus can replicate in several cells, however, DENV prefers immune cells like monocytes, DC and macrophages for replication (Halstead et al., 1977;
King et al., 1999). During a secondary DENV infection a high titer of IgM is produced which binds with DENVs and assists in engulfment by monocytes. Replication of DENV results in apoptosis of infected monocytes, this leads to stimulation of DCs and production of pro-inflammatory cytokines (Bosch et al., 2002; Espina et al., 2003;Palmer et al., 2005).
Diversity of DENV host cells indicates the involvement of several receptors for viral attachment and entry, recent studies resulted in identification of several host cells molecules such as GRP78/BiP, Hsp90, high-affinity laminin receptor, heat-shock protein
70 (Hsp70) and R67 which were responsible for DENV entry in the host cells(Chen et al., 1999; Chen et al., 1997b; Germi et al., 2002; Hilgard and Stockert, 2000;
Jindadamrongwech et al., 2004; Mendoza et al., 2002; Thepparit and Smith, 2004).
DENV also infects human granulocyte precursor cells (myeloid cells) via C-type lectin receptors (CLR). These receptors are C-type lectin domain family 5, member A (CLEC5,
8
Chapter 1 Introduction
MDL-1), intracellular adhesion molecule 3 (ICAM-3) and mannose receptor (MR)
(Fernandez-Garcia et al., 2009; Lozach et al., 2005; Miller et al., 2008; Navarro‐Sanchez et al., 2003; Tassaneetrithep et al., 2003).
Individuals infected with a serotype of DENV produce a lasting immunity however this immunity is specific for that serotype not for other serotypes. If an individual infected with one serotype is infected with another serotype this cross- reactivity can lead to DHF and DSS (Halstead, 2006; Sabin, 1952). Antibodies including
IgG produced during DENV infection in mother can reach fetus, afterward if a new serotype infects the new born this can lead to DHF and DSS.
Recent studies have suggested that pathogenicity is highly variable amongst all four serotypes and the level of virulence achieved is dependent on each individual serotype (Rico-Hesse et al., 1997). Epidemiological data suggests that Asian serotypes were able to produce higher virus titers in mosquitoes as well as in humans which contributed to increasing outbreaks (Cologna et al., 2005). Although vertical transmission is possible in mosquitoes however it is a rare occurrence, the highest rates of vertical transmission is seen in Aedesmediovittatus which acts as a major reservoir for
DENV(Freier and Rosen, 1988).
1.6 DENV IN VITRO CULTURING
Dengue virus can be grown in vitro in several cell lines originating from numerous animals and insects, these cell lines were established by extensive research on different cell lineages like myeloid-derived cells, hepatocytes, lymphocytes and kidney cells (Andrews et al., 1978; Kurane and Ennis, 1988; Kurane et al., 1984). DENV has been isolated from phagocytic cells (monocytes and macrophages),
9
Chapter 1 Introduction
polymorphonuclearleukocytes (PMNs) and leukocytes as these cells engage DENV upon infecting a new human host (Halstead et al., 1977; King et al., 1999;Scott et al., 1980).
Further research has revealed that dendritic cells (DC) along with macrophages having DC-SIGN and mannose receptorsarefavorable for DENV infection and replication
(Kyle et al., 2007). As skin is the initial site of entry of DENV after it bites a human host therefore langerhan cells of skin were also found to be permissive for DENV infection(Schmid et al., 2014).
1.7 ANIMAL MODELS
Animal model studies of DENV has been a challenge for researchers owing to limited replication in wild type (WT) mice and asymptomatic clinical disease in primates other than humans, earliest approaches involved intra-cerebral (IC) inoculation of DENV in suckling mice (Raut et al., 1996). Later adult mice having a normal immune system which were injected which resulted in neurological malfunctions and in severe cases paralysis was observed (Johnson and Roehrig, 1999).
AG129 mice lacking interferon α/β and γ receptors were used for DENV replication by intravenous injections containing high doses of DENV (Johnson and
Roehrig, 1999). This development lead the way for future studies concerning DENV pathogenesis, inhibitor development and antibodies testing (Balsitis et al., 2010; Shresta et al., 2006). Mice and other non human primates lack clinical symptoms and disease manifestations still there are important for virus propagation as DENV can infect and replicate in multiple cells (Bente and Rico-Hesse, 2006; Kuruvilla et al., 2007).
1.8 RNA INTERFERENCE
10
Chapter 1 Introduction
RNA interference (RNAi) is an innate defense and regulatory process of eukaryotic cells for controlling or silencing of gene expression through small double stranded RNA (dsRNA) (Almeida and Allshire, 2005) as shown in Figure 1.4. Small interfering RNAs (siRNAs) are ∼21-22 bp in length with a 3' overhang of 2 nucleotide, this 3' overhang is responsible for RNAi based degradation of target mRNA for achieving gene silencing.
Figure 1.4: A typical siRNApathway:RNAi pathways usually start via small RNAs which can be small interfering RNA (siRNA) or microRNA (miRNA).siRNA pathway
11
Chapter 1 Introduction
start with the cleavage of double stranded RNA (dsRNA) by Dicer enzyme complex, these siRNAs get incorporated into Argonaute 2 (AGO2) and RNAi induced silencing complex (RISC). AGO2 cleaves the sense strand while active RISC uses the anti-sense strand to find its way to target mRNA sites for cleavage (Kim and Rossi, 2007).
1.9 INSILICO ANALYSIS
Molecular characterization of DENV and in depth phylogenetic analysis has resulted in four different serotypes (DENV1, 2, 3 and 4). These serotypes have differences at the genome level which is responsible for serotype diversity(Kuno et al.,
1998). Within serotypes several genotypes are present which result from geographical distribution and mutation amongst viral strains (Araújo et al., 2009; Rico-Hesse, 1990;
Twiddy et al., 2002; Villabona-Arenas and de Andrade Zanotto, 2011). DENV has a
RNA genome with a poor proof reading mechanism; the viral enzyme RNA-dependent
RNA polymerase has a high error rate.
Studies have revealed the fact that during one complete genome replication cycle one mutation occurs and collectively these mutations contribute in diversity amongst serotypes and genotypes (Holmes, 2003; Steinhauer et al., 1992). Mutation studies have shown that the largely deletion mutations are involve in evolution of DENV and emergence of several strains (Sall et al., 2010). The high mutation potential and genetic diversity in the DENV population has contributed to rapid evolution rate (Dunham and
Holmes, 2007; Villabona-Arenas and de Andrade Zanotto, 2011).
Epitope mapping is a laborious process which involves vast peptide screening as well as studying various aspects of immunogenic proteins via proteomics (Muzzi et al.,
2007). Wet lab experiments require large quantities of blood samples in addition factors
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Chapter 1 Introduction
like low prevalence of specific epitopes, assortment of HLA alleles and cost of peptide synthesis makes epitope mapping an uphill task (Sette et al., 2001). To overcome these problems epitopes can be efficiently mapped by online Bioinformatics tools (Vázquez et al., 2002).
1.10 AIM AND OBJECTIVES
Current study was aimed at inhibiting DENV2 and DENV3 in mammalian cell line via RNA interference. A complete lay out of the study includes propagation of
DENV2 and DENV3 both mature and immature viruses. RNAi based inhibitory studies of siRNAs against both DENV2 and DENV3 serotypes. Inhibition of mature DENV2 grown on U-937 DC-SIGN cells. The second phase of study included isolation and propagation of Pakistani DENV2 isolate in suckling mice model.Insilico analysis of local gene sequences for prediction of future vaccine targets.
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Chapter 2
LITERATURE REVIEW
Dengue fever was mentioned in Chinese medical encyclopedia dating back to 992 A.D.
Historical accounts from 18th century suggest the presence of a disease with symptoms resembling DF, epidemics were seen all across the globe from China to Americas.
Tropical and sub tropical regions having higher vector population had sporadic DF outbreaks (Gubler, 1998; Huang et al., 2000). A DF like disease was reported after
World war II in the region of Southeast Asia, patients were mostly children (Rigau-Pérez et al., 1998). Outbreaks were a common occurrence in 1950s, this led to documentation of outbreaks and in 1954 DHF outbreak in Manila was documented for the first time(Monath, 1994).
More than half (3.6 million) of the world's population is living in the tropical and sub-tropical regions of the world, owing to the presence of DENV vector (Aedesaegypti) these regions present a higher potential for DENV outbreaks (Ferreira, 2012; Gubler,
2011; Guzman et al., 2010). According to recent reports approximately 200 million case of DF occur each year, from these cases half million (500,000) progress to complications like DHF and DSS. Annual mortality rate is approximately 20,000 in different regions of the world, many cases are not reported which suggest that the number of cases might be higher (Gubler, 2002; Shepard et al., 2011).
Members of Flavivirus are present all across the planet and several of these viruses are associated with human diseases, some of the prevalent members include
Japanese encephalitis virus (JEV), West Nile virus (WNV), dengue virus (DENV), Tick-
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borne encephalitis viruses (TBEV) and yellow fever virus YFV (Vasilakis and Weaver,
2008). Phylogenetic analysis of viruses in Flavivirus genus revealed that all of these members are closely related based on vector interactions and antigenic properties (Kuno et al., 1998). The vector-borne viruses are further classified into mosquito and tick-borne viruses, three prominent tick-borne are Omsk hemorrhagic fever virus, tick-borne encephalitis virus and Kyasanur Forest disease virus. While mosquito-borne viruses are transmitted largely by Aedes and Culex genera and these mosquitoes are the main reason for global outbreaks of DENV, YFV and JEV (Kuno et al., 1998).
2.1 GENOMIC ORGANIZATION OF DENV
DENV belongs to Flaviviruseshaving a single stranded RNA genome which is approximately 11 kilo bases (Kb), the infectious positive sense RNA is directly translated into viral polyprotein(Boulton and Westaway, 1977; Rice et al., 1985). The diverse features is the presence of a solitary open reading frame (ORF) which encodes a poly- protein which is later cleaved to produce all ten structural as well as non structural proteins. Viral RNA has non coding regions (NCR) or untranslated terminal repeats
(UTR) at both ends, 5' UTR is capped whereas 3'UTR is deficient in a poly (A) tail
(Wengler et al., 1978) Table 2.1. 5' UTR assists in translation of viral RNA and viral replication, RNAi based approaches targeting this region was able to block both of these functions (Deas et al., 2005; Holden et al., 2006). The 3'UTR region is variant amongst
DENV and other Flaviviruses and only a small stretch of nucleotides is conserved
(Markoff, 2003). 3'UTR has also shown to play role in viral RNA translation and replication by interacting with viral replicase enzymes (Chen et al., 1997a).
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Structural region of DENV RNA genome is located near the 5'UTR which encodes three major structural proteins Capsid (C), precursor of matrix (prM) and
Envelope (E) whereas nonstructural (NS) region is the remaining portion of genome stretching till 3'UTR encoding all seven NS proteins (Rice 1985). The poly protein is produced from a single ORF (5'-C-prM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4BNS5-
3’), large scale cleavage of this poly-protein results in the production of structural and NS proteins. All of these processes take place inside the membranes structures located in the cytoplasm of host cell, transportation of proteins is achieved through cellular signal sequences and if these structures are disrupted by external means can lead to inactivation of protein processing(Svitkin et al., 1984). Studies have demonstrated that several structural and nonstructural proteins are cleaved by host enzymes such as signal peptidase, furthermore viral serine protease is also responsible for cleavage of C and major NS proteins (von Heijne, 1984). For virus maturation host enzyme Furin protease cleaves precursor prM into mature M protein inside the Golgi network (Stadler et al.,
1997). Thus host as well as viral enzymes cleaves Flavivirus poly-protein into individual structural and NS proteins for formation of progeny viruses.
2.2 DENV PROTEINS
Amongst structural proteins E is considered as the most important proteins because E protein is involve in receptor binding with host cell receptors for attachment and fusion.
Host antibodies also interact with antigenic epitopes on E protein, consequently most vaccines are derived from E proteins to achieve maximum immunogenicity(Chambers et al., 1990). The M glycoprotein is also immunogenic to some extent however neutralizing antibodies are mostly produce against E protein (Kaufman et al., 1989).
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Envelope is the largest structural protein which is involve in binding with host cell surface receptors and fusion for viral entry (Allison et al., 2001; Crill and Roehrig,
2001). DENV surface is relatively smooth owing to the E homodimers present all across virus surface (Kuhn et al., 2002). Studies relating to structure of DENV revealed that E protein has three domains (EDI, EDII and EDIII) (Rey et al., 1995). These domains have some important functions, ED1 works as a flexible hinge regions which assists in viral fusions.ED2 has two sections (Adams et al., 1995; Guirakhoo et al., 1989). EDIII plays a key role in DENV endocytosis by interacting with host cell receptors, EDIII is also interacts with heparin sulfate receptors of host cells that attachment is followed by fusion and entry (Chu et al., 2005; Huerta et al., 2008; Hung et al., 2004; Krishnan et al., 2007).
Studies have shown that E protein is a type II fusion protein which fuses with cellular endosome membrane via highly hydrophobic region in the EDII (Lescar et al., 2001; Rey et al., 1995).
Pre-membrane (prM/M) is the second structural protein having a size of
19kDa,prM is linked with capsid by a hydrophobic stretch. During DENV maturation prM is converted into matrix (M), the precursor prM contains N-linked glycosylation sites at the N terminal and six conserved cysteine residues which are responsible for disulfide bridging (Chambers et al., 1990; Nowak and Wengler, 1987). Both prM and E have transmembrane domains, these domains perform the role of retention signals and supports heterodimer formation (Lin and Wu, 2005)as shown in Table 2.2. DENV maturation involves cleavage of prM to M this leads to the viral budding via attachment with host cell membrane (Li et al., 2008). Heterodimerization of prM/E results in release of prM resulting in the E homodimers(Stiasny et al., 1996; Wengler, 1989).
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Capsid protein is the smallest structural protein which is basic in nature having a size of 12kDa, C protein is cleaved from polyprotein by NS3/NS2B DENV proteases and host cell signalase and consequently released in cytoplasm (Amberg and Rice, 1999;
Lobigs, 1993). Studies have shown that homodimer form of C protein and DENV nascent
RNA fuse to form nucleocapsid which buds off into the ER Lumen to initiate virus formation by interacting with prM and E proteins (Lindenbach and Rice, 2001; Ma et al.,
2004;Wang et al., 2004) as shown in Table 2.2. C protein is essential for DENV assembly, the importance of C protein becomes obvious with the production of sub-viral particles lacking DENV genome as well as C protein(Ferlenghi et al., 2001).
DENV has seven non structuralproteins; the precise role of these NS protein is still not properly understood. Recent studies suggest that NS protein are responsible for viral replication and encapsidation, moreover these NS are also involve in evading the host antiviral responses (MACKENZIE et al., 1996; Muñoz-Jordán et al., 2003).
NS1 is a 40-46 kDa protein which monomeric and changes to dimeric after few minutes (Falgout et al., 1989).NS1 is highly abundant in cells it's found on cell surface and is also released from the cell. NS1 plays a key role in two major processes, NS1 in association with DENV RNA and human heterogenous nuclear ribo-nucleoprotein
(hnRNPC1/C2) produces nascent mRNA (Noisakran et al., 2008). NS1 is an important mediator in RNA as well as viral replication (Mackenzie et al., 1998).
The precise role of NS2A is not clear nevertheless it is involve in DENV replication. A recent study showed that mutations in NS2A caused hindrance in viral assembly and subsequent replication of DEN (Xie et al., 2013). During DENV infection
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NS2A has shown to inhibit interferon (IFN) signaling assisting DENV to avoid host immune system (Simmons et al., 2010).
NS3 has multiple functions like helicase, protease and genome capping. DENV mRNA is translated into a poly protein; this polyprotein is processed by NS3 protease domain. NS2B act as a co-factor for NS3 and assists NS3 in its protease activity (Erbel et al., 2006). Recent studies have suggested that NS2B/NS3 complex can activate caspases which in turn can lead to apoptosis of host cell (Shafee and AbuBakar, 2003). NS3 has a helicase activity owing to C-terminal amino acids (170-619), this portion of NS3 protein has also shown RTPase and NTPase/helicase activity. The helicase functions by splitting the new DENV RNA from parent template in addition to that the helicase activity results in the unwinding of secondary structures at 3' UTR for starting replication (Gorbalenya et al., 1989).The interaction of hypophosporylated form of DENV NS5 and NS3results in the activation of both RTPase and NTPase. RNA capping is also via involvement of 5' triphosphatase (RTPase) (Bartelma and Padmanabhan, 2002; Yon et al., 2005).
NS4A is one of the smallest DENV protein, owing to its hydrophobia nature it is mostly present in association with host membranes (Miller et al., 2007). Main role of
NS4A is the proper organization of cellular membranes for allowing replication of DENV genome.
NS4B is a hydrophobic protein having a size of 27kDa present throughout the cytoplasm of infected cell; however it is initially found in peri-nuclear regions. NS4B has shown to interact with NS3 helicase region which is indicative of the fact that NS4B is an important component of viral replication (Miller et al., 2006). NS4B has two major roles in DENV RNA replication one of those is assisting in the detachment of NS3 helicase
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from RNA, whereas the second role of NS3 is that it clutches both strands and keeps them separated during RNA replication (Umareddy et al., 2006). NS4B in collaboration with NS4A and NS2A contribute in resisting host IFN activity(Muñoz-Jordán et al.,
2003).
NS5 is the largest of all NS DENV proteins with a size of 104 kDa, detail analysis revealed that NS5 has a conserve sequence amongst DENV serotypes and even some other member of Flaviridae family (Yap et al., 2007). NS5 can be divided into two parts one is involve in capping at the 5' end of DENV genome while the C terminal of NS5 is a
RNA dependent, RNA polymerase that produces intermediate double stranded RNA finally resulting in the production of positive sense RNA for progeny viral particles
(Egloff et al., 2002; Zhou et al., 2007).
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Table 2.1: DENV RNA elements and there functions.
RNA ELEMENTS FUNCTIONS
5' UTR Non canonical translation and viral RNA synthesis
cHP Translation initiation codon selection and viral
viability
5'/3' UAR Viral RNA cyclization and viral viability
5'/3' CS Viral RNA cyclization and viral RNA synthesis
3' UTR Cap dependent non canonical translation and viral
RNA synthesis
VR Translation and viral RNA synthesis
DB1/DB2 Translation and viral RNA synthesis
3'SL Translation and viral RNA synthesis Table
2.2: DENV proteins and there functions.
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VIRAL PROTEIN FUNCTION
C Viral RNA packaging
prM Prevention of premature fusion
E Receptor binding and fusion
NS1 Signal transduction
NS2B NS3 serine protease cofactor
NS3 Helicase, NTPase, 5' triphosphatase and serine protease
NS4B Inhibition of IFN signal transductin
NS5 RDRP and methyltransferase
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2.3 OVER VIEW OF DENGUE VIRUS
Dengue virus infection is the most prevalent mosquito borne viral infection.
DENV belongs to family Flaviviridae having four serotypes i.e. DEN-1, DEN-2, DEN-3 and DEN-4. In humans DENV causes dengue fever (DF) which can develop into severe complications like dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)
(Kyle and Harris, 2008). Reemergence of DENV in recent years has developed into a grave health risk in tropical and subtropical regions of the world (Barrera et al., 2002; da
Glória Teixeira et al., 2002;Sukri et al., 2003). Flaviviruses in general encode for three structural proteins i.e. capsid (C), pre-membrane (prM) and envelope (E) proteins and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
Structural proteins develop into a viral particle while nonstructural proteins play role in viral replication and assembly (Kümmerer and Rice, 2002; Liu et al., 2003).
Anti DENV inhibitors are designed keeping in view potential host and viral targets where various mechanisms can be blocked to control disease progression like enzymatic interactions, viral replication, host and viral receptors interaction, etc.
Antiviral agents that can reduce viral titer many folds can decline high mortality rates associated with DHF/DSS (Noble et al., 2010; Vaughn et al., 2000). Strategies to curb
DENV usually include targeting viral enzymes and replication mechanism, while host machinery can also be targeted with the added advantage of reduced chances for development of viral drug resistance.
Current treatment options include hemorrhagic condition management and fluid therapy with no targeted drugs for viral clearance. As development of effective vaccine has still not been achieved, development of an effective therapeutic agent against DENV
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is vital to bring down annual DENV related mortality rate, a number which usually exceeds the reported one (Whitehead et al., 2007). DF annual epidemiological estimates lack accuracy due to multiple factors, which include large number of unreported cases, absence of proper diagnostic procedures such as determination of serotype and in a few cases diagnostic errors(Farrar, 2008; Range, 2008).
Research in recent years can and has led to the development/identification of possible therapeutic agents for DENV, also advances in drug delivery mechanisms can target the precise area with high efficacy and negligible chances of development of side effects (Pollock et al., 2010). Host factors play a key role in development of DF, a recent study discovered 42 human factors that contribute appreciably in the development of
DENV infection (Sessions et al., 2009). Thus a detail study of these factors can open many avenues for development of anti-dengue therapeutics.
Vaccine approaches against DENV date back to 1929, when for the first time formalin phenol inactivated vaccine was developed. Although this vaccine was unsuccessful still it marks the beginning of vaccine strategies against DENV(Simmons et al., 1931). Later attempts involving live attenuated vaccine were also stopped over growing concerns regarding patient safety (Hotta, 1952; Wisseman et al., 1963). Since
DENV is an enveloped virus, antibodies are produced mostly in response to the highly immunogenic envelope protein (Brinton et al., 1998). Vaccinia vector based vaccine having truncated envelope gene provided complete protection to a DENV challenged monkey (Men et al., 2000). Adenovirus vector vaccine has also been very effective, enhancing immune response in vitro against all four DENV serotypes (Raviprakash et al.,
2008).
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2.4 DENV SEROTYPES AND GENOTYPES
Although DENV has been classified into four major serotypes however each serotype can have several genotypes depending on geographical distribution and place where that strain originated (Rico-Hesse, 1990). DEV1 has five genotypes and members of genotype I are from China, East Africa and parts of Southeast Asia. Genotype II is based on DENV1 from Thailand whereas DENV1 isolated from Malaysia are grouped into genotype III (Goncalvez et al., 2002). DENV1 isolated from Australia and Pacific
Island were placed in genotype IV and lastly genotype V contained DENV1 from West
Africa, South America and some countries from Asia (Wang et al., 2000).
DENV2 has five genotypes originating from various regions of the world, genotype I is further divided into two sub groups Asia I which has Malaysian strain of
DENV2 while Asia II contains DENV2 isolates from Sri Lanka, Taiwan, Philippines and
Vietnam. Genotype II contains strains for several countries, isolates of DENV2 from
Pacific Islands, Indian subcontinent, American and some Caribbean countries make up genotype III. The genotype IV has DENV2 isolates from Asian countries including
Thailand, Vietnam and Americas. Final genotype V has strains from Southeast Asia and parts of West Africa (Lewis et al., 1993).
DENV3 is classified into five genotypes among which genotype I contains strains from Philippines, Indonesia, Malaysia and South Pacific islands. Genotype II is based on strains from Thailand and genotype III is a group of isolates from Samoa, Africa, Sri
Lanka and India. Isolates from Tahiti and Puerto Rico make up genotype IV moreover strains from several Asian countries including China, Malaysia and Philippines are grouped into genotype V (Lanciotti et al., 1994; Wittke et al., 2002). DENV 4 has 4
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genotypes and isolates from Japan, Thailand, Sri Lanka and Philippines are members of genotype I. Strain of DENV2 isolated from Tahiti, Americas, Malaysia and Indonesia are grouped into genotype II while genotype III contains strains from Thailand. Lastly genotype IV contains strains of DENV 4 isolated from wild animals (Lanciotti et al.,
1997).
2.5 PATHOGENESIS
Pathogenesis of DENV is inter-dependent on several factors which can modulate
DF to its complicated manifestations known as DHF/DSS. Worldwide DENV is the leading vector borne viral infection associated with highest mortality rates in children, both DHF/DSS are responsible for this high mortality rate (Anders et al., 2011).
Important factors in DENV enhancing severity of DF are explained below.
2.5.1 High virulence
Studies have shown that several DENV strains have a tendency for high virulence in comparison with normal DENV strains. This phenomena was initially observed by
Rosen and Gubler in 1970s when it was examined that some of the DENV strains caused limited number of DHF/DSS cases while SAME DENV had a higher ratio of DHF/DSS cases (Gubler et al., 1978). A similar case was seen in 1080s when a Southeast Asian strain of DENV2 was introduced in Cuba which lead to an outbreak of DHF in South
America (Guzman et al., 1995). With the onset of genomic sequencing phylogenetic analysis revealed that some strains of DENV were associated with severe DF infections and higher mortality rates (Messer et al., 2003).
2.5.2 Host Innate Immunity
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Humans have a robust immune system and whenever any foreign antigen enters into the body immune response neutralizes the antigen. In humans one of the important component of innate immunity is complement system having several active proteins components.Studies have shown that patients with DSS have increased levels of complement proteins (C3a and C5a) in the plasma (Churdboonchart et al., 1983; Shaio et al., 1992). Complement activation is via antibodies which interact with DENV NS1 protein, this NS1 is present on the surfaces as well as in fluids after being released from
DENV infected cells (Avirutnan et al., 2006; Kurosu et al., 2007). Two complement proteins C5b-C9 which work as a complex are associated with progression of DF into complications like DHF/DSS.
2.5.3 Antibody Dependent Autoimmune Responses in DF
During DF human body mounts an immune response to neutralize DENV, this neutralization takes place by the help of antibodies directed against several viral antigens.
Most of these antibodies are produced against E protein which is the largest DENV structural protein, however a small portion of DENV E protein has a sequence resemblance with numerous host factors including prothrombin, plasminogen and tissue plasminogen factor (Huang et al., 1997; Markoff et al., 1991). Platelets are also a target for cross reactive antibodies which are initially generated against DENV NS1 protein, these cross-reacting antibodies are potent inhibitors of platelet aggregation and can cause severe thrombocytopenia (Chen et al., 2009; Lin et al., 2008a). Amongst several NS1 cross-reactive antibodies the most important antibody is IgM which can activate pro- apoptotic caspase in endothelial cells and this antibody can trigger secretion of inflammatory cytokines i.e. MCP-1, IL-6 and IL-8(Lin et al., 2005).
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2.5.4 Cellular Autoimmunity in DHF/DSS
T cells are crucial part of cellular immunity against DENV as they can clear infected cells and limit DENV replication. Research in T cell studies have demonstrated that although CD8 cells are able to clear DENV however these calls have a potential to cause cross reactivity(An et al., 2004). Highly cross reactive CD8 cells are associated with production of inflammatory cytokines IL-13, TNFα and IFNγ which can cause severe inflammation in human host. These hyper active CD8 cells are short live and after producing pro-inflammatory cytokines these cells die by apoptosis (Dong et al., 2007;
Mongkolsapaya et al., 2006). Studies have shown that these hyper active CD 8 cells are responsible for mass production of pro-inflammatory cytokines however the ability of these cells to produce cytotoxic granules is lost (Hober et al., 1993). Serum analysis of
DHF/DSS patients have revealed soaring levels of pro-inflammatory cytokines (IL-1β,
IL-6, IL-8, TNFα and IFNγ), owing to immuno-pathogenic CD8 cells (Hober et al.,
1993). Initially research on immuno-pathogenesis of DENV revealed that cross reactive
T cells are generated against NS proteins, later on studies conducted on mice models demonstrated that hyper active CD8 cells are produced against both structural (prM and
E) as well as NS (NS1, NS2a and NS3) (Beaumier and Rothman, 2009).
2.5.5 Antibody Associated with Enhancement of DF
Antibodies are important component of human immune response for neutralizing
DENV however some antibodies can further complicate the situation by enhancing DF
(Guzmán et al., 1990). Higher rates of infant mortality have been associated with
DHF/DSS owing to the presence of maternal antibodieswhich have lost ability to neutralize DENV till the age of six months however these antibodies keep reacting with
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DENV until one year (Chau et al., 2009). These antibodies lacking neutralizing potential contribute in ADE. In children older than one year sometimes a prior DENV infection can result in production of non-neutralizing antibodies which can cross react with a subsequent serotype of DENV and lead to DHF/DSS (Halstead et al., 1973; Halstead et al., 1970).
For ADE weakly neutralizing antibodies require immune complex for proper attachment with phagocytes (Halstead and O'rourke, 1977). Fc receptors (FcR) have also been extensively studied for their involvement in ADE, two FcR (FcγRI and FcγRII) were identified for playing crucial roles in development of ADE whereas the significance of FcγRIII in ADE is still not properly understood (Littaua et al., 1990; Unkeless, 1989).
A study on AG129 mice having modified FcRs revealed low virus replication and lack of severe disease (Balsitis et al., 2010). DENV titers go up rapidly during ADE owing to the enhancement of infection in host cells, one of the factor which contributes to elevated rate of virus replication is the conversion of immature virus into viral receptors via
FcR(Balsitis et al., 2010; Libraty et al., 2002).Another possible way of ADE involvement in DHF/DSS is via down regulation of cellular nitric oxide, this results in the inhibition of
STAT1 and IRF1 which in turn limit the innate immunity (Wang et al., 2006).
According to most studies FcR were responsible for ADE however later research revealed that cells lacking FcR were also involved. Research conducted by Halstead and his fellow researcher demonstrated the crucial role of IgG in developing ADE
(Chareonsirisuthigul et al., 2007).
2.6 Viral targets for DENV inhibitors
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Dengue viral proteins play an important role in the pathogenesis and propagation of the virus, these viral proteins are mostly enzymes, that are absent in the host cells thus minimizing the risks associated with inhibitors interfering in host cell mechanisms..
Therefore these proteins can be selectively targeted by inhibitors for viral control and clearance.
2.6.1 Structural proteins’ based inhibition of DENV (Receptor mediated entry inhibition)
DENV envelope protein (E) belongs to class II viral proteins engaged in fusion with host cellular receptors (Harrison, 2005). E protein regulates fusion with numerous host receptors which include glucose regulated protein 78 (GRP78), DC-SIGN, L-SIGN, laminin receptors, mannose receptors and ICAM-3 grabbing non-integrin receptors
(Jindadamrongwech et al., 2004; Miller et al., 2008; Navarro‐Sanchez et al., 2003;
Tassaneetrithep et al., 2003; Thepparit and Smith, 2004). Thus these host receptors can be targeted for inhibiting virus/host interactions leading to infection. Since E protein mediates membrane fusion with host by regulating low pH-induced conformational changes and organizational rearrangements at the surface of virus, this membrane fusion can also be targeted for development of an anti-viral agent.
Structurally DENV E protein has three domains i.e. I, II and III, where domain
III has shown potential for blocking viral entry into the host cell by disruption of viral fusion (Barbas et al., 1997; Liao and Kielian, 2005; Modis et al., 2004). DC-SIGN receptors were blocked by carbohydrates likeplantlectins (HHA) binding agents reducing
DENV infection but due to their specificity to DC-SIGN receptors these agents are inefficient to be used for cells lacking these receptors (Alen et al., 2009).
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2.6.2 Nucleoside based inhibition of DENV
Nucleosides enter the cell via two mechanisms: passive diffusion or through a nucleoside transporter (Pastor-Anglada et al., 2005).Variations have been observed in responses of different cells lines against DENV challenge due to varying levels of nucleoside and nucleotide kinases, therefore for a nucleoside inhibitor approach, multiple clinically relevant cell lines should be evaluated (Leary et al., 2002). A 2´C methyl deaza-adenosine nucleotide inhibitor showed wide range of anti-flaviviral activity targeting specifically RNA dependant RNA polymerase (NS5) (RdRp) of DENV, HCV and other members of Flaviviridae. A recent study found this inhibitor highly effective against HCV-infected chimpanzee model (Carroll et al., 2009; Migliaccio et al., 2003).
DENV was also effectively inhibited by an adenosine analog (NITD008) both in vivo and in vitro studies, this analogue contain two key substitutions. A carbon was substituted at N-7 position of purine and acetylene was added at 2’ position of ribose
(Yin et al., 2009). In a recent study ribavirin a nucleoside analogue has shown promising prospects to inhibit dengue virus NS5 protein. There are also certain other guanosinenucleoside analogues including acyclovir and EICAR that effectively compete with GTP binding to DENV NS5 protein (Benarroch et al., 2004).
2.6.3 Methyltransferases inhibition of DENV
Genome of Flaviviruses has a 5´cap and this capping is achieved by a series of enzyme regulated reactions. The 5´end of viral RNA is hydrolyzed by NS3 tri- phosphatase from tri-phosphate to di-phosphate, while a guanylyl-transferase transfers
GMP to 5´-diphosphate. NS5 methylates N-7 position of guanine, and 3´OH position of first nucleotide ribose that prepares the 5´ cap. DENV serotype 2 deficient in 5´ cap
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methylation has shown inhibition of replication potential (Cleaves and Dubin, 1979;
Egloff et al., 2002; Ray et al., 2006; Wengler and Wengler, 1981; Wengler and Wengler,
1993).
In another approach seventeen mutations were introduced in amino acids of methyltransferase domain targeting specifically amino acids that were directly involved in s-adenosyl-I-methionine binding, or vital amino acids for proper confirmation of NS5 and charged amino acids involved in various interactions (Podvinec et al., 2010). In one of the studies, 10 novel inhibitors of NS5 methyltransferase were identified that interacted with NS5 in a similar fashion. Computer based molecular docking approach was applied to 5 million commercially available compounds in order to screen for inhibitors of methyltransferase, out of which 263 compounds were selected for further testing and it was observed that 10 compounds were effective for inhibition of methyltransferase(Lim et al., 2011). S-adenosyl-homocysteine (SAH) is a chemical derivative that binds to methyltransferase of dengue virus and selectively inhibits its replication, this binding is not related to human enzymes. Dengue virus methyltransferase crystalline structure exhibited that its N6-substituent bound in the cavity caused conformational changes in residues lining the pocket. Therefore, this study was vital since it proved that a virus specific methyltransferase inhibitor could be prepared to inhibit DENV replication (Luzhkov et al., 2007).
Combining computational and experimental screening techniques a new novel inhibitor for dengue methyltransferase has been identified with a previously unknown scaffold. Computer based approaches such as 2D similarity searching, pharmacophore filtering and docking were used to identify compounds that inhibited DENV
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methyltransferase and 15 top hit compounds were selected and were further tested on recombinant methyltransferase. Study resulted in discovery of novel inhibitor for dengue virus that had an IC (50) value of 60 microM(Kroschewski et al., 2008).
2.6.4 Protease (NS2B/NS3) inhibition of DENV
Proteases (host and viral) play a vital role in the viral poly-protein processing.
During recent years RNA virus protease domain has been the target for many antiviral therapies. Protease inhibitors against HCV are in clinical trials and since DENV belongs to the same family, these approaches can therefore be utilized for development of therapeutic agents against DENV (Soriano et al., 2008).
DENV serine protease belongs to Trypsin superfamily having a catalytic triad
(His51, Asp75, Ser135) with more than 50% homology amongst members of genus
Flavivirus(Bazan and Fletterick, 1989; Valle and Falgout, 1998). Serine protease of
DENV has optimal activity at a high pH (pH 9) (Leung et al., 2001) and is resistant to available potent inhibitors of serine proteases. Many factors contribute to the ineffectiveness of protease inhibitors for DENV: the most prominent being weak binding of inhibitor with enzyme, where only aprotinin was found to be effectively inhibit DENV protease by blocking the active site (Ekonomiuk et al., 2009; Mueller et al., 2007;
Tomlinson et al., 2009; Yang et al., 2011).
In a recent study EUDOC docking program based library of small molecules was generated. These compounds exhibited binding to P1 pocket and catalytic site of DENV2
NS3 protease domain. Most favorable compounds obtained through docking were later used for in vitro inhibition of DENV2, these compounds showed high solubility and
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inhibition of DENV2 in cell culture experiments. Final results showed that 2 compounds were most promising for inhibition of protease inhibition (Yin et al., 2006).
BP2109 is another dengue virus protease inhibitor that is associated with viral replication and viral RNA synthesis inhibition (Tomlinson et al., 2009). In another approach different synthesized protease inhibitors, inhibited NS3 protease of dengue virus (Wang et al., 2011). Computer based identified of inhibitors for DENV have also been evaluated for DENV2 protease inhibition. Two of these potential compounds further inhibited viral replication in cell culture experiments (Mueller et al., 2008).
2.6.5 Host enzymes based inhibition of DENV
DENV relies on host enzymatic machinery for its replication and many host factors assist in viral replication. Since host enzymes are vital for various cellular mechanisms therefore a cautious approach is required for minimizing drug induced toxic effects and means for their delivery.
Different inhibitors have been evaluated against host enzymes for dengue virus inhibition. Dengue virus requires host nucleosides for replication and these nucleosides are incorporated into new virus progeny. In a recent study a novel antiviral compound
(NITD-982) has been identified that can inhibit host dihydroorotate dehydrogenase
(DHODH), an enzyme required for pyrimidine biosynthesis. DENV was inhibited via diminishing intracellular pyrimidine pool, a major drawback of this anti-dengue was lack of effectiveness in vivo resulting from exogenous uptake of pyrimidine from diet(Aleshin et al., 2007).
2.6.6 Kinases based inhibition of DENV
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Host kinases contribute in DENV replication by engaging in viral assembly and release from host cell. All four serotypes can be targeted via c-Src kinase inhibitors. In a study conducted in 2007 DENV assembly was blocked by dasatinib c-Src kinase inhibitor. c-Src, AZD0530 and other members of c-Src kinase inhibitors have also proven to be useful and safe anti-viral agents (Chu and Yang, 2007).
A study involving West Nile Virus (WNV) showed that c-Yes a member of Src kinase family was involved in virus maturation as the level of cellular Src family kinase c-yes was up-regulated when infected with WNV. Src Family Kinases (SFK) inhibitor pp2 reduced viral load up to 2-4 log in WNV infected cell lines, thus demonstrating that
Src family kinases have a pivotal role in viral replication and assembly inside the host.
RNA interference mediated down regulation of c-yes also resulted in decline in WNV titers suggesting that SFK are important host enzymes utilized by virus for its replication
(Hirsch et al., 2005).
2.6.7 Glucosidase based inhibition of DENV
Glucosidases are a group of enzymes that catalyzeglucoside hydrolysis, folding and glycosylation of various DENV structural and nonstructural proteins (E, prM and
NS1). In order to perform these functions DENV requires host glucosidase. Inhibition of host endoplasmic reticulum -glucosidase prevented folding of Pre-membrane and
Envelope glycoprotein of DENV (Hirsch et al., 2005). Such glucosidases can be potential targets as therapeutic against DENV. Deoxynojirimycin (DNJ) is an effective inhibitor of
Endoplasmic reticulum (ER) -glucosidase that inhibits posttranslational modification in a dose dependent manner. Castanospermin is an -glucosidase inhibitor that potentially
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disrupts the folding of the structural proteins prM and E of all the four serotypes of
DENV (Whitby et al., 2005; Wu et al., 2002).
Iminocyclitol compounds have been tested effectively as antiviral compounds that affect the endoplasmic-reticular glucosidase mediated morphogenesis of DENV. These compounds specifically target budding viruses and show limited toxicity, these modifications can later be applied for development of anti-viral agents that can target multiple viruses (Gu et al., 2007).
2.6.8 Cytokines and receptors based inhibition of DENV
Immune system is involved in the progression of DF to DHF and DSS. Cytokine and chemokine including TNFα, IL-8, MMP-9 and VEGF/VEGF receptor have shown significant involvement in development of complications that lead to DHF and DSS
(Luplerdlop et al., 2006; Shresta et al., 2006; Souza et al., 2009; Srikiatkhachorn et al.,
2007). Recent studies attribute platelet-activating factor (PAF) with DENV pathogenesis.
Platelet-activating factor receptor (PAFR) has an important role in DENV pathogenesis and in a study it was observed that mice lacking PAFR showed decreased thrombocytopenia, hem-concentration, low levels of cytokines and a longer duration for manifestation of symptoms (Bai et al., 2007).
For limiting DENV pathogenesis regulatory mechanisms are required. In mouse models, antibodies produced against these cytokines provided protection but further research is required for their possible use in humans. Receptor mediated entry inhibition can also play a pivotal role in the inhibition of dengue virus.
2.6.9 Computational design of peptide inhibitors against DENV
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Viral structural proteins mediate viral entry into the host cell via fusing with host cell receptors. Inhibitors blocking this fusion process can retard progression of infection.
In recent years a few inhibitors were designed and evaluated against beta sheet envelope
(E) proteins and cell attachment of DENV. These inhibitor peptides have shown promising results to inhibit DENV (Hrobowski et al., 2005; Rees et al., 2008). However, crystal structures of E protein both pre-fusion and post fusion were not exploited in most of the approaches for development of Flavivirus E protein directed inhibitors (Heinz et al., 1991; Kanai et al., 2006;Modis et al., 2003).
In a recent study inhibitor peptides were designed using residue-specific all-atom probability discriminatory function (RAPDF). X-ray diffraction structure of DENV-2 envelope protein served as a template for making mutant structures yielding mutant peptides (Zhang et al., 2004b). Residual side chains were substituted in all peptides by backbone-dependent side chain rotamer library and linear repulsive steric energy term provided by SCWRL version 3.0 (Bower et al., 1997). These peptides were successful in inhibiting DENV infection up to a certain extent.
2.7 Inhibition of DENV by siRNA
The production of siRNAs in eukaryotic cells occur by cleavage of long dsRNA sequences by endonuclease Dicer which is a complex protein having TAR-RNA binding protein (TRBP) (Zhang et al., 2004a). The next step involves the transfer of siRNA from
Dicer to RNA-induced silencing complex (RISC), RISC contains Argonaute 2 (Ago) protein having the potential of cleaving target mRNA strand. Ago are vital for proper gene silencing and in humans Ago-2 perform this cleavage (Liu et al., 2004; Meister et al., 2004).
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Thermodynamic properties of siRNA targeting specific mRNA are vital for selection and attachment with RISC, mRNA molecule complementary to guiding RNA is cleaved by Ago-2 (Khvorova et al., 2003; Schwarz et al., 2003). One other class of RNAi is represented by 60-70 base pair hairpin shaped MicroRNAs (miRNAs) produced from precursor pri-miRNAs within the nucleus.
Production of miRNAs is achieved by complex known as Drosha-DGCR8 (Han et al., 2004; Lee et al., 2003). RNAse III Dicer excises the loop and consequently one strand is presented to RISC, this whole process takes place in the cytoplasm of the cell.
The miRNA shares a 3' UTRcomplementarity with target mRNA which is finally degraded for gene silencing (Bagga et al., 2005).
RNAi dependent specific RNA degradation involves double stranded RNAs
(dsRNA) which are abundantly present in the cytoplasm of eukaryotic cells (Elbashir et al., 2001; Fire et al., 1998; Montgomery et al., 1998). Gene silencing is achieved by the formation of RNA induced silencing complexes (siRNA) in association with various cellular factors (Hammond et al., 2000;Meister and Tuschl, 2004; Mello and Conte,
2004). Studies suggest that DENV could also be effectively inhibited by RNAi(Joost
Haasnoot et al., 2003; Sanchez-Vargas et al., 2004). It has also been suggested that mosquito’s continual infection without any pathogenic observations can attribute to
RNAi involvement (Sánchez-Vargas et al., 2009). Additionally, inside the host cytoplasm during the un-coating and replication processes, ssRNA viral genomes can be targeted by
RNAi(Uchil and Satchidanandam, 2003).
In a recent study siRNA produced against DENV glycoprotein gene precursors inhibited DENV replication and resulted in increased cell survival rate (Wu et al., 2010).
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In Another study an engineered Sindbis virus containing 290 bp region of DENV serotype 2 prMregion produced resistance against DENV type 2 challenge. dsRNA were designed for DENV serotype 1 in a different study achieving reduction in viral load but it was less efficient as compared to Sindbis-induced silencing (Adelman et al., 2001;
Caplen et al., 2002). A more detailed analysis is required for the development of potential siRNA based inhibitors of dengue virus with multiple targets directed against more than one serotype.
2.8 VACCINE DEVELOPMENT FOR DENV
Development of vaccine against DENV is still a challenge for scientists across the globe. Presence of multiple serotypes pose a major problem as protection against a particular serotype can lead to a more severe condition in the form of DHF/DSS on co- infection with a different serotype due to phenomena known as antibody dependent enhancement (ADE) (Boonnak et al., 2008; Whitehead et al., 2003). A possible solution to this problem is development of a tetravalent vaccine which can provide protection against all four serotypes (DENV1-DENV4) (Konishi et al., 2006; Raviprakash et al.,
2008;Simmons et al., 1931). Historically efforts regarding development of DENV vaccine date back to early 20th century but these approaches failed to provide a suitable vaccine and in 1929 Formalin inactivated virus approach did not satisfy safety concerns
(Guirakhoo et al., 2004; Hotta, 1952; Wisseman et al., 1963).
Recent advances in molecular virology and immunology have opened new avenues in the field of vaccine development, latest approaches include sub unit vaccines,
DNA vaccines, chimeric vaccines and viral vector based vaccines.
2.8.1 Chimeric Vaccine
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Chimeric vaccines contain fusion of two viral proteins resulting in high levels of protection. In a recent study a chimeric vaccine containing Yellow fever (YF) and DENV was prepared, Envelope (E) and prM region of YF were replaced by wild type (WT)
DENV representing all four serotypes. Chimeric vaccine viruses were electroporated into
Vero cells for production of chimeric viruses, 92% of monkeys survived when challenged after vaccination with chimeric YF/DENV vaccine (Trent et al., 2010).
Vaccines obtained from Vero cells contain higher amounts of cellular proteins,
DNA and cell debris, therefore a stringent process is required for purification of this formulation. Human diploid cell lines provide a better alternative with comparatively low levels of cellular debris (Raviprakash et al., 2008).
2.8.2 DNA vaccine
DNA vaccines provide added advantage of intracellular antigen processing and are safe as compared to live attenuated virus vaccines. Few concerns regarding DNA vaccines are; poor intake resulting in reduced antigen expression and weak immune response to incorporated structural proteins of virus. Alternatively addition of adjuvant may enhance immunity but is associated with increased cost related to manufacturing. In a recent study DNA vaccine in combination with recombinant viral vector vaccine was developed to tackle issues involving immunogenicity and gene delivery (Chen et al.,
2007b).
DENV serotype1 Envelope (E) and prM genes were expressed in Venezuelan
Equine Encephalitis (VEE) virus vector comprising of two doses of naked DNA with a third dose of VEE dengue particles, induced complete protection in macaques
(Ramanathan et al., 2009). Envelope conserved domain (Domain III) expressed in
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mammalian expression vector has provided immunity against all the four serotypes in mice (McArthur et al., 2008).
Monkeys and mice have also been subjected to numerous DENV DNA based vaccines in an effort to reach a suitable candidate vaccine for humans (De Paula et al.,
2008; Konishi et al., 2000). Drawbacks of DNA vaccines are weak immune responses and high cost burdens and these factors demise the chances for the production of cost effective and potent DNA vaccine in near future (Wang et al., 1998; Wang et al., 2001).
2.8.3Live attenuated vaccines
Live attenuated vaccines (LAV) have been successful against Yellow fever virus
(YFV) and Japanese Encephalitis (JE) Virus. LAVs provide long term immunity and are cost effective as compared to DNA and Chimeric vaccines. Among other advantages,
LAV activate both types of immune responses i.e.humoral and cell mediated for a long period of time thus making LAVs ideal candidates for protection against DENV
(Edelman, 2007; Innis and Eckels, 2003).
Efforts for development of LAV for DENV have encountered a few problems from the beginning like low levels of immune activation and side effects that hampered the progress in development. For more than the last two decades monovalent LAV candidates have been propagated on primary and diploid cell cultures and later evaluated in humans to reach a final effective low cost vaccine (Edelman et al., 2003; Sun et al.,
2003).
A final break through was achieved by Halstead and Marchette in 2003 by multiple passaging of monovalent DENV LAV on primary dog kidney cell line (Halstead and Marchette, 2003). Monovalent DENV LAVs obtained from several passages on dog
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kidney cell line were combined to make a tetravalent vaccine which was tested in phase I and phase II trials in adults and children from Thailand and America which was effective and provided protection. However certain unreported complications occurred in phase III trials which ultimately led to the suspension of this DENV LAV (Edelman et al., 2003;
SABCHAREON et al., 2004;Sabchareon et al., 2002). Walter Reed Army Institute of
Research has successfully reported the production of LAV based vaccine that elicits immune response against all the four serotypes of DENV.
2.8.4Subuint vaccine
Subunit vaccines have also proved to be safer in comparison to live attenuated vaccines but are associated with weak immune stimulation mostly relying on adjuvant or other molecules for boosting immune response adequate for protection (Hem and
HogenEsch, 2007; Ishii and Akira, 2007; Webster and Hill, 2003). In a recent approach an immunogenic portion of DENV Envelope protein (E) was combined with five different adjuvants and effective immune activation was observed with all five formulations (Putnak et al., 2005).
A tetravalent vaccine having bivalent adenovirus constructs (CAdVax-Den12 and
CAdVax-Den34) provided a long-term protection in macaque monkeys by production of neutralizing antibodies against all four serotypes (Raviprakash et al., 2008). In a different study a recombinant tetravalent vaccine having consensus domain III of DENV Envelope protein (E) in combination with aluminum phosphate elicited higher antibody response along with memory immunity in Blab/c mice (Leng et al., 2009).
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2.9 Epitope variation a challenge for DENV vaccine design
DENV is a highly variable virus this variance has resulted in failure of multiple attempts for development of a tetravalent DENV vaccine (Rothman, 2004). Variability varies at the serotype level with high inter-serotype and low intra-serotype variance(Livingston et al., 1995). This serotype specific variance is the main reason for multiple peptide ligands spanning the whole DENV poly-protein with differences of one or several amino acids(Sloan-Lancaster and Allen, 1996), diversity of host immune responses is attributed to these epitope variations (Mongkolsapaya et al., 2006; Welsh and Rothman, 2003).
Advances in immunology have greatly increased the understanding of antigen variation and host immune response associated with different peptide ligands. Antigen variation can result in suppressed, enhanced or neutral host immune response which further increases the need for analysis of antigenic variance for reaching a potent vaccine candidate (Takahashi et al., 1989). For resolving the concerns regarding antigenic variance of DENV a bioinformatics approach can be implemented for ruling out cross reactive and low immunogenic epitopes. A strategy involving T-cell based epitopic prediction will enhance the efficacy of future vaccine tremendously.
2.9.1 DENV T Cell antigenic diversity analysis
Host T cells are targets for mapping and analysis Helper and cytotoxic T lymphocytes mediate cellular immune responses via the T-cell receptors (TCR) that recognize T-cell epitopes presented on cell surfaces by HLA molecules. HLA class I molecules, expressed on the surface of most nucleated cells, present endogenous
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epitopes, synthesized and processed in the 26 cytoplasm, to CD8+ cytotoxic T
Lymphocytes (CTLs) that eventually kill the infected cells(Bjorkman and Parham, 1990;
Shastri et al., 2002).
CD8+ cells are important in conferring immune response against intracellular viruses. On the other hand, HLA class II molecules display exogenously derived epitopes on the surface of professional antigen presenting cells (APCs), such as dendritic cells, B- cells and macrophages, for immune recognition by CD4+ helper T cells. Activated helper
T cells produce secondary signals for activation of both T cells and B cells (Esser et al.,
2003; Pulendran and Ahmed, 2006;Zinkernagel, 2004)
2.9.2 T cell epitope mapping of DENV
Immune studies have demonstrated that both structural and non-structural proteins are able induce T cell mediated immune response (Bashyam et al., 2006; Simmons et al.,
2005). Predominantly helper T-cell epitopic regions are present on the structural proteins where as cytotoxic T-cell epitopes are located on NS proteins (Roehrig, 2003). Studies have shown that human leukocyte antigen (HLA) alleles are the main sites for binding of
DENV T cell epitopes. Association of DENV T cells epitopes with 12 HLA alleles has been established, most of theses epitopes are located on NS3 protein(Screaton and
Mongkolsapaya, 2006; Welsh and Rothman, 2003).
2.9.3 Insilico analysis of DENV T cell epitopes
Recently several studies have shown bioinformatics based DENV T cell epitopes mapping which was later proved by wet lab experiments, therefore bioinformatics is seen as a reliable tool for T cell mapping (Lin et al., 2008b; Wang et
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al., 2008). Advancements in the field of bioinformatics can be utilized by researchers working on host immune interactions and use this knowledge for development of anti- dengue vaccines and therapeutics.
2.9.4 B cell epitope mapping antigenic diversity analysis
Research conducted on DF patients revealed that infection resulted in activation of B cell response after approximately 4 days post infection (Polyreactive, 2011). DENV structural proteins along with NS1 are responsible for host antibody response therefore, identification of B cell epitopes is vital for designing effective vaccine and drugs against
DENV. Characterization of B cell epitopes will also pave the way for development of rapid diagnostic kits and reagents for detection of DENV (Beltramello et al., 2010; Wu et al., 2003). Some of the B cell epitopes were indentified for highly immunogenic DENV proteins (E, prM, C, NS1 and NS4a) however, with high rate of mutations and the presence of several strains makes it an uphill task to fully identify B cell epitopes (Brien et al., 2010; Lin et al., 2012; Steidel et al., 2012).
2.9.5Insilico analysis of DENV B cell epitopes
Previous approaches directed at B cell epitope prediction were focusing at E, NS1 and NS5 proteins (Chiou et al., 2012; Sun et al., 2012). Later studies revealed the importance of C and prM proteins in production of antibody responses, there is still a long way to go for complete B cell epitope mapping of DENV (Dejnirattisai et al.,
2010a). Initially B cell epitopes were identified via techniques for antisera analysis like overlapping synthetic peptides (PEPSCAN) (Aaskov et al., 1989; Innis et al., 1989).
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Several antigenic regions were recognized by recombinant technology as well as protein cleavage by enzymes (Megret et al., 1992; Roehrig et al., 1998).
B cell epitopes can also be identified by displaying fusion peptide on the surface of a phage know as Phage display. This technique has been widely used because it allows conformational changes in the antigenic protein as seen in the virus itself (D'Mello et al.,
1997; Fu et al., 1997;Young et al., 1997). There is a lack of serotype based epitope mapping which is a major factor for achieving a uniform immune response against each serotype in a tetravalent vaccine. Furthermore, emergence of new strains makes B cell epitope mapping a vital component of a future tetravalent DENV vaccine.
Although experimental techniques were successful in defining some B cell epitopes located on DENV proteins however, there are a number of limitations of wet laboratory experiments. Computer based algorithms were able to overcome these limitations and these softwares were able to map B cell epitopes in a very short time with minimum effort(Yang and Yu, 2009; Yasser and Honavar, 2010).
In comparison with T cell epitope prediction, B cell epitope prediction is much intricate owing to a high variability of epitope length as recently described by Kringelum et al reporting that average length of epitopes was 15 amino acid residues (Kringelum et al., 2013). This study explained the fact that exact length of conformational B cell epitope is still not clear however, overall average was 15 residues. The software based epitope mapping has certain limitations like having limited number of epitopes and secondly inability to detect of random peptides as B cell epitopes(Chen et al., 2007a;
EL‐Manzalawy et al., 2008; Sweredoski and Baldi, 2009).
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DF is one of the major global health risk across the globe therefore, anti-dengue vaccine and drug development is vital for safety of people living all over the world.
DENV vaccine development faces major difficulties like antibody-dependent enhancement (ADE) and multiple serotypes, these factors led to the failure of several previous vaccinal strategies (Sabchareon et al., 2012). It will be worthwhile to use an alternative approach utilizing B cell epitope mapping minimizing weak links like cross- reacting antibody responses and variable immune response (Crill et al., 2012;
Dejnirattisai et al., 2010a).
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Chapter 5
DISCUSSION
Amongst arthropods borne viral infections the leading cause is Dengue virus.
According to recent estimates annual cases range from 100 to 200 million and with approximately 2 billion people living in high risk zones (Murray et al., 2013). Children living in these endemic regions are at high risk owing to disease severity and high mortality associated with DHF/DSS (Poovorawan et al., 2006). These factors further increase need for development of a vaccine and inhibitors for protecting general population and reducing annual mortality amongst children and adults.Molecular research conducted in viral replication and protein synthesis revealed several potential targets for anti-viral therapeutic development.
According to some recent studies, inhibition of target enzymes and proteins stemmed viral growth. siRNA also provides a very safe alternative to chemotherapeutic agents because of its limited side effects and its specificity for viral genomes. However limitations like lack of effective delivery mechanisms, time dependent reduction in RNA interference and expensiveness are major hurdles in the use of siRNA as possible anti- dengue therapeutic agent.
Last twenty years have seen DENV becoming a major global threat therefore; drastic measures are required for limiting this viral pathogen. Scientists have been working on different vaccine and therapeutic approaches for against DENV since the emergence of the disease. However advances in molecular biology helped in understanding various aspects of DENV replication and host interactions, this
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information led to the development of new inhibitors and vaccines. The propagation of
DENV in mosquitoes as well as humans was explained by a number of biologist which suggested that blood meal of an infected human individual results in replication of DENV in mosquitoes gut region(Hidari and Suzuki, 2011). After achieving high titers DENV travels to the salivary gland and it is transmitted to a healthy person via a new blood meal. Inside the human host DENV is taken up by antigen presenting cells (APCs) via endocytosis involving cellular protein clathrin(Sun and Kochel, 2013). DENV entry was explained in depth by Alhoot et al 2011 and suggested that clathrin mediated endocytosis was crucial for DENV internalization in the host cell (Alhoot et al., 2011). Receptor studies revealed that CD14 receptor expressed on the membranes of immune cells play central role in binding and endocytosis of DENV (Alhoot et al., 2011). In one of the inhibitory approach CD14 expression was suppressed by siRNAs which led to massive reduction in the expression of cellular CD14 receptors. When immune cells with limited expression of CD14 were presented with DENV challenge it was observed that internalization of DENV was drastically reduced (Alhoot et al., 2011). Although RNAi is a very effective tool for gene silencing however there are some limitations in RNAi technology, one of these limitation is host cell responses for degrading dsRNA. To tackle this concern a study was conducted which was able to enhance the knock down efficiency of siRNA and provided protection against host cell responses (Fischer and James, 2004).
In mosquitoes DENV can effectively replicate in several locations this is one of the reason which lead to the development of C6/36 cell line (Sakoonwatanyoo et al.,
2006). These C6/36 cells are mosquito cells which allow replication of DENV and yield high titer of DENV in vitrostudies. Xinweiet al in 2010 conducted a study for inhibiting
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DENV replication in C6/36 cells, when C6/36 cells treated with siRNAwere challenged with DENV a huge drop in DENV RNA was observed in addition to reduction in viral titer cell survival rate increased tremendously (Wu et al., 2010).
With the recent advancements in the field of molecular biology,siRNA technology has become a leading tool in gene therapy with applications like up regulation and down regulation of certain target genes. Based on this enormous potential siRNA technology is being extensively used for anti-virus drug development. With advantages like specificity, minimum or no toxicity and efficient mode of action siRNAs are one of the important components of biopharmaceuticals. Recent studies suggest that siRNAs can be used for blocking virus transmission (Joost Haasnoot et al., 2003). DENV carrying a single stranded RNA as the genome in the cytoplasm is a probable target for different
RNAi based inhibition mechanisms.
Arboviruses with RNA genome require an intermediate dsRNA molecule which can trigger host cell RNAi pathway (Blair and Olson, 2015). Cellular enzyme DICER chopsdsRNA and generates several small interfering RNAs, these 20-24 base pair siRNAs interact with argonate containing RISC (RNA induced silencing complex). This complex causes the unwinding of dsRNA and one of the strand of this dsRNA serves as a target locator, with the help of this sequence RISC binds with the target mRNA. In the next step the endonuclease activity takes place and cuts the target mRNA resulting in suppression of that specific gene (Dunoyer et al., 2004). As RNAi has been a part of innate immunity of most eukaryotic cells therefore this aspect was utilized by biologist for development of anti-viral inhibitors. This discovery led to the application of RNAi in several experiments involving gene silencing and pathogen inhibition. In recent times
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several human viruses were successfully targeted by RNAi which proves that RNAi is an effective tool in modern therapeutics (Bogerd et al., 2014).
In the current study we observed that synthetic siRNAswhich targeting the highly conserved regions of DENV3 genome can effectively inhibit DENV3 in Vero-81 cell lines. These conserved regions of DENV genome are potential targets for effective antiviral drug designing and targeting, moreover this information may be useful for future studies involving genome based inhibition of DENV. Three regions (3' UTR, 5' UTR and structural genes) were specifically targeted in this study since these regions were found to be highly conserved and also play a key role in DENV replication and assembly.3'UTR is approximately 450 nucleotide in length without a poly A tail with several conserve structures. These folds at 3' UTR results in the formation of conserve looped structures known as stem loop (3'SL), in depth studies conducted on the properties of this 3' SL in different arbovirusesrevealed that 3'SL is a key component of DENV replication (Alvarez et al., 2005a). In comparison to SL other components of 3'UTR are still not explained in detail and the exact function of other conserve regions are still a mystery (Wei et al.,
2009). In addition to SL regions various softwares have been able to predict two further folded structures A2 and A3. When these two folds were thoroughly studied via available algorithms, it was observed that both folds had conserved regions CS2 and RCS2
(Manzano et al., 2011). 3'UTR is significantly important in DENV replication this was proved by experiments in which deletions in the 3'UTR greatly hindered DENV replication and also resulted in defective virion formation (Tilgner et al., 2005). The exact role of 3'UTR in DENV replication is still not fully known, inhibitors could be designed only if the whole mechanism is revealed. Thus, 3'UTR has critical role in
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DENV replication and disruption of conserve domains in the 3'UTR will result in impaired DENV replication and dysfunctional virus production.
5'UTR is highly conserved in all the serotypes, it has contains approximately 100 base pairs. In depth analysis revealed that 5'UTR has a essentialrole in DENV replication
(Filomatori et al., 2006). Studies have shown that 5'UTR elements form a stem loop secondary structures and these structures are conserved amongst several members of arboviruses(Gebhard et al., 2011). Deletions in the 5'UTR region resulted in defective protein production and disruption of normal cell replication. It was seen that 5'UTR elements were responsible for binding of RNA with host proteins that are responsible for translation of protein (Sirigulpanit et al., 2007). Thus 5'UTR is pivotal for DENV replication but experiments have also suggested that 5'UTR is also involved in viral assembly (Roby et al., 2014).
All six siRNAs targeting DENV3 genome underwent strict scrutiny and only those siRNAs were selected which were able to fulfill all vital parameters for siRNA designing. Regions targeted included 3'UTR, 5'UTR and structural genes (C, prM and E) in DENV3 genome, all three regions are vital for maintaining replication cycle of
DENV3. siRNA designed from 3'UTR showed a significant reduction in the cellular
DENV3 level, which was confirmed by IFA and real time qPCR. A previous study conducted by Diego et al also demonstrated that deletions in 3'UTR resulted in several folds decrease in DENV titer. Results showed DENV3UTR3'siRNA2 was highly effective in limiting DENV3 replication when compared with other five siRNAs which were similarly tested for inhibition study.
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Foci counts revealed that DENV3UTR5'siRNA designed from 5UTR was also able to reduce DENV3 up to 65% 48 hours post DENV3 challenge. Synthetic siRNAs(DENV3SsiRNA1 and DENV3SsiRNA2)targeting the structural region had a limited triumph in limiting viral replication. As previously discussed UTRs are key elements in viral replication and genome organization, this can be one of the explanation for higher inhibition efficacy of siRNAs designed to target UTRs in DENV genome.
RNA disruption at UTRs via RNAi can significantly hinder viral replication and this can lead to reduce and faulty genome replication as well as protein synthesis.
Experiments conducted on DENV3 in Vero-81 cells demonstrated that synthetic siRNAs can effectively inhibit DENV3 and proved to be an efficient molecular tool for development of anti-dengue inhibitor. Although there are concerns regarding short half life of siRNAs and targeted delivery, however scientists are developing new technologies to overcome these issues. Yuen et al were able to achieve targeted delivery via combining nanotechnology and liposomes, this approach was successful in vivo models. Futures studies involving siRNAs designed in this study will elucidate the role of molecular medicine against DENV.
Previously most RNAi approaches were based on random targeting of DENV genome as well as host factors associated with entry and replication of DENV. This approach was useful for inhibiting DENV however there was still need for identifying highly conserved regions that can be efficiently targeted for development of lasting inhibitors. siRNAs were designed from conserved regions located in DENV2 genome for inhibition studies. These conserved regions were selected for persistent effectiveness of
RNAi, as other regions could undergo mutations which can render the siRNAsineffective.
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This information regarding DENV2conserved regions would help future endeavors in the anti-dengue drug development and vaccine production.
Most of the previous research concerning DENV therapeutics and vaccine formulation was conducted by utilizing DENVs propagated in C6/36 cells. That is why
C6/36 cells are most abundantly used cell line for DENV propagation, however progeny
DENVs are a diverse population of immature and mature DENV due to inefficient Furin related conversion of prMto M (Richter et al., 2014; Zybert et al., 2008). This mixed population is later on used for anti-dengue therapeutic testing as well as neutralization studies involving vaccine candidates.
Dendritic cells (DCs) are important component of our immune system and provide immunity against several pathogens however these DCs serve as target sites for
DENV replication (Marovich et al., 2001). DENV can effectively replicate in DCs, consequently one of the study was specifically aimed for targeting DENVs in infected
DCs. A complex of peptide and siRNA was introduced in infected DCs for targeting
DENV E gene; this peptide and nucleic acid fusion significantly decrease DENV replication. This fusion complex was also tested in mice models grafted with human cells results showed that this complex was able to knock down DENV and DENV challenged mice survived (Subramanya et al., 2010).Although studies have shown that immature and mature DENVs are infectious and both respond similarly to anti viral therapeutics(Zybert et al., 2008). However, in human body replication of DENV mostly occurs in DCs yielding mostly mature virions. Therefore, to maximize the efficacy of siRNA for future in vivo studies experiments were done on mature DENVs. Mature DENV2s were propagated in U937 DC-SIGN cells; these cells had adequate Furin to yield higher
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proportion of mature DENV2. To confirm that the population of DENV2s derived from
DC SIGNS was mostly mature western blot was performed for detection of prMprotein, samples showing insignificant levels of prM were selected for further experimentations.
Mature DENV2 were targeted by six siRNAs designed from UTRs (3' and 5'), these regions were highly conserved and had central role in DENV replication and assembly. Results revealed that DENV2SsiRNA2 was able to reduce DENV2 significantly, 85-90% reduction in DENV2titer was observed in cells transfected by
DENV2SsiRNA2. Promising results were also shown by DENV2UTR3'siRNA2 which was able to drop DENV2 titer up to 55%. However remaining four siRNAs did not had any significant effect on DENV2 titers, foci counts revealed that these four siRNAs had similar number in comparison with control groups. Thus only two siRNAs
(DENV2SsiRNA2 and DENV2UTR3'siRNA2 were effective in limiting DENV replication. These results show that mature DENV2 can be effectively targeted by RNAi, with appropriate delivery mechanism and sustained release RNAi can be an effective anti-viral therapeutic agent in future.
Currently several studies are being conducted for development of therapeutic agents utilizing RNAi technology but several factors can lead to reduced efficiency of these siRNA derived approaches. Some of these include short half life and a swift degradation in the cells. This rapid unavailability can lead to recurrence as well as ineffectiveness in complete knock out of pathogen. One of the major concern regarding siRNAtherapy is targeted delivery because it is vital for siRNA to reach target cells to knock out gene of interest. For targeted delivery researchers are focusing on liposome
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and nanotechnology these approaches have yielded promising results and several of these highly specific siRNAs have made it to clinical trials.
Cross-protective tetravalent vaccine with a constant immune can be a major breakthrough against DF/DHF, since a protective vaccine employed before the onset of rainy season will ensure safety of the general population. Numerous attempts for reaching a DENVvaccine have resulted in failure because of multiple factors including cross- reactivity amongst serotypes (Mahalingam et al., 2013). Latest approaches in vaccine development like DNA vaccines and sub-unit vaccine proved to be useful and many of these vaccinal candidates are in clinical trial phases (Kanesa-Thasan et al., 2003).
Tetravalent vaccines can also provide protection against all four serotypes. These approaches exploiting both virus and host targets can ultimately lead to the development of DENV inhibitors and vaccines as treatment and prophylactic options.
Dengue virus has three structural genes which are essential for viral stability and interaction with host cell via receptor mediated endocytosis. In humans majority of antibodies are produced against E protein, as E is highly immunogenic that is why it is one of the major component of vaccine strategies (Lin et al., 2012).DENV structural proteins play a key role in binding with host cell receptors as well as maintaining virus integrity. Although all three proteins are immunogenic however, majority of antibodies are produced against E protein. Antibodies are also generated against C and prM but studies have revealed that several prM antibodies are cross-reactive weakly neutralizing.
Two types are of antibodies are produced against DENV structural proteins strongly neutralizing antibodies predominantly targeting the structural region and cross reactive weakly neutralizing antibodies generated against the prM/M regions
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(Dejnirattisai et al., 2010b). Thus, structural proteins play a central role in maintaining the viral integrity and targeting structural genes will result in dysfunctional virion production lacking stability and infectivity. Therefore, a vaccine candidate should have only those eptitopes which can generate strongly neutralizing antibodies for providing maximum protection against DENV. A perfect vaccine should provide cross protection against all serotypes from every corner of globe. With the discovery of fifth DENV serotype and several regional variants the importance of phylogenetics has boosted tremendously, there is an urgent need for evaluating these changes for reaching a global
DENV vaccine.
For evaluating the linkages of Pakistani DENV2 structural genes with worldwide sequences insilico study was performed. Phylogenetic trees revealed that Pakistani
DENV2 is similar to Indian DENV2 strain which points to the fact that geographical proximity can result in the spread of infections in related populations. In 2010 outbreak, thousands of cases were reported in the city of Lahore which is located near the Indian border and several hundred people travel across both countries via Lahore, which could have led to the simultaneously outbreak in both countries. Moreover, this data will help future researchers working on the development of universal DENV vaccine to ensure that vaccine is equally effective for both countries of this region.
Antigenic sites located on structural proteins induce the host immune response, these include both B and T cell epitopes. However, most vaccines rely on humoral immune response for neutralizing of a specific pathogen for this reason B cell epitopes of
Pakistani DENV2 isolates were predicted in the current study. Generally there are two main reasons for B cell epitope prediction, for development of vaccine or a diagnostic kit.
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Viral infections can be diagnosed by using antibodies targeting antigenic domains of a pathogen. Small peptide based vaccines can be used for prophylaxis and curative purposes, as these peptides are highly antigenic in nature. By applying these technique problems like production of cross-reactive weakly neutralizing antibodies can be addressed without the possibility of enhancing infection. In the present study, insilico analysis comprising of features like inter-molecular contact, antigenicity, residue solvent accessibility, flexibility, surface accessibility and spatial distribution were performed on all three structural DENV2 proteins. By using these linear epitopes monoclonal neutralizing antibodies can be generated and thus result in the production of an effective
DENV vaccine.
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Chapter 6
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Research Papers
Papers Accepted/Published
UmmarRaheel and Najam Us SaharSadafZaidi (2014). Inhibition of dengue virus 3 in
mammalian cell culture by synthetic small interfering RNAs targeting highly conserved
sequences. Tropical J Pharmaceutical Research. 13(10): 1621-1627. (IF:1)
UmmarRaheel, Muhsin Jamal and NajamusSaharSadafZaidi (2015). A molecular
approach designed to limit the replication of mature DENV2 in host cells. Accepted in
Viral Immunology(IF:1.7)
UmmarRaheel, Muhammad Faheem, Muhammad NasirRiaz, NaghmanaKanwal,
FarakhJaved, NajamusSaharSadafZaidi, IshtiaqQadri.Dengue fever in
IndianSubcontinent an overview. J Infect DevCtries. 2011 Apr 26;5(4):239-47. (IF: 1.2)
UmmarRaheel, Muhammad Faheem, Muhammad NasirRiaz,
HashaamAkhtarSadafZaidi, IshtiaqQadri.Dengue virus: Advances in therapeutic
approaches and vaccine development”World Scientific Journal” Dengue Edition. (IF:2.7)
Muhammad Faheem,UmmarRaheel, Muhammad NasirRiaz, NaghmanaKanwal,
FarakhJaved, NajamusSaharSadafZaidi, IshtiaqQadri. A Molecular Evaluation ofDengue
Virus Pathogenesis and Its Latest Vaccine Strategies. MolBiol Rep. 2011
Aug;38(6):3731-40. Epub 2010 Nov 24.(IF:2.1)
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