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Infection by dandy fever viruses is accounted as one of the major public wellness jobs of more than 100 states in tropical and semitropical countries. Each twelvemonth, an estimated 50 million of septic instances and over 500,000 instances of dandy fever hemorrhagic febrility and dandy fever daze syndrome or DHF/DSS were reported ( 1 ) . Dengue febrility and dandy fever hemorrhagic febrility are caused by dandy fever virus. Aedes aegypti and Aedes albopictus are used as a mosquito vectors. Dengue virus is a positive sense individual stranded RNA and a member of the genus Flavivirus in the household Flaviviridae. Its genome is about 11 Kb in length. The mature virion consists of three structural proteins and seven nonstructural proteins ( 2 ) . Dengue viruses are comprised of four serotypes ( DEN-1, 2, 3, and 4 ) which all four serotypes can do terrible disease ( 3 ) . Clinical manifestation of dengue infection is runing from symptomless to diagnostic infection. The diagnostic infection may come on into feverish unwellness to dengue febrility or to dangerous disease ( DHF/DSS ) ( 4 ) .

It is widely accepted that badness of dengue infection is determined by both viral factors and host factors. In the instance of viral factors, the different strains of dandy fever virus were reported to do the different clinical result. The well known grounds is studied in American genotype and Southeast Asian genotype of DEN-2 in which Southeast Asian genotype virus is found to tie in with terrible disease ( DHF/DSS ) while the American genotype virus can do merely mild disease ( 5, 6, 7 ) . However, the result of dandy fever virus infection besides depends on host factors due to the fact that terrible signifier of dandy fever virus infection occurs in dandy fever immune person more than in dengue non-immune person. This grounds indicates that host unsusceptibility is really of import. Several hypothesizes were used as the theoretical account to explicate functions of host unsusceptibility on the terrible disease in dengue infection and one of those hypothesis is antibody dependent sweetening ( ADE ) . ADE infection is the phenomenon in which preexistent antibody heighten viral reproduction instead than neutralizes the viruses ( 8 ) . ADE occurs when virus-antibody complex binds to FcR on the FcR-bearing cells ensuing in the addition of viral entry and viral production ( 9 ) . Several viral pathogens can use ADE to ease the infection and to increase virus production. In Ross River virus ( RRV ) , infection with subneutralizing antibody to RRV can stamp down antiviral cistron look which help virus to retroflex freely in macrophage cells, in vitro ( 10 ) . Therefore, ADE phenomenon becomes one of the jobs for dengue vaccinum development because cross-reactive antibody may heighten the viral burden ensuing in the addition of disease patterned advance ( 11 ) .

Disease badness in dandy fever virus infection positively correlates to the high viral burden. Then, the inquiry is, does ADE infection facilitate virus production. Halstead and co-worker observed that most badness instances in dengue infection normally occur with the patient sing dengue virus infection ( 12 ) . Furthermore, they farther demonstrated that infection with composites between dandy fever virus and subneutralizing antibody from terrible disease patients could heighten viremia in Rhesus monkeys ( 13 ) . A similar phenomenon has been demonstrated in an in vitro assay utilizing FcR bearing cells ( 15,16,17,18 ) . The heightening mechanism is found to originate at the interaction between Fc?RI or Fc?RII and virus-IgG composite ( 19, 20 ) . This interaction stimulates negative regulators of intracellular innate immune response, therefore, the first line of intracellular defence are suppressed ( ) . This mechanism creates an appropriate biological environment for dandy fever reproduction ensuing in increasing viral production. Recently, computational theoretical account based on epidemic theory suggested that ADE helps the dandy fever viruses spread faster than other co-circulating dandy fever viruses that did non see sweetening ( 24 ) . This grounds implies to us that ADE infection at least inpart may increase dandy fever virus fittingness. However, the word picture of dandy fever virus that released from ADE infection has non been good studied. We hypothesize that infection via dengue virus-antibody composites increase dandy fever virus fittingness. Therefore, several factors that determine viral fittingness were compared between viruses produced from DENV-ADE infection and DENV non-immune serum infection.

Chapter II

OBJECTIVEES

The chief aim of this thesis is to look into that heightening antibody could increase fittingness of dandy fever viruses. Therefore, this experiment is divided into two sub-objective which are to analyze the efficiency of reproduction of dandy fever viruses in ADE infection in comparing with those in DENV infection and to look into the feature of dandy fever viruses produced from ADE infection versus viruses from DENV infection.

Chapter III

LITERATURE REVIEW

Dengue virus

Dengue virus is the infective agent which causes dandy fever febrility and dandy fever hemorrhagic febrility. Ades aegypti and Aedes albopictus mosquitoes are used as vectors in the transmittal to human. Dengue virus has spherical virion which contains a positive sense single-stranded RNA about 11 Kb in length. The genome organisation of dandy fever virus is 5′-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3 ‘ . A structural protein in mature dandy fever virus consists of envelope glycoprotein ( E ) , membrane protein ( M ) and capsid protein ( C ) . The envelop glycoprotein and membrane protein are located on the outer surface of virion. The envelope glycoprotein comprises of three spheres which are sphere I, domain II and domain III. The sphere III of dandy fever virus plays function in the binding to receptor on permissive cells and induces humeral immune response during dengue infection ( ) . Mutant in sphere III of E protein is involved in the virulency of dandy fever virus. In vitro, aminic acerb permutation in at E 390 in sphere III part was showed to diminish the replicative efficiency of dandy fever virus in monocyte-derived macrophage ( ) . Similarly, aminic acerb permutation from Asp to His at E 390 was showed to increase neurovirulence in mice ( ) . In add-on to the envelope glycoprotein, dandy fever virus surface is besides composed of membrane protein. In the immature atom, membrane proteins are present as PrM protein and so it is cleaved by host peptidase furin. The map of PrM protein is to protect E protein to blend when immature atoms are transported through the acidic environment of the trans-Golgi web in secretory tract ( ) . The mirid bug protein is indispensable for the ripening of viral atom and nucleocapsid formation which consists of the multiples transcripts of C protein environing a individual viral RNA genome ( ) . Furthermore, dandy fever virus contains seven non-structural proteins which are NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 that involved in the dandy fever reproduction ( ) . NS1 protein is non found in the viral atom alternatively it is released into extracellular during infection. NS1 protein has been reported to be cofactor for viral RNA reproduction and colocalization of double-strand RNA replicative signifier ( 6, 7 ) . NS3 protein is indispensable for viral polyprotein processing, RNA reproduction, and capping of viral genomic RNA. The N-terminal part contains viral peptidase that requires the NS2B protein for peptidase activity to treat the viral polyprotein. The C-terminal part service as the RNA helicase/NTPase which responsible for wind offing a double-stranded RNA replicative signifier and RNA replicative intermediate during viral reproduction ( 17,18 ) . The 5 ‘ triphosphatase activity of NS3 protein and NS5 methytranferase are involved in the capping of viral RNA ( ) . NS5 protein has three spheres which are the N-terminal S-adenosyl methionine methyltransferase ( MTase ) sphere, atomic localisation sequence ( NLS ) , and RNA dependent RNA polymerase sphere ( 20 ) . The N-terminal S-adenosyl methionine methyltransferase ( MTase ) sphere is responsible for both guanine N-7 and ribose 2’-O methylations which are required for formation of viral RNA cap construction ( 21, 22 ) . The atomic localisation sequences ( NLS ) is recognized by cellular factors to let protein to transport into the karyon ( 23 ) . RNA dependent RNA polymerase is located in The C-terminal sphere. The minus-strand RNA serves as a templet for the RNA dependant RNA polymerase to synthesise plus-strand genomic RNA ( 24, 25 ) . The precise map of NS2A, NS2B, NS4A and NS4B remains ill-defined. NS2A and NS4A are believed to tie in in viral reproduction. NS2A was reported to adhere the 3 ‘ untranslated part ( UTR ) of viral RNA and to the other constituent of reproduction composite ( 26 ) . NS2B is necessary for NS3 protein to exhibit its proteolytic activity ( ) .

Dengue virus reproduction

In order to reproduction, dengue virus can come in to host cell by receptor-mediated endocytosis. The acidification of the endosome allows the merger of viral membrane to vesicular membrane ensuing in releasing of viral genomic RNA into the cytol ( ) . The viral protein is so translated straight from the positive-sense RNA as a individual polyprotein that is treating by viral and host peptidases ( ) . The negative-strand viral RNA is synthesized from positive-strand viral RNA and used as the templet for the production of the viral genomic RNA ( ) . The viral formation occurs in the endoplasmic Reticulum to organize the immature virion. Then, it is transported into trans-Golgi web ( TGN ) where it is cleaved by host peptidase furin to bring forth the mature and released out of the host cell by host secretory tract ( )

The diverseness of dandy fever virus

Dengue virus is RNA virus which is the member of flavivirus genus of Flaviviridae household. There are four serotypes of dandy fever virus which are DEN-1, DEN-2, DEN-3, and DEN-4. All four serotypes of dandy fever virus can do terrible disease ( 3 ) . Dengue virus most likely arises from sylvatic strain. The rhythm of transmittal exists in the wood of Asia and Africa between non-human Primatess and Aedes mosquitoes. Cross-species transmittal from non-human Primatess to worlds may happen due to the rapid addition in human population, the widespread urbanisation, and the modern transit. However, the mechanism of cross-species transmittal remains unknown ( 4 ) . The development of dandy fever virus started when DENV-4 was the first to diverge, followed by DEN-2, and the concluding spilt between DEN-1 and DEN-3. Splitting of DENV into 4 serotypes may be due to geographic divider or ecological divider in different archpriest populations so that the four serotypes evolved independently ( 5 ) . In add-on to split dandy fever virus into four serotypes, each serotype of dandy fever virus can be branched in to several genotypes. Based on nucleotide sequences of the envelope ( E ) cistron, DEN-1 viruses have been divided into two genotypes, DEN-3 viruses into five genotypes, DEN-4 viruses into one genotype and DEN-2 viruses into six genotypes ( ) . However, the apprehension of the planetary scattering and evolutionary history of those genotypes remain uncomplete ( ) . Then, DEN-2 is best studied for familial diverseness. DEN-2 can be classified into 6 genotype that are American genotype, American/Asian genotype, Asiatic 1 genotype, Asiatic 2 genotype, and Cosmopolitan genotype. These genotypes frequently have different geographical distributions. For illustration, Cosmopolitan genotype has a distribution covering the tropical universe, Asiatic 1 and Asiatic 2 genotypes are merely found in Asiatic population while the American genotype disperses chiefly in the Americas ( ) . Two major factors are associated with familial diverseness of dandy fever viruses that are mutant and intra-serotypic recombination. Mutant occurs because viral RNA-dependent RNA polymerase deficiencies of proofreading mechanism and creates dynamic distributions of non-identical but closely related mutant genome or quasispecies ( ) . Clonal sequencing of dandy fever viruses from plasma of patients reveal that dandy fever virus exist as a quesispecies in vivo ( ) . All four serotypes of dandy fever virus have a average permutation rate about 10-3 nucleotide permutations per site per twelvemonth ( ) . Intra-serotypic recombination besides arises because the polymerase enzyme switches between parental viral molecules during reproduction ( ) . Analysis of dandy fever virus cistron sequences of samples from patients was identified as recombination bespeaking that there were recombination within DEN-1 strain in natural population of dandy fever virus ( ) . Recombination has been demonstrated in the other member of the Flavividae such as hepatitis C virus and pestiviruses ( ) .

Clinical manifestation of dandy fever virus infection

Clinical manifestation of dandy fever virus infection can change from symptomless to terrible infection with hemorrhage and daze ( DHF/DSS ) . The manifestation of diagnostic signifier of dengue infection can run from uniform febrility, dandy fever febrility, dandy fever hemorrhagic febrility and dandy fever daze syndrome. Undifferentiated fever normally occurs in primary infection but may follow in secondary infection. Clinically, it is non difference from other viral infection. Dengue febrility ( DF ) follows either primary or secondary infection. It is characterized by utmost febrility, concern, retro-orbital hurting, articulation and muscular hurting. A roseola may besides happen about three to four yearss after oncoming of the febrility. Hemorrhagic manifestation is uncommon in dandy fever febrility ( 1 ) . However, bleeding can besides sometime occur in dandy fever febrility patient ( 2 ) . Dengue hemorrhagic febrility ( DHF ) is a terrible signifier of dandy fever virus infection. It normally follows in secondary dandy fever infection but sometimes occur in primary infections. The unwellness of DHF usually starts with suddenly high febrility accompanied by terrible concerns aspecially in retro-orbital country, anorexia, facial flushing, acute abdominal hurting, emesis and other symptom similar to those DF. DHF is characterized by plasma escape due to increasing of vascular permeableness, thrombopenia, bleeding, and in the most terrible instance, daze. Dengue hemorrhagic febrility is divided into four classs of badness. Grade I, haematological manifestation is merely positive tourniquet trial. Grade II, it has self-generated shed blooding in the tegument or from mucosal surfaces in add-on to the manifestation of class I. Dengue daze syndrome ( DSS ) refers to Rate III and Grade IV of DHF which daze is present. DHF with circulative failure seen as pulse force per unit area and hypotension is DHF class III. In DHF class IV, there is profound circulatory prostration with an undetectable blood force per unit area and pulsation. When the circulatory prostration is present, DHF is associated with high mortality ( 3, 4,5 ) .

The epidemiology of dandy fever

Dengue is public wellness job in many states worldwide with more than 50 million instances of dengue infection and over 500,000 instances of DHF were reported ( 1 ) . It is believed that a pandemic of dandy fever started from Southeast Asia after World War II. The motion of military personnels makes the ecology changed which contributes the distribution of the mosquito vector ( 2 ) . The first eruption of DHF occurred in Manila Philippines in 1954 and so distribute to several states in the Region. After that, Southeast Asian became the hyperendemic countries because there is co-circulating of four serotypes of dandy fever viruses ( 3 ) . At present, the epidemic of dandy fever is reported in many states such as Thailand, Indonesia, Sri Lanka, Vietnam, Singapore, India, and Myanmar ( ) . Thailand is hyperendemic country of dandy fever viruses ( 4, 5, 6 ) . DHF were foremost reported in 1958 which over 200 deceases were reported. After that, the big epidemic occurred in 1987 with 174,285cases and about 1,000 deceases. Currently ( 7 ) , DF and DHF have been reported at least 10,000 instances per twelvemonth and it has been taking cause of hospitalization of kids in Thailand. Furthermore, four serotypes of dandy fever viruses have been isolated in DHF instances ( 8 ) . In America, DHF instances were foremost reported in America part during the dengue epidemic in Cuba, 1981 which an estimated 350,000 instances of DHF and 150 deceases were reported ( 9 ) . This eruption caused by the debut of Southeast Asian genotype of DEN-2 ( 10 ) . After that, DHF were reported in many states in Americas. In 1989, the 2nd eruption of DHF occurred in Venezuela with 3,108 instances of DHF and 73 deceases ( 11 ) . The go arounding serotypes of dandy fever virus in this eruption were DEN-1, 2 and 4 ( 12 ) . In 1994-1997, the big eruption of dandy fever occurred in America parts. There were reported of DHF instances in many states of America after the re-introduction of new strain of DEN-3 which caused of DHF epidemics in Sri Lanka and India in the 1980 ( 13, 14, 15, 16 ) . At present, dengue epidemic state of affairs in America parts is non different from those in Asia ( 17 ) . Recently, a big eruption occurred in Brazil with 120,570 instances and 647 of DHF were reported. The dominant circulating serotype is DEN-3 and DEN-2 ( 18 ) . However, all four serotype of dandy fever virus have been reported to do DHF worldwide. The incidence of DHF is more often found in patients with secondary infection than primary infection ( 19, 20 ) . The increasing DHF/DSS instances have been reported in every twelvemonth and no effectual vaccinums are available. Therefore, dengue vector control grogram and the pathogenesis surveies are the pressing demand to restrict the dandy fever disease.

The pathogenesis

The clinical result of dandy fever virus infection depends on both host factors and viral factors. Several hypotheses have been proposed to explicate the pathogenesis of DHF/DSS in dandy fever virus infection such as the virulency strains of dandy fever virus or the host unsusceptibility.

Viral factors

Differences strains of dandy fever virus were reported to do differences clinical result of dandy fever virus infection. The virulency of dandy fever virus was observed in both epidemiological surveies and molecular surveies. The well known grounds was studied in American genotype viruses and Southeast Asian genotype viruses of dandy fever virus serotype 2. American genotype viruses are referred to low virulency while Southeast Asian genotype viruses associate high virulency. Before 1981, there was no study of DHF instances in America parts. In 1981, there was introduced of Southeast Asia genotype viruses which result in the DHF instances were foremost reported in Cuba ( ) . Then, Halstead et Al. studied the serum of samples from patient before and after the epidemic in Peru, 1995. They found that in secondary infection no instances of DHF were found with American genotype virus infection. This suggests that the American genotype did non do dandy fever hemorrhagic febrility and dandy fever daze syndrome ( ) . Molecular surveies besides supported low virulency of American genotype viruses. Pryor et al investigated amino acid difference between American genotype and Southeast Asian genotype, they found that replicate efficiency of dandy fever virus was decreased in monocyte-derived macrophages when amino acid permutation occurs at E-390 ( ) . Similarly, mutant at E-390, the 3 ‘ NTRs and 5 ‘ NTRs sequence of American genotype were introduced into of the Southeat Asian genotype virus ensuing in diminishing virus end product in cell civilization ( ) .

Host factors

Several hypothesizes were used as the theoretical account to explicate functions of host unsusceptibility on the terrible disease in dengue infection and one of those hypothesis is antibody dependent sweetening ( ADE ) . Antibody dependent sweetening ( ADE ) is formulated to explicate the determination that terrible manifestation of DHF/DSS occurs in patient in secondary dandy fever virus infection that has different serotype from the old 1. During heterotypic secondary dandy fever infection, preexisting antibody recognizes the infecting virus and signifiers an antigen-antibody composite, which is so bound to and internalized by Ig Fc receptor on bearing cells. This mechanism consequences in heightening the entry of virus into the host cells and increase viral reproduction ( ) . Several viral pathogens can use ADE to ease the infection and to increase virus production. Ross River virus infection ( RRV ) , subneutralizing of anti-RRV IgG has been shown to heighten the infection of RRV monocyte and macrophage cells ( ) . This sweetening is associated by Fc receptor. Based on molecular studied found that infection with subneutralizing antibody to RRV can stamp down antiviral cistron look which contribute virus to retroflex freely ( ) . In HIV infection, heightening antibody has been reported to increase HIV infection. The sweetening of HIV infection was demonstrated by both complement receptor and Fc receptor ( ) . In add-on heightening antibody is besides the major obstruction in vaccinum development. In respiratory syncytial virus ( RSV ) vaccinum was reported to increase when infection with heightening antibody. In vitro studied demonstrated that the figure of septic cells was increased when subneutralizing antibodies from carnal immunized with the formalin-inactivated ( FI ) respiratory syncytial virus ( RSV ) vaccinum were co-infected with RSV in monocyte cell lines ( ) . In dengue virus infection, ADE was foremost described by Halstead and co-worker when they found that most of terrible instances in dengue infection normally occur in heterotypic secondary infection. They farther demonstrated that infection with composites between dandy fever virus and subneutralizing antibody from terrible disease patients could heighten viremia in Rhesus monkeys. ADE of dandy fever infection was besides investigated in mice when infection with subneutralizing serotype-specific antibody or subneutralizing serotype-cross-reactive antibody to mice. They found that both subneutralizing serotype-specific antibody and subneutralizing serotype-cross-reactive antibody can do deadly disease in mice. This suggests that dandy fever virus can utilize subneutralizing antibody to heighten infection. In vitro, dengue infection with diluted serum and monoclonal antibody has been demonstrated to increase viral production in several cell type such as monocytes, macrophages and dendritic cells ( ) . Littaua R et Al found that Fc?RII was used as the go-between in ADE of dandy fever infection ( ) . Similarly, Fc?RI was besides reported to intercede sweetening of dengue infection in monocyte cells. This consequence can propose that The ADE mechanism of dengue infection is mediate by the interaction between FcR and virus-IgG composite. Furthermore, ADE of dandy fever virus infection has affected to stamp down innate immune which is the first line of intracellular defence mechanism. Based on molecular studied revealed that the interaction between FcR and virus-IgG composite have affected to excite DAK and Atg5-Atg12 that down-regulate of MDA-5 and RIG-1 activation ensuing in suppressing the type I IFN production ( ) .This mechanism creates an appropriate biological environment for dandy fever reproduction ensuing in increasing viral production. Recently, Cummings et Al used computational theoretical account studied the impact of heightening antibody in epidemiology of dengue viral serotypes. They found that ADE helps the dandy fever viruses spread faster than other co-circulating serotypes that did non see sweetening. In add-on to heightening antibody, several host factors have been reported to find the pathogenesis of dandy fever virus infection such as the memory T-cell response and storm of cytokines. In secondary infection, the enlargement cross-reactive memory T cells which have high eagerness to old infection but low eagerness to current infection consequence in delayed viral clearance ( ) .The consequence of antibody dependent sweetening leads to high viral burden and increases antigen presentation. The interaction of antigen-resenting cell with memory T-cell induced proliferation and the production of proimflamatory cytokines such as IFN? and TNF? . These cytokines can hold direct effects to vascular endothelial cell. Furthermore, it has been reported that the storm of cytokines in dengue infection could lend of vascular escape in DHF/DSS patients ( ) . Several cytokines have been observed to increase in patient with DHF such as IL-10, IL-6, IL-8, TNF-? and IFN-? . For illustrations, TNF-? and IL-6 could consequence to increase the vascular permeableness ( ) . IL-10 degrees has been correlated with thrombocyte decay in dandy fever virus infection and may be down modulating lymph cell and thrombocyte map ( ) .

Viral fittingness

Fitness is the parametric quantity to specify the replicative version of being in its environment ( ) . In viral development, fittingness is a parametric quantity to order the endurance of viruses when its environment is changed. Viruss gain fittingness when a mutation can increase the chance to last in the altered environment while some mutation can non last ensuing in fittingness loss ( ) .Several viral pathogens are RNA virus which has high mutant rate in their genome ( ) . High mutant rate of RNA virus is caused by the deficiency of proofreading mechanism of RNA dependent RNA polymerase. This mechanism creates the heterogenous population which called quasispecies and each population in quasispecies has different fittingness ( ) . Furthermore, the quasispecies of viral pathogens are significantly job in the medical intervention. In biological environment, host immune system and antiviral drug therapy are the of import selective force per unit area to drive the development of virus in order to last in its environment ( ) . The quasispecies in HIV create antiviral drug opposition which has higher fittingness than wild type ensuing in job in vaccinum development and drug therapy ( ) . Antigenic impetus and antigenic displacement in grippe A viruses can do virus less susceptible to immune response. The amino acid alteration in glycoprotein hemagglutinin ( HA ) part which is the mark of neutralizing antibodies make virus hedging from host immune system ( ) . Recently, neuraminidase opposition mutations of grippe A viruses were observed in oseltamivir-treated patients which may increase the chance in the transmittal ( ) . High familial diverseness of Hepatitis C virus creates the job in vaccinum development. In chronic HCV infection, the familial fluctuation of HCV is higher than those in non-chronic infection ( ) . Familial fluctuation of Hepatitis C virus besides contributes virus to get away from host immune acknowledgment. The hypervariable part HVR1 within E cistron that is epitope site for neutralizing antibody has been reported to tie in in result of infection. Based on studied in patients, high fluctuation in HVR1 part is found in patient with come oning to chronic infection while non-chronic infection is significantly stable. In the instance of dandy fever virus, indirect grounds indicates that the familial fluctuation contribute to increase fittingness and to bring forth more deadly strain of dandy fever virus. Each viral strain could of course differ in virulency. In the hereafter, we might be exposed to viruses with an expanded scope of infective belongingss.

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