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Infection by dengue viruses is accounted as one of the major public health problems of more than 100 countries in tropical and subtropical areas. Each year, an estimated 50 million of infected cases and over 500,000 cases of dengue hemorrhagic fever and dengue shock syndrome or DHF/DSS were reported (1). Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Aedes aegypti and Aedes albopictus are used as a mosquito vectors. Dengue virus is a positive sense single stranded RNA and a member of the genus Flavivirus in the family Flaviviridae. Its genome is approximately 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 cause severe disease (3). Clinical manifestation of dengue infection is ranging from asymptomatic to symptomatic infection. The symptomatic infection may progress into febrile illness to dengue fever or to life-threatening disease (DHF/DSS) (4).
It is widely accepted that severity of dengue infection is determined by both viral factors and host factors. In the case of viral factors, the different strains of dengue virus were reported to cause the different clinical outcome. The well known evidence is studied in American genotype and Southeast Asian genotype of DEN-2 in which Southeast Asian genotype virus is found to associate with severe disease (DHF/DSS) while the American genotype virus can cause only mild disease (5, 6, 7). However, the outcome of dengue virus infection also depends on host factors due to the fact that severe form of dengue virus infection occurs in dengue immune individual more than in dengue non-immune individual. This evidence indicates that host immunity is very important. Several hypothesizes were used as the model to explain roles of host immunity on the severe disease in dengue infection and one of those hypothesis is antibody dependent enhancement (ADE). ADE infection is the phenomenon in which pre-existing antibody enhance viral replication rather than neutralizes the viruses (8). ADE occurs when virus-antibody complex binds to FcR on the FcR-bearing cells resulting in the increase of viral entry and viral production (9). Several viral pathogens can utilize ADE to facilitate the infection and to increase virus production. In Ross River virus (RRV), infection with subneutralizing antibody to RRV can suppress antiviral gene expression which help virus to replicate freely in macrophage cells, in vitro (10). Thus, ADE phenomenon becomes one of the problems for dengue vaccine development because cross-reactive antibody may enhance the viral load resulting in the increase of disease progression (11).
Disease severity in dengue virus infection positively correlates to the high viral load. Then, the question is, does ADE infection facilitate virus production. Halstead and colleague observed that most severity cases in dengue infection usually occur with the patient experiencing dengue virus infection (12). Moreover, they further demonstrated that infection with complexes between dengue virus and subneutralizing antibody from severe disease patients could enhance viraemia in Rhesus monkeys (13). A similar phenomenon has been demonstrated in an in vitro assay using FcR bearing cells (15,16,17,18). The enhancing mechanism is found to initiate at the interaction between FcγRI or FcγRII and virus-IgG complex (19, 20). This interaction stimulates negative regulators of intracellular innate immune response, thus, the first line of intracellular defense are suppressed (). This mechanism creates an appropriate biological environment for dengue replication resulting in increasing viral production. Recently, computational model based on epidemic theory suggested that ADE helps the dengue viruses spread faster than other co-circulating dengue viruses that did not experience enhancement (24). This evidence implies to us that ADE infection at least inpart may increase dengue virus fitness. However, the characterization of dengue virus that released from ADE infection has not been well studied. We hypothesize that infection via dengue virus-antibody complexes increase dengue virus fitness. Thus, several factors that determine viral fitness were compared between viruses produced from DENV-ADE infection and DENV non-immune serum infection.
The main objective of this thesis is to investigate that enhancing antibody could increase fitness of dengue viruses. Thus, this experiment is divided into two sub-objective which are to study the efficiency of replication of dengue viruses in ADE infection in comparison with those in DENV infection and to investigate the characteristic of dengue viruses produced from ADE infection versus viruses from DENV infection.
Dengue virus is the infectious agent which causes dengue fever and dengue hemorrhagic fever. Ades aegypti and Aedes albopictus mosquitoes are used as vectors in the transmission to human. Dengue virus has spherical virion which contains a positive sense single-stranded RNA approximately 11 Kb in length. The genome organization of dengue virus is 5'-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3'. A structural protein in mature dengue 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 domains which are domain I, domain II and domain III. The domain III of dengue virus plays role in the binding to receptor on permissive cells and induces humeral immune response during dengue infection (). Mutation in domain III of E protein is involved in the virulence of dengue virus. In vitro, amino acid substitution in at E 390 in domain III region was showed to decrease the replicative efficiency of dengue virus in monocyte-derived macrophage (). Similarly, amino acid substitution from Asp to His at E 390 was showed to increase neurovirulence in mice (). In addition to the envelope glycoprotein, dengue virus surface is also composed of membrane protein. In the immature particle, membrane proteins are present as PrM protein and then it is cleaved by host protease furin. The function of PrM protein is to protect E protein to fuse when immature particles are transported through the acidic environment of the trans-Golgi network in secretory pathway (). The capsid protein is essential for the maturation of viral particle and nucleocapsid formation which consists of the multiples copies of C protein surrounding a single viral RNA genome (). Moreover, dengue virus contains seven non-structural proteins which are NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 that involved in the dengue replication (). NS1 protein is not found in the viral particle instead it is released into extracellular during infection. NS1 protein has been reported to be cofactor for viral RNA replication and colocalization of double-strand RNA replicative form (6, 7). NS3 protein is essential for viral polyprotein processing, RNA replication, and capping of viral genomic RNA. The N-terminal region contains viral protease that requires the NS2B protein for protease activity to process the viral polyprotein. The C-terminal region serve as the RNA helicase/NTPase which responsible for unwinding a double-stranded RNA replicative form and RNA replicative intermediate during viral replication (17,18). The 5' triphosphatase activity of NS3 protein and NS5 methytranferase are involved in the capping of viral RNA (). NS5 protein has three domains which are the N-terminal S-adenosyl methionine methyltransferase (MTase) domain, nuclear localization sequence (NLS), and RNA dependent RNA polymerase domain (20). The N-terminal S-adenosyl methionine methyltransferase (MTase) domain is responsible for both guanine N-7 and ribose 2'-O methylations which are required for formation of viral RNA cap structure (21, 22). The nuclear localization sequences (NLS) is recognized by cellular factors to allow protein to transport into the nucleus (23). RNA dependent RNA polymerase is located in The C-terminal domain. The minus-strand RNA serves as a template for the RNA dependent RNA polymerase to synthesize plus-strand genomic RNA (24, 25). The precise function of NS2A, NS2B, NS4A and NS4B remains unclear. NS2A and NS4A are believed to associate in viral replication. NS2A was reported to bind the 3' untranslated region (UTR) of viral RNA and to the other component of replication complex (26). NS2B is necessary for NS3 protein to exhibit its proteolytic activity ().
Dengue virus replication
In order to replication, dengue virus can enter to host cell by receptor-mediated endocytosis. The acidification of the endosome allows the fusion of viral membrane to vesicular membrane resulting in releasing of viral genomic RNA into the cytoplasm (). The viral protein is then translated directly from the positive-sense RNA as a single polyprotein that is processing by viral and host proteases (). The negative-strand viral RNA is synthesized from positive-strand viral RNA and used as the template for the production of the viral genomic RNA (). The viral formation occurs in the endoplasmic reticulum to form the immature virion. Then, it is transported into trans-Golgi network (TGN) where it is cleaved by host protease furin to generate the mature and released out of the host cell by host secretory pathway ()
The diversity of dengue virus
Dengue virus is RNA virus which is the member of flavivirus genus of Flaviviridae family. There are four serotypes of dengue virus which are DEN-1, DEN-2, DEN-3, and DEN-4. All four serotypes of dengue virus can cause severe disease (3). Dengue virus most likely arises from sylvatic strain. The cycle of transmission exists in the forest of Asia and Africa between non-human primates and Aedes mosquitoes. Cross-species transmission from non-human primates to humans may occur due to the rapid increase in human population, the widespread urbanization, and the modern transportation. However, the mechanism of cross-species transmission remains unknown (4). The evolution of dengue virus started when DENV-4 was the first to diverge, followed by DEN-2, and the final spilt between DEN-1 and DEN-3. Splitting of DENV into 4 serotypes may be due to geographic partition or ecological partition in different primate populations so that the four serotypes evolved independently (5). In addition to divide dengue virus into four serotypes, each serotype of dengue virus can be branched in to several genotypes. Based on nucleotide sequences of the envelope (E) gene, 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 understanding of the global dispersion and evolutionary history of those genotypes remain incomplete (). Then, DEN-2 is best studied for genetic diversity. DEN-2 can be classified into 6 genotype that are American genotype, American/Asian genotype, Asian 1 genotype, Asian 2 genotype, and Cosmopolitan genotype. These genotypes often have different geographical distributions. For example, Cosmopolitan genotype has a distribution covering the tropical world, Asian 1 and Asian 2 genotypes are only found in Asian population while the American genotype disperses mainly in the Americas (). Two major factors are associated with genetic diversity of dengue viruses that are mutation and intra-serotypic recombination. Mutation occurs because viral RNA-dependent RNA polymerase lacks of proofreading mechanism and creates dynamic distributions of non-identical but closely related mutation genome or quasispecies (). Clonal sequencing of dengue viruses from plasma of patients reveal that dengue virus exist as a quesispecies in vivo (). All four serotypes of dengue virus have a mean substitution rate approximately 10-3 nucleotide substitutions per site per year (). Intra-serotypic recombination also arises because the polymerase enzyme switches between parental viral molecules during replication (). Analysis of dengue virus gene sequences of samples from patients was identified as recombination indicating that there were recombination within DEN-1 strain in natural population of dengue virus (). Recombination has been demonstrated in the other member of the Flavividae such as hepatitis C virus and pestiviruses ().
Clinical manifestation of dengue virus infection
Clinical manifestation of dengue virus infection can vary from asymptomatic to severe infection with bleeding and shock (DHF/DSS). The manifestation of symptomatic form of dengue infection can range from undifferentiated fever, dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Undifferentiated fever usually occurs in primary infection but may follow in secondary infection. Clinically, it is not difference from other viral infection. Dengue fever (DF) follows either primary or secondary infection. It is characterized by extreme fever, headache, retro-orbital pain, joint and muscular pain. A rash may also occur about three to four days after onset of the fever. Hemorrhagic manifestation is uncommon in dengue fever (1). However, hemorrhage can also sometime occur in dengue fever patient (2). Dengue hemorrhagic fever (DHF) is a severe form of dengue virus infection. It usually follows in secondary dengue infection but sometimes occur in primary infections. The illness of DHF normally starts with abruptly high fever accompanied by severe headaches aspecially in retro-orbital area, anorexia, facial flushing, acute abdominal pain, vomiting and other symptom similar to those DF. DHF is characterized by plasma leakage due to increasing of vascular permeability, thrombocytopenia, hemorrhage, and in the most severe case, shock. Dengue hemorrhagic fever is divided into four grades of severity. Grade I, hematologic manifestation is only positive tourniquet test. Grade II, it has spontaneous bleeding in the skin or from mucosal surfaces in addition to the manifestation of grade I. Dengue shock syndrome (DSS) refers to Grade III and Grade IV of DHF which shock is present. DHF with circulatory failure seen as pulse pressure and hypotension is DHF grade III. In DHF grade IV, there is profound circulatory collapse with an undetectable blood pressure and pulse. When the circulatory collapse is present, DHF is associated with high mortality (3, 4,5 ).
The epidemiology of dengue
Dengue is public health problem in many countries worldwide with more than 50 million cases of dengue infection and over 500,000 cases of DHF were reported (1). It is believed that a pandemic of dengue started from Southeast Asia after World War II. The movement of troops makes the ecology changed which contributes the distribution of the mosquito vector (2). The first outbreak of DHF occurred in Manila Philippines in 1954 and then spread to several countries in the Region. After that, Southeast Asian became the hyperendemic areas because there is co-circulating of four serotypes of dengue viruses (3). At present, the epidemic of dengue is reported in many countries such as Thailand, Indonesia, Sri Lanka, Vietnam, Singapore, India, and Myanmar (). Thailand is hyperendemic area of dengue viruses (4, 5, 6). DHF were first reported in 1958 which over 200 deaths were reported. After that, the large epidemic occurred in 1987 with 174,285cases and about 1,000 deaths. Currently (7), DF and DHF have been reported at least 10,000 cases per year and it has been leading cause of hospitalisation of children in Thailand. Moreover, four serotypes of dengue viruses have been isolated in DHF cases (8). In America, DHF cases were first reported in America region during the dengue epidemic in Cuba, 1981 which an estimated 350,000 cases of DHF and 150 deaths were reported (9). This outbreak caused by the introduction of Southeast Asian genotype of DEN-2 (10). After that, DHF were reported in many countries in Americas. In 1989, the second outbreak of DHF occurred in Venezuela with 3,108 cases of DHF and 73 deaths (11). The circulating serotypes of dengue virus in this outbreak were DEN-1, 2 and 4 (12). In 1994-1997, the large outbreak of dengue occurred in America regions. There were reported of DHF cases in many countries 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 situation in America regions is not different from those in Asia (17). Recently, a large outbreak occurred in Brazil with 120,570 cases and 647 of DHF were reported. The dominant circulating serotype is DEN-3 and DEN-2 (18). However, all four serotype of dengue virus have been reported to cause DHF worldwide. The incidence of DHF is more frequently found in patients with secondary infection than primary infection (19, 20). The increasing DHF/DSS cases have been reported in every year and no effective vaccines are available. Therefore, dengue vector control grogram and the pathogenesis studies are the urgent need to limit the dengue disease.
The clinical outcome of dengue virus infection depends on both host factors and viral factors. Several hypotheses have been proposed to explain the pathogenesis of DHF/DSS in dengue virus infection such as the virulence strains of dengue virus or the host immunity.
Differences strains of dengue virus were reported to cause differences clinical outcome of dengue virus infection. The virulence of dengue virus was observed in both epidemiological studies and molecular studies. The well known evidence was studied in American genotype viruses and Southeast Asian genotype viruses of dengue virus serotype 2. American genotype viruses are referred to low virulence while Southeast Asian genotype viruses associate high virulence. Before 1981, there was no report of DHF cases in America regions. In 1981, there was introduced of Southeast Asia genotype viruses which result in the DHF cases were first 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 cases of DHF were found with American genotype virus infection. This suggests that the American genotype did not cause dengue hemorrhagic fever and dengue shock syndrome (). Molecular studies also supported low virulence of American genotype viruses. Pryor et al investigated amino acid difference between American genotype and Southeast Asian genotype, they found that replicate efficiency of dengue virus was decreased in monocyte-derived macrophages when amino acid substitution occurs at E-390 (). Similarly, mutation at E-390, the 3' NTRs and 5' NTRs sequence of American genotype were introduced into of the Southeat Asian genotype virus resulting in decreasing virus output in cell culture ().
Several hypothesizes were used as the model to explain roles of host immunity on the severe disease in dengue infection and one of those hypothesis is antibody dependent enhancement (ADE). Antibody dependent enhancement (ADE) is formulated to explain the finding that severe manifestation of DHF/DSS occurs in patient in secondary dengue virus infection that has different serotype from the previous one. During heterotypic secondary dengue infection, preexisting antibody recognizes the infecting virus and forms an antigen-antibody complex, which is then bound to and internalized by immunoglobulin Fc receptor on bearing cells. This mechanism results in enhancing the entry of virus into the host cells and increase viral replication (). Several viral pathogens can utilize ADE to facilitate the infection and to increase virus production. Ross River virus infection (RRV), subneutralizing of anti-RRV IgG has been shown to enhance the infection of RRV monocyte and macrophage cells (). This enhancement is associated by Fc receptor. Based on molecular studied found that infection with subneutralizing antibody to RRV can suppress antiviral gene expression which contribute virus to replicate freely (). In HIV infection, enhancing antibody has been reported to increase HIV infection. The enhancement of HIV infection was demonstrated by both complement receptor and Fc receptor (). In addition enhancing antibody is also the major obstacle in vaccine development. In respiratory syncytial virus (RSV) vaccine was reported to increase when infection with enhancing antibody. In vitro studied demonstrated that the number of infected cells was increased when subneutralizing antibodies from animal immunized with the formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine were co-infected with RSV in monocyte cell lines (). In dengue virus infection, ADE was first described by Halstead and colleague when they found that most of severe cases in dengue infection usually occur in heterotypic secondary infection. They further demonstrated that infection with complexes between dengue virus and subneutralizing antibody from severe disease patients could enhance viraemia in Rhesus monkeys. ADE of dengue infection was also 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 cause lethal disease in mice. This suggests that dengue virus can use subneutralizing antibody to enhance 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 FcγRII was used as the mediator in ADE of dengue infection (). Similarly, FcγRI was also reported to mediate enhancement of dengue infection in monocyte cells. This result can suggest that The ADE mechanism of dengue infection is mediate by the interaction between FcR and virus-IgG complex. Moreover, ADE of dengue virus infection has affected to suppress innate immune which is the first line of intracellular defense mechanism. Based on molecular studied revealed that the interaction between FcR and virus-IgG complex have affected to stimulate DAK and Atg5-Atg12 that down-regulate of MDA-5 and RIG-1 activation resulting in inhibiting the type I IFN production ().This mechanism creates an appropriate biological environment for dengue replication resulting in increasing viral production. Recently, Cummings et al used computational model studied the impact of enhancing antibody in epidemiology of dengue viral serotypes. They found that ADE helps the dengue viruses spread faster than other co-circulating serotypes that did not experience enhancement. In addition to enhancing antibody, several host factors have been reported to determine the pathogenesis of dengue virus infection such as the memory T-cell response and storm of cytokines. In secondary infection, the expansion cross-reactive memory T cells which have high avidity to previous infection but low avidity to current infection result in delayed viral clearance ().The effect of antibody dependent enhancement leads to high viral load and increases antigen presentation. The interaction of antigen-resenting cell with memory T-cell induced proliferation and the production of proimflamatory cytokines such as IFNγ and TNFα. These cytokines can have direct effects to vascular endothelial cell. Moreover, it has been reported that the storm of cytokines in dengue infection could contribute of vascular leakage in DHF/DSS patients (). Several cytokines have been observed to increase in patient with DHF such as IL-10, IL-6, IL-8, TNF-α and IFN-γ. For examples, TNF-α and IL-6 could effect to increase the vascular permeability (). IL-10 levels has been correlated with platelet decay in dengue virus infection and may be down regulating lymphocyte and platelet function ().
Fitness is the parameter to define the replicative adaptation of organism in its environment (). In viral evolution, fitness is a parameter to dictate the survival of viruses when its environment is changed. Viruses gain fitness when a mutant can increase the opportunity to survive in the altered environment while some mutant can not survive resulting in fitness loss ().Several viral pathogens are RNA virus which has high mutation rate in their genome (). High mutation rate of RNA virus is caused by the lack of proofreading mechanism of RNA dependent RNA polymerase. This mechanism creates the heterogeneous population which called quasispecies and each population in quasispecies has different fitness (). Moreover, the quasispecies of viral pathogens are significantly problem in the medical treatment. In biological environment, host immune system and antiviral drug therapy are the important selective pressure to drive the evolution of virus in order to survive in its environment (). The quasispecies in HIV create antiviral drug resistance which has higher fitness than wild type resulting in problem in vaccine development and drug therapy (). Antigenic drift and antigenic shift in influenza A viruses can make virus less susceptible to immune response. The amino acid change in glycoprotein hemagglutinin (HA) region which is the target of neutralizing antibodies make virus evading from host immune system (). Recently, neuraminidase resistance mutants of influenza A viruses were observed in oseltamivir-treated patients which may increase the opportunity in the transmission (). High genetic diversity of Hepatitis C virus creates the problem in vaccine development. In chronic HCV infection, the genetic variation of HCV is higher than those in non-chronic infection (). Genetic variation of Hepatitis C virus also contributes virus to escape from host immune recognition. The hypervariable region HVR1 within E gene that is epitope site for neutralizing antibody has been reported to associate in outcome of infection. Based on studied in patients, high variation in HVR1 region is found in patient with progressing to chronic infection while non-chronic infection is significantly stable. In the case of dengue virus, indirect evidence indicates that the genetic variation contribute to increase fitness and to produce more virulent strain of dengue virus. Each viral strain could naturally differ in virulence. In the future, we might be exposed to viruses with an expanded range of pathogenic properties.