Showing papers on "Dengue virus published in 2003"

A Ligand-Binding Pocket in the Dengue Virus Envelope Glycoprotein.

Abstract: Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a β-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.

DC-SIGN (CD209) Mediates Dengue Virus Infection of Human Dendritic Cells

Abstract: Dengue virus is a single-stranded, enveloped RNA virus that productively infects human dendritic cells (DCs) primarily at the immature stage of their differentiation. We now find that all four serotypes of dengue use DC-SIGN (CD209), a C-type lectin, to infect dendritic cells. THP-1 cells become susceptible to dengue infection after transfection of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), or its homologue L-SIGN, whereas the infection of dendritic cells is blocked by anti–DC-SIGN antibodies and not by antibodies to other molecules on these cells. Viruses produced by dendritic cells are infectious for DC-SIGN– and L-SIGN–bearing THP-1 cells and other permissive cell lines. Therefore, DC-SIGN may be considered as a new target for designing therapies that block dengue infection.

Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever.

Abstract: Dengue virus presents a growing threat to public health in the developing world. Four major serotypes of dengue virus have been characterized, and epidemiological evidence shows that dengue hemorrhagic fever (DHF), the more serious manifestation of the disease, occurs more frequently upon reinfection with a second serotype. We have studied dengue virus–specific T-cell responses in Thai children. During acute infection, few dengue-responsive CD8+ T cells were recovered; most of those present showed an activated phenotype and were undergoing programmed cell death. Many dengue-specific T cells were of low affinity for the infecting virus and showed higher affinity for other, probably previously encountered strains. Profound T-cell activation and death may contribute to the systemic disturbances leading to DHF, and original antigenic sin in the T-cell responses may suppress or delay viral elimination, leading to higher viral loads and increased immunopathology.

Inhibition of interferon signaling by dengue virus

Abstract: Dengue virus is a worldwide-distributed mosquito-borne flavivirus with a positive strand RNA genome. Its transcribed polyprotein is cleaved by host- and virus-encoded peptidases into 10 proteins, some of which are of unknown function. Although dengue virus-infected cells seem to be resistant to the antiviral action of IFN, the viral products that mediate this resistance are unknown. Therefore, we have analyzed the ability of the 10 dengue virus-encoded proteins to antagonize the IFN response. We found that expression in human A549 cells of the dengue virus nonstructural proteins NS2A, NS4A, or NS4B enhances replication of an IFN-sensitive virus. Moreover, expression of NS4B and, to a lesser extent, of NS2A and NS4A proteins results in down-regulation of IFN-β-stimulated gene expression. Cells expressing NS4B or infected with dengue virus do not exhibit nuclear signal transducer and activator of transcription (STAT) 1 on treatment with IFN-β or IFN-γ, indicating that NS4B might be involved in blocking IFN signaling during dengue virus infections. This protein, encoded by a positive strand RNA virus, is implicated as an IFN-signaling inhibitor.

Dendritic‐cell‐specific ICAM3‐grabbing non‐integrin is essential for the productive infection of human dendritic cells by mosquito‐cell‐derived dengue viruses

Abstract: Dengue virus (DV) is a mosquito-borne flavivirus that causes haemorrhagic fever in humans. DV primarily targets immature dendritic cells (DCs) after a bite by an infected mosquito vector. Here, we analysed the interactions between DV and human-monocyte-derived DCs at the level of virus entry. We show that the DC-specific ICAM3-grabbing non-integrin (DC-SIGN) molecule, a cell-surface, mannose-specific, C-type lectin, binds mosquito-cell-derived DVs and allows viral replication. Conclusive evidence for the involvement of DC-SIGN in DV infection was obtained by the inhibition of viral infection by anti-DC-SIGN antibodies and by the soluble tetrameric ectodomain of DC-SIGN. Our data show that DC-SIGN functions as a DV-binding lectin by interacting with the DV envelope glycoprotein. Mosquito-cell-derived DVs may have differential infectivity for DC-SIGN-expressing cells. We suggest that the differential use of DC-SIGN by viral envelope glycoproteins may account for the immunopathogenesis of DVs.

Origin and Evolution of Japanese Encephalitis Virus in Southeast Asia

Abstract: Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.

Emergence and Global Spread of a Dengue Serotype 3, Subtype III Virus

Abstract: Over the past two decades, dengue virus serotype 3 (DENV-3) has caused unexpected epidemics of dengue hemorrhagic fever (DHF) in Sri Lanka, East Africa, and Latin America. We used a phylogenetic approach to evaluate the roles of virus evolution and transport in the emergence of these outbreaks. Isolates from these geographically distant epidemics are closely related and belong to DENV-3, subtype III, which originated in the Indian subcontinent. The emergence of DHF in Sri Lanka in 1989 correlated with the appearance there of a new DENV-3, subtype III variant. This variant likely spread from the Indian subcontinent into Africa in the 1980s and from Africa into Latin America in the mid-1990s. DENV-3, subtype III isolates from mild and severe disease outbreaks formed genetically distinct groups, which suggests a role for viral genetics in DHF.

Microevolution and virulence of dengue viruses.

Abstract: The evolution of dengue viruses has had a major impact on their virulence for humans and on the epidemiology of dengue disease around the world. Although antigenic and genetic differences in virus strains had become evident, it is mainly due to the lack of animal models of disease that has made it difficult to detect differences in virulence of dengue viruses. However, phylogenetic studies of many different dengue virus samples have led to the association between specific genotypes (within serotypes) and the presentation of more or less severe disease. Currently, dengue viruses can be classified as being of epidemiologically low, medium, or high impact; i.e., some viruses may remain in sylvatic cycles of little or low transmissibility to humans, others produce dengue fever (DF) only, and some genotypes have been associated with the potential to cause the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) in addition to DF. Although the factors that contribute to dengue virus epidemiology are complex, studies have suggested that specific viral structures may contribute to increased replication in human target cells and to increased transmission by the mosquito vector; however, the immune status and possibly the genetic background of the host are also determinants of virulence or disease presentation. As to the question of whether dengue viruses are evolving toward virulence as they continue to spread throughout the world, phylogenetic and epidemiological analyses suggest that the more virulent genotypes are now displacing those that have lower epidemiological impact; there is no evidence for the transmission of antigenically aberrant, new strains.

Serotype-specific dengue virus circulation and dengue disease in bangkok, thailand from 1973 to 1999

Abstract: Dengue virus circulation and association with epidemics and severe dengue disease were studied in hospitalized children with suspected dengue at the Queen Sirikit National Institute of Child Health in Bangkok, Thailand, from 1973 to 1999. Dengue serology was performed on all patients and viral isolation attempted on laboratory-confirmed patients. Acute dengue was diagnosed in 15,569 children and virus isolated from 4,846. DEN-3 was the most frequent serotype in primary dengue (49% of all isolates), DEN-2 in secondary and in dengue hemorrhagic fever (37% and 35%, respectively). The predominant dengue serotype varied by year: DEN-1 from 1990-92, DEN-2 from 1973-86 and 1988-89; DEN-3 in 1987 and 1995-99; and DEN-4 from 1993-94. Only DEN-3 was associated with severe outbreak years. Our findings illustrate the uniqueness of each serotype in producing epidemics and severe disease and underscore the importance of long-term surveillance of dengue serotypes in understanding the epidemiology of these viruses.

Antigenic structure of flavivirus proteins.

Abstract: The increased activity of Dengue virus in the tropical regions of the world and the recent movement of West Nile virus from the eastern to the western hemisphere emphasize the fact that vector-borne flaviviruses are medically important emerging infectious diseases These facts warrant continued efforts to decode all facets of flavivirus immunology This chapter reviews current understanding of the antigenic fine structure of flaviviral structural and nonstructural (NS) proteins and their involvement in B- and T-cell host responses The virion structural glycoprotein E elicits both virus-neutralizing antibodies and antiviral TH-cell responses Consistent with the current hypothesis of the MHC class I pathway of protein processing, immunodominant flaviviral TC-cell epitopes mainly reside on the NS proteins To prepare effective and inexpensive subunit vaccines, we will need to continue to better understand these structure–function relationships of flavivirus proteins

Inferring the rate and time-scale of dengue virus evolution

Abstract: Dengue is often referred to as an emerging disease because of the rapid increases in incidence and prevalence that have been observed in recent decades. To understand the rate at which genetic diversification occurs in dengue virus and to infer the time-scale of its evolution, we employed a maximum likelihood method that uses information about times of virus sampling to estimate the rate of molecular evolution in a large number of viral envelope (E) gene sequences and to place bounds around the dates of appearance of all serotypes and specific genotypes. Our analysis reveals that dengue virus generally evolves according to a molecular clock, although some serotype-specific and genotype-specific rate differences were observed, and that its origin is more recent than previously suggested, with the virus appearing approximately 1,000 years ago. Furthermore, we estimate that the zoonotic transfer of dengue from sylvatic (monkey) to sustained human transmission occurred between 125 and 320 years ago, that the current global genetic diversity in the four serotypes of dengue virus only appeared during the past century, and that the recent rise in genetic diversity can be loosely correlated both to human activities such as population growth, urbanization, and mass transport and to the emergence of dengue hemorrhagic fever as a major disease problem.

Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus.

Abstract: A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 107 PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

American genotype structures decrease dengue virus output from human monocytes and dendritic cells.

Abstract: The dengue virus type 2 structures probably involved in human virulence were previously defined by sequencing the complete genome of both American and Southeast (SE) Asian genotype templates in patient serum (K. C. Leitmeyer et al., J. Virol. 73:4738-4747, 1999). We have now evaluated the effects of introducing a mutation in the envelope glycoprotein (E) gene and/or replacement of 5'- and 3'-nontranslated regions on dengue virus replication in human primary cell cultures. A series of chimeric infectious clones were generated containing different combinations of American and SE Asian genotype sequences. Some of the chimeric viruses had altered plaque morphology in mammalian cells; however, they replicated at similar rates in mosquito cells as measured by quantitative reverse transcription-PCR and plaque assay. Although susceptibility to virus infection varied from donor to donor in experiments using human macrophage and dendritic cells, we were able to measure consistent differences in viral RNA output per infected cell. Using this measurement, we demonstrated that the chimeric virus containing the E mutation had a lower virus output compared to the parental infectious clone. A larger reduction in virus output was observed for the triple mutant and the wild-type, American genotype virus from which chimeric inserts were derived. It appears that the three changes function synergistically, although the E mutation alone gives a lower output compared to the 5'- and 3'-terminal mutations. The data suggest that these changes may be responsible for decreased dengue virus replication in human target cells and for virulence characteristics during infection.

A model of dengue fever

Abstract: Background Dengue is a disease which is now endemic in more than 100 countries of Africa, America, Asia and the Western Pacific. It is transmitted to the man by mosquitoes (Aedes) and exists in two forms: Dengue Fever and Dengue Haemorrhagic Fever. The disease can be contracted by one of the four different viruses. Moreover, immunity is acquired only to the serotype contracted and a contact with a second serotype becomes more dangerous.

Selection-driven evolution of emergent dengue virus.

Abstract: In the last four decades the incidence of dengue fever has increased 30-fold worldwide, and over half the world's population is now threatened with infection from one or more of four co-circulating viral serotypes (DEN-1 through DEN-4). To determine the role of viral molecular evolution in emergent disease dynamics, we sequenced 40% of the genome of 82 DEN-4 isolates collected from Puerto Rico over the 20 years since the onset of endemic dengue on the island. Isolates were derived from years with varying levels of DEN-4 prevalence. Over our sampling period there were marked evolutionary shifts in DEN-4 viral populations circulating in Puerto Rico; viral lineages were temporally clustered and the most common genotype at a particular sampling time often arose from a previously rare lineage. Expressed changes in structural genes did not appear to drive this lineage turnover, even though these regions include primary determinants of viral antigenic properties. Instead, recent dengue evolution can be attributed in part to positive selection on the nonstructural gene 2A (NS2A), whose functions may include replication efficiency and antigenicity. During the latest and most severe DEN-4 epidemic in Puerto Rico, in 1998, viruses were distinguished by three amino acid changes in NS2A that were fixed far faster than expected by drift alone. Our study therefore demonstrates viral genetic turnover within a focal population and the potential importance of adaptive evolution in viral epidemic expansion.

Antibodies from dengue patient sera cross-react with endothelial cells and induce damage

Abstract: Dengue virus infection causes a wide range of diseases from the mild febrile illness dengue fever to the life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and hemorrhagic syndrome are the clinical features associated with dengue infection, yet the mechanisms remain unclear. In this study, the cross-reactivity of dengue patient sera with endothelial cells was demonstrated. There were higher percentages of endothelial cells reactive with dengue hemorrhagic fever/dengue shock syndrome patient sera than those with dengue fever patient sera. The percentages of endothelial cells reactive with patient serum IgM were higher than those with IgG. Further studies showed that the endothelial cell binding activity was inhibited by pretreatment with dengue virus nonstructural protein 1 (NS1). The antibodies against NS1 produced after dengue virus infection may, at least in part, account for the cross-reactivity of patient sera with endothelial cells. Furthermore, dengue patient sera induced endothelial cell apoptosis via a caspase-dependent pathway that was also inhibited by NS1 pretreatment. In addition to apoptosis, patient sera caused cell lysis in the presence of complement, and DHF/DSS patient sera showed higher percentages of cytotoxicity than dengue fever patient sera. Thus, the generation of cross-reactive autoantibodies against endothelial cells would lead to their dysfunction, which may play a role in the pathogenesis of dengue virus infection.

Patterns of Intra- and Interhost Nonsynonymous Variation Reveal Strong Purifying Selection in Dengue Virus

Abstract: Considerable uncertainty surrounds the evolutionary rates of and selection pressures acting on arthropod-borne RNA viruses (arboviruses). In particular, it is unclear why arboviruses such as dengue virus show substantial genetic variation within individual humans and mosquitoes yet low long-term rates of amino acid substitution. To address this question, I compared patterns of nonsynonymous variation in populations of dengue virus sampled at different levels of evolutionary divergence. Although nonsynonymous variation was abundant in viral populations within individual humans, there was a marked reduction in the frequency of nonsynonymous mutations in interhost comparisons. Moreover, intrahost genetic variation corresponded to a random pattern of mutation, and most of the sites that exhibited nonsynonymous variation within hosts were invariant at deeper phylogenetic levels. This loss of long-term nonsynonymous variation is the signature of extensive purifying selection such that more than 90% of all nonsynonymous mutations are deleterious. Consequently, although arboviruses are able to successfully adapt to diverse cell types, they are characterized by a high rate of deleterious mutation.

Coexistence of different serotypes of dengue virus.

Abstract: We formulate a non-linear system of differential equations that models the dynamics of dengue fever This disease is produced by any of the four serotypes of dengue arbovirus Each serotype produces permanent immunity to it, but only a certain degree of cross-immunity to heterologous serotypes In our model we consider the relation between two serotypes Our interest is to analyze the factors that allow the invasion and persistence of different serotypes in the human population Analysis of the model reveals the existence of four equilibrium points, which belong to the region of biological interest One of the equilibrium points corresponds to the disease-free state, the other three equilibria correspond to the two states where just one serotype is present, and the state where both serotypes coexist, respectively We discuss conditions for the asymptotic stability of equilibria, supported by analytical and numerical methods We find that coexistence of both serotypes is possible for a large range of parameters

Dengue 2 PDK-53 Virus as a Chimeric Carrier for Tetravalent Dengue Vaccine Development

Abstract: Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK2 cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice. Chimeric D2/1, D2/3, and D2/4 viruses replicated efficiently in Vero cells and were immunogenic in AG129 mice. Chimeric D2/1 viruses protected adult AG129 mice against lethal D1 virus challenge. Two tetravalent virus formulations, comprised of either PDK53-E- or PDK53-V-vectored viruses, elicited neutralizing antibody titers in mice against all four dengue serotypes. These antibody titers were similar to the titers elicited by monovalent immunizations, suggesting that viral interference did not occur in recipients of the tetravalent formulations. The results of this study demonstrate that the unique attenuation loci of D2 PDK-53 virus make it an attractive vector for the development of live attenuated flavivirus vaccines.

Flavivirus Capsid Is a Dimeric Alpha-Helical Protein

Abstract: The Flaviviridae family of enveloped RNA viruses causes significant disease in both humans and agriculturally important animals. Flavivirus, the largest of the three genera of Flaviviridae, comprises over 70 viruses, mostly arthropod transmitted, including yellow fever virus (YF), dengue virus (DEN), West Nile virus, and tick-borne encephalitis virus (TBE) (15). The mature flavivirus particle is spherical with a diameter of 50 nm and contains multiple copies of three different structural proteins (C, M, and E), a host-derived membrane bilayer, and a single copy of a positive-sense RNA genome of approximately 11,000 nucleotides. The RNA genome is translated from a single open reading frame generating a polyprotein that is processed by viral and host proteases to yield the three structural proteins located at the N terminus followed by at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (23). The nonstructural proteins associate to form the viral replicase complex, for which several enzymatic functions have been identified, including protease, helicase, methyltransferase, and RNA-dependent RNA polymerase (15). The replicase complex is associated with intracellular membranes of the infected host and induces discrete membrane structures (16). Recent evidence suggests that the process of particle assembly may be coupled to genome replication (6, 12). This coupling of replication and virus assembly has made analysis of flavivirus assembly difficult, and thus, little is known about this important aspect of the virus life cycle. The structure of DEN, recently determined by cryoelectron microscopy (cryo-EM) and three-dimensional image reconstruction, has elucidated the molecular organization of the end product of the flavivirus assembly pathway (11). The organization of the E protein within the DEN particle was determined by modeling the atomic resolution structure of TBE E protein (22) into the outer density of the cryo-EM reconstruction. The model consists of a herringbone arrangement of E proteins, with three E protein monomers in the asymmetric unit of the virus, but lacking the anticipated T=3 icosahedral symmetry. The M protein is thought to reside just below E with both M and E being associated with the host-derived lipid bilayer. Internal to the lipid bilayer is the nucleocapsid core (NC) of the virus, consisting of multiple copies of capsid (C) protein that surround a single copy of the viral RNA genome. The density observed for the NC is roughly half of that observed for the outer density and suggests disorder or movement of the NC within the virus particle. The location of the NC within the virus particle indicates that the C protein directly contacts the genome RNA. A common phenomenon during flavivirus infection is the production and release into the extracellular medium of virus-like particles (VLPs) (24). NC is not present in VLPs, rendering them noninfectious. VLPs have also been produced in several heterologous expression systems in which only prM and E are present (1, 4, 9, 20, 21). Thus, the ability of E, in the presence of prM, to form a closed spherical protein shell is an intrinsic property of these two proteins and is independent of NC formation. However, the production of infectious particles requires the formation of NC, suggesting that an early stage of virus assembly involves the interaction of C protein with the genome RNA (7). The NC presumably then interacts with prM and/or E, resulting in the formation of a virus particle upon concomitant budding into the lumen of the endoplasmic reticulum (15). Unfortunately, details concerning the interactions of C protein with either the RNA genome or prM and E are not discernible in the cryo-EM reconstruction of DEN. Given the role of C in promoting encapsidation of the viral RNA and subsequent assembly of infectious virus particles, it is important to understand the structural and biophysical properties of the C protein that allow it to function in the flavivirus life cycle.

Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections.

Abstract: We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.

A Live, Attenuated Dengue Virus Type 1 Vaccine Candidate with a 30-Nucleotide Deletion in the 3′ Untranslated Region Is Highly Attenuated and Immunogenic in Monkeys

Abstract: The Δ30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3′ untranslated region (3′ UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1Δ30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4Δ30 vaccine candidate. rDEN1Δ30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3′ UTR, and both vaccines were highly protective against challenge with wt DEN1. Both rDEN1Δ30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1Δ30 nor rDEN1mutF showed restricted replication following intrathoracic inoculation in the mosquito Toxorhynchites splendens. The ability of the Δ30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the Δ30 mutation into wt DEN viruses belonging to each of the four serotypes.

Amplification of the sylvatic cycle of dengue virus type 2, Senegal, 1999-2000: entomologic findings and epidemiologic considerations.

Abstract: After 8 years of silence, dengue virus serotype 2 (DENV-2) reemerged in southeastern Senegal in 1999. Sixty-four DENV-2 strains were isolated in 1999 and 9 strains in 2000 from mosquitoes captured in the forest gallery and surrounding villages. Isolates were obtained from previously described vectors, Aedes furcifer, Ae. taylori, Ae. luteocephalus, and--for the first time in Senegal--from Ae. aegypti and Ae. vittatus. A retrospective analysis of sylvatic DENV-2 outbreaks in Senegal during the last 28 years of entomologic investigations shows that amplifications are periodic, with intervening, silent intervals of 5-8 years. No correlation was found between sylvatic DENV-2 emergence and rainfall amount. For sylvatic DENV-2 vectors, rainfall seems to particularly affect virus amplification that occurs at the end of the rainy season, from October to November. Data obtained from investigation of preimaginal (i.e., nonadult) mosquitoes suggest a secondary transmission cycle involving mosquitoes other than those identified previously as vectors.

Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells

Abstract: Endothelial cells are permissive to dengue virus (DV) infection in vitro, although their importance as targets of DV infection in vivo remains a subject of debate To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays DD identified eight differentially expressed cDNAs, including inhibitor of apoptosis-1, 2′-5′ oligoadenylate synthetase (OAS), a 2′-5′ OAS-like (OASL) gene, galectin-9, myxovirus protein A (MxA), regulator of G-protein signaling, endothelial and smooth muscle cell-derived neuropilin-like protein, and phospholipid scramblase 1 Microarray analysis of 22,000 human genes confirmed these findings and identified an additional 269 genes that were induced and 126 that were repressed more than fourfold after DV infection Broad functional responses that were activated included the stress, defense, immune, cell adhesion, wounding, inflammatory, and antiviral pathways These changes in gene expression were seen after infection of HUVECs with either laboratory-adapted virus or with virus isolated directly from plasma of DV-infected patients Tumor necrosis factor alpha, OASL, and MxA and h-IAP1 genes were induced within the first 8 to 12 h after infection, suggesting a direct effect of DV infection These global analyses of DV effects on cellular gene expression identify potentially novel mechanisms involved in dengue disease manifestations such as hemostatic disturbance

The extent of homologous recombination in members of the genus Flavivirus

Abstract: The family Flaviviridae includes important human pathogens, such as dengue (DEN) virus, yellow fever (YF) virus and hepatitis C virus, many of which have emerged or re-emerged in recent years. Until recently, flavivirus evolution was thought to proceed in a clonal manner, with diversity generated mainly through the accumulation of mutational changes. However, this assumption has now been shown to be invalid, with homologous recombination demonstrated in all three genera of the Flaviviridae. Since recombination has important implications for the study of virus evolution, a survey of recombination in the viruses of the genus Flavivirus was carried out. Using envelope gene sequence data and a combination of graphical and phylogenetic analyses, hitherto unreported recombination in Japanese encephalitis virus and St Louis encephalitis virus was detected, as well as further recombinants in DEN virus. However, no evidence for recombination was found in West Nile or YF viruses, or in the tick-borne flavivirus group. It is proposed that the difference between the mosquito- and tick-borne viruses can be accounted for by their differing modes of transmission, whilst the variation among the mosquito-borne flaviviruses reflects both the ecology of the particular host and vector species and also bias in the sampling process.

Efficiency of dengue serotype 2 virus strains to infect and disseminate in aedes aegypti

Abstract: Dengue serotype 2 (DEN-2) viruses with the potential to cause dengue hemorrhagic fever have been shown to belong to the Southeast (SE) Asian genotype. These viruses appear to be rapidly displacing the American genotype of DEN-2 in the Western Hemisphere. To determine whether distinct genotypes of DEN-2 virus are better adapted to mosquito transmission, we classified 15 viral strains of DEN-2 phylogenetically and compared their ability to infect and disseminate in different populations of Aedes aegypti mosquitoes. Envelope gene nucleotide sequence analysis confirmed that six strains belonged to the American genotype and nine strains were of the SE Asian genotype. The overall rate of disseminated infection in mosquitoes from Texas was 27% for the SE Asian genotype versus 9% for the American genotype. This pattern of infection was similar in another population of mosquitoes sampled from southern Mexico (30% versus 13%). Together, these findings suggest that Ae. aegypti tends to be more susceptible to infection by DEN-2 viruses of the SE Asian genotype than to those of the American genotype, and this may have epidemiologic implications.