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PubMed2006

Bacterially expressed and refolded envelope protein (domain III) of dengue virus type-4 binds heparan sulfate.

An arboviral infection like dengue fever/dengue hemorrhagic fever (DHF) with high morbidity and mortality rate are extensively prevalent in several parts of the world. Global efforts have been directed towards development of vaccine for prevention of dengue. However, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. Here we report molecular interaction of domain III of envelope protein of dengue virus type-4 with heparan sulfate. A codon optimized synthetic gene encoding domain III of dengue virus type-4 envelope protein was expressed in Escherichia coli and purified under denaturing conditions, refolded and purified to homogeneity. Refolded Den4-DIII was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified protein was recognized in Western analyses by monoclonal antibody specific for the 6x His tag as well as the H241 monoclonal antibody. The in vitro refolded recombinant protein preparation was biologically functional and found to bind cell free heparan sulfate. This is the first report providing molecular evidence on binding of dengue-4 envelope protein to heparan sulfate. We developed a homology model of dengue-4 envelope protein (domain III) and mapped the possible amino acid residues critical for binding to heparan sulfate. Domain III envelope protein of dengue virus is a lead vaccine candidate. Our findings further the understanding on biology of dengue virus and will help in development of bioassay for the proposed vaccine candidate.

Blotting, Western↗
PubMed2005

Imported dengue fever in Austria 1990-2005.

Dengue is the most important human viral disease transmitted by an arthropod vector. The steadily increasing numbers of tourists visiting endemic areas coupled with the present resurgence of dengue, raises the risk of exposure for large numbers of travelers and imported dengue cases are increasingly observed in non-endemic countries. We aimed to study the epidemiology, clinical manifestations and laboratory findings in imported dengue at a City of Vienna hospital. Medical records of 93 patients (age: 17-68 years, 43f, 50m) with imported dengue in Vienna between 1990 and April 2005 were analyzed retrospectively. Forty-eight (52%) were classified as confirmed and 45 (48%) as probable dengue, according to the CDC criteria. The patients acquired the infection in South East Asia (56%), the Indian subcontinent (18%), Africa (10%) and Oceania (3%). The most important symptoms were fever, headache, arthralgia and myalgia, nausea and vomiting, diarrhea, chills, extreme fatigue and dizziness. A rash was observed in 43%, and lymphadenopathy in 22%. Laboratory findings were thrombocytopenia, leukopenia and elevated hepatic enzymes. Eighteen patients showed hemorrhagic manifestations, and 7 fulfilled the criteria of dengue hemorrhagic fever; 1 of them had dengue shock syndrome. Case fatality rate was nil. Dengue has to be considered in all febrile travelers returning from endemic areas. Prompt diagnosis and symptomatic treatment is warranted and should prevent patients from unnecessary and potentially harmful diagnostic and therapeutic procedures.

Adolescent↗
PubMed1999

The use of Toxorhynchites splendens for identification and quantitation of serotypes contained in the tetravalent live attenuated dengue vaccine.

Assurance of identity and quantity is an indispensable part of quality control in the manufacture of vaccines. Dengue-1 PDK13, dengue-2 PDK53, dengue-3 PGMK30F3 and dengue-4 PDK48 in the live attenuated tetravalent dengue vaccine were assayed by identification and quantitation in a mosquito system (Toxorhynchites splendens). Each serotype of dengue virus was identified by dengue specific monoclonal antibodies in the indirect fluorescent antibody test. Virus content was estimated by calculating the 50% mosquito infectious dose (MID50). Differences from 0 to +/-0.5 log10 were observed between the original monovalent titer and that from the blend which showed no significant difference at 95% confidence limit (P < 0.05). This result indicates that there is no interference between dengue serotypes in mosquitoes infected by intrathoracic inoculation with the virus mixture. It can be also concluded that this mosquito system can be used as an effective measure for infectivity titration of each component in the tetravalent dengue vaccine.

Animals↗
PubMed1999

Fatal dengue hemorrhagic fever in Cuba, 1997.

OBJECTIVES: After more than 15 years without dengue activity, a dengue II epidemic was reported in Cuba in 1997. Three thousand and twelve serologically confirmed cases were reported, with 205 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) cases and 12 fatalities. This report presents the clinical, serologic, and virologic findings in the 12 fatal DHF/DSS cases. METHODS: Serum and necropsy samples were studied by viral isolation in C636 cell line and polymerase chain reaction. Serum samples were tested by IgM capture enzyme-linked immunoassay (ELISA) and ELISA inhibition method (EIM). RESULTS: All 12 cases were classified as DHF/DSS according to the Pan American Health Organization Guidelines for Control and Prevention of Dengue and Dengue Hemorrhagic Fever in the Americas. All patients were older than 15 years. Women were more frequently affected. The symptoms and signs presented by these patients were similar to those previously described in DHF/DSS cases. Clinical deterioration occurred on average at day 3.75. Abdominal pain and persistent vomiting were the earliest and most frequent warning signs. Dengue infection was confirmed in all cases. IgM antibodies were detected in 11 of 12 cases, all of them with a secondary infection. Dengue II virus was detected by viral isolation in 12 samples and by polymerase chain reaction in 17. Virus or RNA was detected in various tissues, including kidney, heart, lung, and brain. CONCLUSION: The clinical, pathologic, and laboratory features of 12 cases of fatal dengue hemorrhagic fever were reviewed. The results obtained demonstrate that adults with a primary dengue infection are at risk of developing the severe disease (DHF) if they are infected with a different serotype.

Adolescent↗
PubMed2002

Biosensor for dengue virus detection: sensitive, rapid, and serotype specific.

A serotype-specific RNA biosensor was developed for the rapid detection of Dengue virus (serotypes 1-4) in blood samples. After RNA amplification, the biosensor allows the rapid detection of Dengue virus RNA in only 15 min. In addition, the biosensor is portable, inexpensive, and very easy to use, making it an ideal detection system for point-of-care and field applications. The biosensor is coupled to the isothermal nucleic acid sequence-based amplification (NASBA) technique with which small amounts of virus RNA are amplified using a simple water bath. During the NASBA reaction, a generic sequence is attached to all RNA molecules as described earlier (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). It has been shown earlier that Dengue virus can be detected specifically using two DNA probes: a first probe hybridized with the attached generic sequence and, therefore, bound to every amplified RNA molecule; and a second probe either bound to all four Dengue virus serotypes or chosen to be specific for only one serotype. These probes were utilized in the biosensor described in this publication. For a generic Dengue virus biosensor, the second probe is complementary to a conserved region found in all Dengue serotypes. For identification of the individual Dengue virus serotypes, four serotype-specific probes were developed (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtiff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The generic DNA probe (reporter probe) is coupled to the outside of dye-encapsulating liposomes. The conserved or Dengue serotype specific probes (capture probes) are immobilized on a polyethersulfone membrane strip. Liposomes are mixed with amplified target sequence and are then applied to the membrane. The mixture is allowed to migrate along the test strip, and the liposome-target sequence complexes are immobilized in the capture zone via hybridization of the capture probe with target sequence. The amount of liposomes present in the immobilized complex is directly proportional to the amount of target sequence present in the sample and can be quantified using a portable reflectometer. The different biosensor components have been optimized with respect to sensitivity and, foremost, specificity toward the different serotypes. An excellent correlation to a laboratory-based detection system was demonstrated. Finally, the assay was tested using a limited number of clinical human serum samples. Although Dengue serotypes 1, 2 and 4 were identified correctly, serotype 3 displayed low cross-reactivity with biosensors designed for detection of serotypes 1 and 4.

Animals↗
PubMed1992

The predisposing and protective factors against dengue virus transmission by mosquito vector.

An outbreak of dengue fever occurred in Taiwan between 1987 and 1988. The highest attack rate among adults was estimated at 5.6% in the city of Kao-hsiung. A case-control study was carried out to determine the risks of contracting dengue infection and to identify protective factors against the infection. One hundred dengue patients of the authors' hospital who were diagnosed by virologic or serologic tests constituted the case group. Each dengue patient was matched to a control patient of the same age and sex who had been diagnosed as suffering from a non-vector-borne disease on the same day as the dengue patient. Of the household protective measures against dengue infection prior to the occurrence of illness, the adjusted odds ratio, estimated by stratified analysis, was lower for people who lived in screened houses (odds ratio = 0.58, 95% confidence interval 0.36-0.92) as compared with inhabitants of unscreened houses. The odds ratio was as low as 0.18 (95% confidence interval 0.06-0.56) for people whose homes were fully screened with door screens opening outwardly. Patients who lived near markets and/or open sewers or ditches were running a risk of dengue infection 1.8 (95% confidence interval 1.3-2.4) times higher than those who lived elsewhere. To control dengue outbreaks, the authors recommend that special attention should be devoted to the reduction of outdoor vector sources. Full screening, especially outwardly opening screen doors, seems to be an individual's best protection against dengue fever.

Adolescent↗
PubMed2005

Concurrent outbreak of leptospirosis and dengue in Mumbai, India, 2002.

This prospective study was undertaken to investigate the possibility of a concurrent outbreak of leptospirosis and dengue and to describe the clinical illnesses. From 20 June to 14 November 2002, children who presented to our hospital with a suspected diagnosis of leptospirosis or dengue were admitted. In every child with suspected leptospirosis, a screening latex agglutination test was carried out to detect anti-Leptospira antibodies. The diagnosis of leptospirosis was confirmed by a positive enzyme-linked immunosorbent assay (ELISA) test or microagglutination test. The diagnosis of dengue was confirmed by a positive IgM antibody capture ELISA test. Clinical features in the leptospirosis and leptospirosis-negative groups, and dengue and dengue-negative groups were analysed. Of 90 children screened, 15 (16.7 per cent) had leptospirosis. Two children with Weil's disease died and the remaining 13 responded well to intravenous penicillin. Five clinical features were significantly associated with leptospirosis, namely conjunctival suffusion (p=0.007), haemorrhage (p=0.020), abdominal pain (p=0.011), hepatosplenomegaly (p=0.044), and oedema (p=0.007). As the number of these five features concomitantly present increased, the chances of the child having leptospirosis also increased significantly (p<0.0001). Of 90 children screened, 16 (17.8 per cent) had dengue. All responded well to the treatment and went home. Two clinical features were significantly associated with dengue, namely arthralgia (p=0.020) and thrombocytopenia (p=0.001). If both these features were present, the chances of the child having dengue increased significantly (p=0.001). Our study shows that a concurrent outbreak of leptospirosis and dengue had occurred in the slums of Mumbai city.

Age Distribution↗
PubMed1988

Production of interferon alpha by dengue virus-infected human monocytes.

Human monocytes appear to be very important in the pathogenesis of dengue infection. They are thought to be the most active sites of virus replication during dengue infection. We have analysed interferon (IFN) production by dengue virus from peripheral blood mononuclear cells (PBMC). IFN activity was first detected at 12 h after infection of monocytes and reached a maximum level by 48 h. Non-adherent PBMC depleted of monocytes did not produce detectable levels of IFN, and did not contain dengue antigen-positive cells after exposure to dengue virus. The IFN produced was characterized as IFN-alpha by neutralization tests using specific antisera to HuIFN-alpha, HuIFN-beta and HuIFN-gamma, and by radioimmunoassay. The culture fluids of dengue virus-infected monocytes, which contained IFN-alpha, were able to inhibit infection of human monocytes by dengue virus. These results suggest that IFN-alpha produced by dengue virus-infected monocytes may play an important role in controlling primary dengue virus infection.

Antigens, Viral↗
PubMed1970

Dengue virus plaque development in simian cell systems. II. Agar variables and effect of chemical additives.

Dengue type 2 virus, strain New Guinea B, plaqued with equal facility and titer under overlays containing six different grades of commercial agar in the LLC-MK(2) cell system. Doubling the agar volume on LLC-MK(2) cell monolayers increased the plaque development time of dengue type 1, strain Hawaii. Storage of agar at 56 C reduced or totally abolished dengue type 4, strain H-241, plaque titer in LLC-MK(2) cells. The influence of six known virus plaque-enhancing compounds on plaque development of all dengue virus serotypes was studied in two continuous simian kidney cell lines, LLC-MK(2) and Vero. In the absence of any chemical additive, plaque development of all dengue serotypes was more rapid (4 to 10 days) in the LLC-MK(2) line than in the Vero line (6 to 13 days). Increased plaque development time of type 1, strain Hawaii, by pancreatin and plaque-size doubling of dengue types 1 and 4 was the only advantage conferred by the addition of six chemical additives in the LLC-MK(2) cell system. Dengue types 1 and 6 failed to plaque in the Vero cell system unless aided by a plaque-enhancing compound; plaques of dengue types 2, 3, 4, and 5 appeared sooner (2 days) and were increased in plaque diameter. The optimal DEAE concentration for plaquing dengue type 1, strain Hawaii, was 100 mug/ml; plaque development either failed at lower concentrations or was inhibited at higher (200 mug/ml) concentrations.

Agar↗
PubMed2000

Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus.

Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.

Antibodies, Monoclonal↗
PubMed1981

In vitro virulence marker: growth of dengue-2 virus in human leukocyte suspension cultures.

We wished to find a simple, biologically relevant method to evaluate the virulence of dengue viruses for human beings. Since cells of mononuclear phagocyte lineage may be important sites of dengue infection in primates, we evaluated the permissiveness of these cells to dengue virus as a correlate of virus virulence. Two wild-type, large-plaque, monkey-virulent dengue-2 virus strains and two small-plaque, monkey-avirulent dengue-2 virus strains were evaluated for their ability to replicate in human peripheral blood leukocyte cultures supplemented with enhancing antibody. One of the small-plaque strains was demonstrated to have reduced virulence for man. Wild-type dengue-2 viruses replicated readily in peripheral blood leukocyte suspension cultures, whereas small-plaque dengue-2 strains did not. Differences between our data and results obtained by other workers employing adherent peripheral blood leukocytes are discussed. Antibody-enhanced growth of dengue virus in suspension cultures of human peripheral blood leukocytes gives promise of being a simple in vitro system for characterizing dengue virus virulence.

Cells, Cultured↗
PubMed1989

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes.

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.

Antibodies, Monoclonal↗
PubMed1993

Dengue virus protein recognition by virus-specific murine CD8+ cytotoxic T lymphocytes.

The identification of the protein targets for dengue virus-specific T lymphocytes may be useful for planning the development of subunit vaccines against dengue. We studied the recognition by murine dengue virus-specific major histocompatibility complex class I-restricted, CD8+ cytotoxic T lymphocytes (CTL) of dengue virus proteins using recombinant vaccinia viruses containing segments of the dengue virus genome. CTL from H-2k mice recognized a single serotype-cross-reactive epitope on the nonstructural (NS) protein NS3. CTL from H-2b mice recognized a serotype-cross-reactive epitope that was localized to NS4a or NS4b. CTL from H-2d mice recognized at least three epitopes: a serotype-specific epitope on one of the structural proteins, a serotype-cross-reactive epitope on NS3, and a serotype-cross-reactive epitope on NS1 or NS2a. Our findings demonstrate the limited recognition of dengue virus proteins by CTL from three inbred mouse strains and the predominance of CTL epitopes on dengue virus nonstructural proteins, particularly NS3. Since human dengue virus-specific CTL show similar patterns of recognition, these findings suggest that nonstructural proteins should be considered in designing vaccines against dengue.

Animals↗
PubMed1997

Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts.

Aedes↗
PubMed2001

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection.

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.

Animals↗
PubMed2001

Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis.

The ability of dendritic cells (DCs) to shape the adaptive immune response to viral infection is mediated largely by their maturation and activation state as determined by the surface expression of HLA molecules, costimulatory molecules, and cytokine production. Dengue is an emerging arboviral disease where the severity of illness is influenced by the adaptive immune response to the virus. In this report, we have demonstrated that dengue virus infects and replicates in immature human myeloid DCs. Exposure to live dengue virus led to maturation and activation of both the infected and surrounding, uninfected DCs and stimulated production of tumor necrosis factor alpha (TNF-alpha) and alpha interferon (IFN-alpha). Activation of the dengue virus-infected DCs was blunted compared to the surrounding, uninfected DCs, and dengue virus infection induced low-level release of interleukin-12 p70 (IL-12 p70), a key cytokine in the development of cell-mediated immunity (CMI). Upon the addition of IFN-gamma, there was enhanced activation of dengue virus-infected DCs and enhanced dengue virus-induced IL-12 p70 release. The data suggest a model whereby DCs are the early, primary target of dengue virus in natural infection and the vigor of CMI is modulated by the relative presence or absence of IFN-gamma in the microenvironment surrounding the virus-infected DCs. These findings are relevant to understanding the pathogenesis of dengue hemorrhagic fever and the design of new vaccination and therapeutic strategies.

Antibodies, Viral↗
PubMed2005

Knowledge and attitudes in Puerto Rico concerning dengue prevention.

OBJECTIVE: Dengue has been endemic in Puerto Rico for three decades. Multiple educational and community-based efforts have been developed to inform the population about dengue prevention. We undertook this study to understand the community members' knowledge, attitudes, and practices related to dengue prevention and to elicit their ideas for future prevention campaigns. METHODS: A qualitative study based on grounded theory analysis was conducted between February and May of 2001. The study involved a total of 34 participants in four group interviews who had been identified through the Puerto Rico dengue surveillance system. RESULTS: In general, participants had correct knowledge about dengue prevention, but they did not associate the mosquitoes inside their houses with Aedes aegypti. Participants insisted that "neighbors" needed to control larval habitats, and the participants also asked the Government to fumigate. CONCLUSIONS: The patterns of knowledge and opinion that emerged in the discussions can be arranged along an axis going from high levels of correct knowledge to low levels of correct knowledge about dengue and dengue hemorrhagic fever and related practices. There were few participants at either extreme. Three themes explained these patterns: misconceptions about dengue (based on previously delivered information), the "invisibility" of dengue (as compared to other diseases), and responsibility (individual and Government). Four strategies for preventive behaviors were recommended: developing community groups to identify community priorities on prevention, developing volunteer groups to deliver prevention messages, making house visits to demonstrate specific control measures, and conducting a complementary media campaign to support these strategies.

Dengue↗
PubMed1985

Dengue transmission in two Puerto Rican communities in 1982.

Paired serologic, entomologic, and environmental surveys were performed in two Puerto Rican communities, Salinas and Manatí, in summer and fall 1982. Paired samples on 434 persons in Salinas and 324 persons in Manatí showed recent dengue infection rates of 35% and 26%, respectively. Ae. aegypti larval indices were higher in Salinas than in Manatí but were relatively high throughout both communities. Breteau indices in neighborhoods ranged from 43 to 172, and infection rates in the neighborhoods were 22% to 45%. With a multivariate technique, we analyzed possible associations of environmental variables with dengue incidence and prevalence of dengue antibody. Wood-constructed housing and low socioeconomic status were among the variables significantly associated with dengue incidence. Predictors of dengue antibody prevalence included socioeconomic level, tree height, shade, and window and door screens. Recent dengue infections clustered within the sampled members of households (P less than 0.05, binomial test). An estimated 35% of dengue infections were symptomatic, and no serious illnesses were reported. The potential for high dengue infection rates in Puerto Rico will continue unless substantial reductions in vector populations are achieved. Targeting dengue surveillance and vector control activities in areas with demonstrated environmental risk factors may limit transmission during future outbreaks.

Adolescent↗