Dengue 2 Research Articles

English

The potential risk of non-human primates in Senegal to be natural hosts for arboviruses of importance for human has been assessed. A total of 58 wild monkeys, including 14 Erythrocebus patas and 44 Chlorocebus sabaeus, were trapped at three sites within forest galleries and the nearby village of Ngari, in the Kedougou area, Southeastern Senegal. Blood samples were taken and sera analyzed by enzyme-linked immunosorbent assay (ELISA) for the presence of Yellow Fever (YF) and/or Dengue 2 (DEN-2) reacting antibodies. An overall yellow fever seroprevalence of 22.4% was found, including 5.2% and 17.2% YF IgG positive E. patas (3/58) and C. sabaeus (10/58) respectively. Three of the positive C. sabaeus were trapped near Ngari village, and the others in forest galleries. Also, 12.0% of the primates tested positive including 5.2% of E. patas and 6.9% of C. sabaeus, all of them were from the forest galleries. Ultimately Cercopithecidae act as potential amplificatory reservoir hosts for YF virus and, seroconversion observed within wild C. sabaeus and E. patas demonstrates also an active DENV-2 virus circulation within non-human primates in Senegal. The present study addresses and discusses new insight of both viruses’ natural enzootic cycles. Key words: Yellow fever, Dengue, monkeys, Senegal.

Insecticide susceptibility of Aedes aegypti populations from Senegal and Cape Verde Archipelago.

BackgroundTwo concomitant dengue 3 (DEN-3) epidemics occurred in Cape Verde Archipelago and Senegal between September and October 2009. Aedes aegypti was identified as the vector of these epidemics as several DEN-3 virus strains were isolated from this species in both countries. The susceptibility to pyrethroids, organochlorine, organophosphates and carbamate was investigated in two field strains of Aedes aegypti from both countries using WHO diagnostic bioassay kits in order to monitor their the current status of insecticide susceptibility.FindingsThe two tested strains were highly resistant to DDT. The Cape Verde strain was found to be susceptible to all others tested insecticides except for propoxur 0.1%, which needs further investigation. The Dakar strain was susceptible to fenitrothion 1% and permethrin 0.75%, but displayed reduced susceptibility to deltamethrin, lambda-cyhalothrin and propoxur.ConclusionsAs base-line results, our observations stress a careful management of insecticide use for the control of Ae. aegypti. Indeed, they indicate that DDT is no longer efficient for the control of Ae. aegypti populations in Cape Verde and Dakar and further suggest a thorough follow-up of propoxur susceptibility status in both sites and that of deltamethrin and lambda-cyhalothrin in Ae. aegypti populations in Dakar. Thus, regular monitoring of susceptibility is greatly needed as well as the knowing if this observed resistance/susceptibility is focal or not and for observed resistance, the use of biochemical methods is needed with detailed comparison of resistance levels over a large geographic area.KeywordsAedes aegypti, Insecticides, Susceptibility, Cape Verde, Senegal

Dissemination in Pigtailed Macaques after Primary Infection of Dengue-3 Virus

Nonhuman primates (NHPs) play as indispensable animal model in biomedical research for studying a variety of human health issues, diseases and disorders, therapies, and preventive strategies. Since the immunological and physiological responses of NHPs, at some extent, to experimental viral infections are similar to humans, it is possible that studies of dengue infection in NHPs may aid understanding of dengue infection in humans. In this study,we used pigtailed macaques ( Macaca nemestrina ) as the experimental animal to study dengue-3 (DEN-3) virus infection.We evaluated DEN-3 viral distribution and replication sites after a primary infection in all collected tissues. Sequential localization in tissue of DEN-3 virus was studied in pigtailed macaques euthanized three days post viral inoculation (10 pfu mL ). Pigtailed macaque that was inoculated subcutaneously or intravenously; showed the highest viremia (62.94 pfu mL and 58.62 pfu mL ) detected by one step reverse transcription real time PCR. The virus inoculated in pigtailed macaques by subcutaneous injection was rapidly disseminated from the inoculation site to the lymph nodes, adrenal glands, kidneys, heart, thyroid, liver, prostate gland, and seminal vesicles. Meanwhile, dissemination of dengue virus in pigtailed macaques inoculated intravenously was detected in lymph nodes, thymus, salivary glands, liver, and prostate gland. This study suggested that the above mentioned-tissue specimens are involved or affected by DEN-3 virus replication and the route of infection seemed to have influenced the virus dissemination.

Chimeric Tick-Borne Encephalitis/Dengue Virus Is Attenuated inIxodes scapularisTicks andAedes aegyptiMosquitoes

In an effort to derive an efficacious live attenuated vaccine against tick-borne encephalitis, we generated a chimeric virus bearing the structural protein genes of a Far Eastern subtype of tick-borne encephalitis virus (TBEV) on the genetic background of recombinant dengue 4 (DEN4) virus. Introduction of attenuating mutations into the TBEV envelope protein gene, as well as the DEN4 NS5 protein gene and 3' noncoding region in the chimeric genome, results in decreased neurovirulence and neuroinvasiveness in mice, and restricted replication in mouse brain. Since TBEV and DEN4 viruses are transmitted in nature by ticks and mosquitoes, respectively, it was of interest to investigate the infectivity of the chimeric virus for both arthropod vectors. Therefore, parental and chimeric viruses were tested for growth in mosquito and tick cells and for oral infection in vivo. Although all chimeric viruses demonstrated moderate levels of replication in C6/36 mosquito cells, they were unable to replicate in ISE6 tick cells. Further, the chimeric viruses were unable to infect or replicate in Aedes aegypti mosquitoes and Ixodes scapularis tick larvae. The poor infectivity for both potential vectors reinforces the safety of chimeric virus-based vaccine candidates for the environment and for use in humans.

Establishment and characterization of a new Aedes aegypti (L.) (Diptera: Culicidae) cell line with special emphasis on virus susceptibility

A new cell line from the neonate larvae of Aedes aegypti (L) mosquito was established and characterized. The cell line at the 50th passage (P) level consisted of three prominent cell types, i.e., epithelial-like cells (92%), fibroblast-like cells (7%), and giant cells ( approximately 1%). Karyological analysis showed diploid (2n = 6) number of chromosomes in >75% cells at P-50. The growth kinetics studied at 52nd passage level showed approximately tenfold increase in cell number over a 10-d study period. The species specificity studies using DNA amplification fingerprinting profile analysis using RAPD primers demonstrated 100% homology with the host profile showing the integrity of the cell line. Electron microscopy revealed the absence of mycoplasma or other adventitious agents. The cell line supported the multiplication of seven arboviruses, i.e., Chikungunya (CHIK), Japanese encephalitis, West Nile, dengue 2 (DEN-2), Chandipura, vesicular stomatitis, and Chittoor viruses. The cell line did not replicate Ganjam and Kaisodi viruses. CHIK virus yield in the new cell line was approximately 3log and 0.5log 50% tissue culture infective dose (TCID(50))/mL higher than Vero E6 and C6/36 cell lines, respectively. In the case of DEN-2 virus, it yielded 1log TCID(50)/mL higher than Vero E6, but lesser than C6/36 cell line. Due to its high susceptibility to a broad spectrum of viruses, the new cell line may find application in virus isolation during epidemics and in antigen production.

Substitution or deletion mutations between nt 54 and 70 in the 5′ non-coding region of dengue type 2 virus produce variable effects on virus viability

A C57U nucleotide mutation in a predicted RNA stem structure (nt 11-16/56-61) of the 5' non-coding region (5'NCR) of dengue 2 (DEN-2) 16681 virus is partially attenuating, but unstable during serial passage of certain candidate DEN-2 PDK-53-based vaccine viruses containing this mutation. Here, 11 different mutations (one or more point substitution and/or deletion) between nt 54 and 70 in the 5'NCR of the pD2/IC-30P-A (16681) infectious clone are described. Four mutants were infectious. Three mutants with single point substitutions replicated well in cell culture and exhibited variable neurovirulence in mice. Constructs containing multiple substitutions or any deletions failed to produce infectious viruses. Unexpectedly, a double C57U+G58C mutant replicated as efficiently as D2/IC-30P-A virus, and was more neurovirulent for newborn ICR mice. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 5'NCR-C57U mutation.

Implications of previous subclinical dengue infection but not virus load in dengue hemorrhagic fever

In a study comparing the virus load and immune reaction between patients with primary and secondary dengue-2 (DEN-2) infections in a hospital-based analysis, we found that 40.7% (55/135) of the 135 patients had secondary DEN-2 infection following a DEN-2 outbreak in southern Taiwan. Most of the secondary infections had subclinical primary dengue infections (78.2%; 43/55). Patients with secondary DEN-2 infections had lower platelet counts, and blood interferon-alpha and virus load, but significantly higher interleukin-10 (P=0.030) and anti-DEN-1 neutralization titers (P=0.013) than those with primary infection. Patients with secondary DEN-2 infection also had a higher rate of dengue hemorrhagic fever (DHF) (61.7% vs. 36.3%). A previous subclinical dengue infection is involved in the secondary DEN-2 infection associated with altered immune reaction and higher DHF rate, but lower blood virus load.

SEROPREVALENCE AND DISTRIBUTION OF FLAVIVIRIDAE, TOGAVIRIDAE, AND BUNYAVIRIDAE ARBOVIRAL INFECTIONS IN RURAL CAMEROONIAN ADULTS

Arboviruses from the families Flaviviridae, Togaviridae, and Bunyaviridae are suspected to cause widespread morbidity in sub-Saharan African populations, but little research been done to document the burden and distribution of these pathogens. We tested serum samples from 256 Cameroonian adults from nine rural villages for the presence of Dengue-2 (DEN-2), West Nile (WN), Yellow fever (YF), Chikungunya (CHIK), O'nyong-nyong (ONN), Sindbis (SIN), and Tahyna (TAH) infection using standard plaque-reduction neutralization tests (PRNT). Of these samples, 12.5% were DEN-2 positive, 6.6% were WN positive, 26.9% were YF positive, 46.5% were CHIK seropositive, 47.7% were ONN positive, 7.8% were SIN positive, and 36.3% were TAH positive. DEN-2, YF, and CHIK seroprevalence rates were lower among individuals living in dwellings with grass or thatched roofs versus corrugated tin and in villages isolated from urban centers. Seroprevalence rates of YF and CHIK increased with age. These results suggest that inter-epidemic arboviral infection is common in central African populations.

Live Attenuated Chimeric Yellow Fever Dengue Type 2 (ChimeriVax™-DEN2) Vaccine: Phase I Clinical Trial for Safety and Immunogenicity: Effect of Yellow Fever Pre-immunity in Induction of Cross Neutralizing Antibody Responses to All

A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax™-DEN2) in comparison to that of YF vaccine (YF-VAX®). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax™-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAXâ by the subcutaneous route (SC). To determine the effect of YF pre-immunity on the ChimeriVaxTM-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax™-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX® and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVaxTM-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX® seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3, and 4 were observed in YF naïve subjects inoculated with either ChimeriVax™-DEN2 or YF-VAX®. In contrast, 100% of YF immune subjects inoculated with ChimeriVax™-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2, and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that 1) the safety and immunogenicity profile of the ChimeriVax™-DEN2 vaccine is consistent with that of YF-VAX®, and 2) pre-immunity to YF virus does not interfere with ChimeriVaxTM-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.

Non-structural proteins of dengue 2 virus offer limited protection to interferon-deficient mice after dengue 2 virus challenge

Chimeric (D2/WN) viruses containing the pre-membrane (prM) and envelope (E) proteins of West Nile virus (WN virus) and the capsid (C) and non-structural proteins of dengue 2 (DEN2) virus were used to evaluate the protective immunity elicited by either the flaviviral E protein or non-structural proteins. AG129 interferon-deficient mice, previously shown to be protected against lethal DEN1 or DEN2 viral infection after vaccination with a wild-type or candidate vaccine strain of DEN1 or DEN2 virus, respectively, were immunized with chimeric D2/WN virus and then challenged with DEN2 virus. D2/WN chimeric viruses were non-pathogenic in AG129 mice. These viruses elicited little anti-DEN E antibody, high levels of anti-DEN NS1 antibody and no or very low levels of DEN2 virus-neutralizing antibodies. Only 15% of D2/WN-immunized mice survived challenge with DEN2 virus. However, their mean survival time increased by 11-14 days over non-immunized controls. These results suggest that, whilst the non-structural proteins were able to enhance mean survival times of AG129 mice, this protection was not as effective as protection mediated by the E protein.

Dengue 2 genotypes in the state of Oaxaca, Mexico

To genetically characterize dengue 2 (DEN-2) viruses in Oaxaca, Mexico, the C protein, and a portion of the prM protein genes of 8 isolates from the 2001 DEN epidemic were sequenced. The sequences were compared to those of prototype DEN-2 viruses from various parts of the world. Phylogenetic analysis suggested that the 2001 isolates of DEN-2 were of the American/Asian genotype and were most similar to the Jamaica and Venezuelan isolates MARA3, LARD1996 and LARD1910. Molecular analyses confirmed the origin of the isolates. This study indicates that DEN-2 strains of American/Asian genotype probably from Southeast Asian are circulating in Oaxaca.

Determining genetic stabilities of chimeric dengue vaccine candidates based on dengue 2 PDK-53 virus by sequencing and quantitative TaqMAMA

The genetic stabilities of the three attenuation loci of the candidate dengue 2 (D2) PDK-53 vaccine virus were evaluated for the PDK-53 virus and PDK-53-vectored chimeric D2/1, D2/3, and D2/4 viruses following 10 sequential passages in Vero cells. Sequencing revealed that the dominant NS1–53-Asp and the NS3–250-Val attenuation loci were extremely stable, whereas reversion occurred at the 5′NCR-57-U locus in 10 of the 18 viral lineages tested. A more sensitive and quantitative assay, the TaqMan mismatch amplification mutation assay (TaqMAMA), was employed to more finely discriminate the level of reversion at the 5′NCR-57 locus. This rapid genetic assay permitted detection of ≤1% reversion of 5′NCR-57 U-to-C in viral populations. By TaqMAMA, various levels of reversion at 5′NCR-57 were detected in all 18 of the PDK-53-based viral lineages tested at Vero passage 10, but only 3 lineages had reversion levels >80% in the viral population. Chimeric viruses based on the PDK-53-V (all three mutations present) genetic background were more stable than those developed in the PDK-53-E (5′NCR and NS1 mutations present) background. The TaqMAMA can be applied in quality control analyses to ensure that attenuated vaccine seeds contain undetectable or minimal levels of reversion at a given attenuation locus.

Simultaneous infection with dengue 2 and 3 viruses in a Chinese patient return from Sri Lanka

Dengue is an acute viral disease transmitted by the Aedes aegypti and Aedes albopictus mosquito, which are present in most tropical urban areas of the world. There are four antigenically distinct serotypes, designated dengue-1 (DEN-1), dengue-2 (DEN-2), dengue-3 (DEN-3) and dengue-4 (DEN-4). Dengue outbreaks have occurred in several regions in Asia, involving four serotypes of dengue 1, 2, 3 and 4. In review of the few cases of dual infection documented in the literature, we report here a case of simultaneous infection with DEN-2 and DEN-3 in a Chinese patient return from Sri Lanka. The dual infection was identified by type-specific indirect immunofluorescence assay and confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence determination. This is the first documented case of simultaneous infection with serotype of DEN-2 and DEN-3 in China.

A fragment of the envelope protein from dengue-1 virus, fused in two different sites of the meningococcal P64k protein carrier, induces a functional immune response in mice.

Previously we have reported the capacity of the fusion protein PD3, composed of the P64k protein and the envelope (E) fragment from amino acids (aa) 286-426 of dengue-2 virus (DEN-2), to induce a functional immune response in mice against the homologous virus. In that case, the E fragment was inserted within the lipoyl-binding domain of the meningococcal P64k protein. In the present study, to test the functionality of the same E region from dengue-1 (DEN-1), a similar construct was made. Furthermore, another alternative of fusion protein was also constructed where the same E fragment from DEN-1 was fused to the C-terminus of the P64k protein. The recombinant proteins obtained (PD11 and PD10) were semi-purified and analysed for their antigenicity, immunogenicity and the ability to protect mice against lethal challenge. Both molecules exhibited the same recognition patterns against anti-DEN-1 polyclonal antibodies. In addition, when administered to mice, they elicited high levels of neutralizing antibodies and induced significant protection against lethal challenge with DEN-1 after intracerebral inoculation. These results reveal the availability of two sites within the P64k for the further insertion of DEN fragments, enabling a construct carrying two fragments from heterologous serotypes within the same molecule of this protein carrier.

Complete coding sequences of dengue-1 viruses from Paraguay and Argentina

We have determined the complete coding sequences of six dengue-1 (DEN-1) viruses isolated from Paraguay and Argentina in 2000 from patients with dengue fever. Sequences of strains 259par00, 280par00, 295arg00, 297arg00 and 301arg00 can encode a polyprotein of 3392 amino acids. Strain 293arg00 circulated as a “wild type+deletion mutant” quasispecies, with a subpopulation characterized by a 3-nucleotide deletion in the NS4A region. This variant, which would encode a three amino acid change in the NS4A protein, was found as a minority population in one additional partially-sequenced isolate from the same outbreak. These six South American strains group into two different clades of the “American–African” DEN-1 genotype—one clade is most closely related to strains isolated from Brazil in 1997, the other to a Peruvian strain isolated in 1991 for which only partial sequence information is available. DEN-1 viruses isolated worldwide comprise at least four different genotypes according to previously defined classification criteria.

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

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-MK(2) 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.

Diverse dengue type 2 virus populations contain recombinant and both parental viruses in a single mosquito host.

Envelope (E) protein genes sampled from populations of dengue 2 (DEN-2) virus in individual Aedes aegypti mosquitoes and in serum from dengue patients were copied to cDNA, cloned, and sequenced. The nucleotide sequences of the E genes in more than 70% of the clones differed from the consensus sequence for the corresponding virus population at up to 11 sites, and 24 of the 94 clones contained at least one stop codon. Virus populations recovered up to 2 years apart yielded clones with similar polymorphisms in the E gene. For one mosquito, the clones obtained fell into two genotypes. One group of sequences was closely related to those of viruses recovered from dengue patients in the same locality (Yangon, Myanmar) since 1995 and were classified as Asian 1 genotype. The second group were Cosmopolitan genotype viruses which were also circulating in Yangon in 2000 and which were related to DEN-2 viruses sampled from southern China in 1999. Finally, one clone was identified as a recombinant genome composed of portions of these two "parental" genotypes. This is the first report of recombinant and parental dengue viruses in a single host.

Molecular evolution and phylogeny of dengue type 4 virus in the caribbean

We sequenced the E gene and adjacent prM/M and NS1 junctions (1940 bp) of 48 Dengue-4 (DEN-4) isolates collected between 1981 and 1999 from 8 Caribbean islands and from 7 South and Central American countries. Phylogenetic analysis confirms a single introduction in the early 1980s and a high degree of gene flow resulting in a pattern of evolution defined more by time period than geographic origin, especially within the Caribbean basin. A modern Caribbean clade consisting of four distinct lineages has arisen, comprised of isolates from Caribbean islands and nearby regions of South America. This clade is defined by three amino acid substitutions in the E (aa 163 and 351) and NS1 (aa 52) proteins. These findings highlight the importance of migration and gene flow in dengue viral change and suggest that efforts to understand disease dynamics in the Caribbean basin need to focus at regional, rather than local scales.

Phylogenetic relationships of dengue-1 viruses from Argentina and Paraguay.

We sequenced the Capsid-pre Membrane (C/prM) and the Envelope-Nonstructural protein 1 (E/NS1) regions of 24 recent isolates of dengue-1 (DEN-1) from South America. This included 12 Argentinean and 11 Paraguayan DEN-1 strains isolated in 2000 plus a Paraguayan strain isolated in 1988. These sequences were compared with published sequences of DEN-1 isolated worldwide to determine the origin of these isolates. Pairwise comparisons of strains from Paraguay and Argentina revealed a nucleotide divergence of 0-5% in the E/NS1 region and 0-3% in the C/prM region. Our results showed that these viruses belong to the same genotype, but can be separated into two clades. Interestingly, both clades circulated simultaneously in the same geographic area during the 2000 outbreaks. Amino acid differences were found between both clades in the C/prM region at position 100 (Lys vs. Arg) and in the E/NS1 region at positions 722 (Ala vs. Thr). Although the geographic movement of DEN-1 virus can not be unequivocally traced from the genetic relationship determined here, our results suggest that the recent epidemics in Argentina and Paraguay were due to the re-emergence of a previously circulating strain, or to the virus circulating unnoticed, rather than to the introduction of a new genotype.

The reappearance of dengue-3 and a subsequent dengue-4 and dengue-1 epidemic in Puerto Rico in 1998.

In January 1998, dengue-3 (DEN)-3 (group III genotype) was detected in Puerto Rico after an absence of 20 years. Public health officials intensified education efforts to promote community participation in dengue control. Virologic surveillance revealed an unexpected paradox: DEN-4 and DEN-1 produced a large epidemic overlaying the DEN-3 epidemic. In 1998 there were 17,000 reported cases of dengue (4.8/1,000 persons), and among all virus isolations (n = 960), DEN-4 (419, 43.6%), DEN-1 (337, 35.1%), and DEN-2 (143, 14.9%) were detected much more frequently than DEN-3 (61, 6%). Age group-specific attack rates were highest for persons 10-19 years old, followed by infants less than a year of age. Nineteen fatal cases (median = 37 years old, range = 8 months to 90 years) had a positive laboratory diagnosis of dengue. Among DEN-3 cases no fatalities were documented, 50 were hospitalized, and 10 of 48 (21%) fulfilled the criteria for dengue hemorrhagic fever (four had primary infections and six had secondary infections). During 1999, DEN-3 became the predominant serotype isolated (182 of 310 isolations, 59%). The reappearance of DEN-3 and its subsequent circulation from 1999 to 2001 produced no changes in dengue incidence that could have been detected in the absence of virologic surveillance.