The dengue and dengue hemorrhagic fever epidemic in Puerto Rico, 1994-1995.

From June 1, 1994 to May 31, 1995 a total of 24,700 cases of dengue (7.01/1,000 population) were reported to the laboratory-based surveillance system in Puerto Rico (1991-1994, annual average: 2.55/1,000). Dengue virus 2 predominated. The earliest indicator of epidemic activity was the virus isolation rate in May 1994 (14.0% versus 5.7% average). The male-to-female ratio among cases was 1:1.1; 65.4% were younger than 30 years (the 10 to 19 year age group had the highest incidence, 11.8/1,000). At least 5,687 cases (23.0%) showed a hemorrhagic manifestation; 4,662 (18.9%) were hospitalized, and 40 died (0.2%; 10 laboratory-positive). Two cases documented by laboratory were transmitted by unusual routes--intrapartum and through a bone marrow transplant. Among 2,004 hospitalized cases reported by infection control nurses, 139 (6.9%) fulfilled the criteria for dengue hemorrhagic fever (DHF) and another 13 cases (0.6%) had dengue shock syndrome. This epidemic produced the largest number of hospitalizations, DHF cases, and deaths from any dengue epidemic in Puerto Rico. Severity did not change throughout the year. Surveillance capabilities were maintained by temporary, simplified reporting methods, none of which could be recommended as the single method of choice for surveillance; each must be used (on site, or as a service available from a reference laboratory) at the right time in the epidemic cycle. The utility of comparisons of current and previous data underscores the value of long-term surveillance. Our analysis was unable to document whether significantly increased transmission occurred more often in cities where the water supply was rationed or where the local landfill was closed.

Dengue activity in Puerto Rico during an interepidemic period (1995-1997).

From 1995 to 1997 dengue was reported in Puerto Rico at an average annual rate of 1.75/1,000 population, compared to 6.73 in 1994, an epidemic year. Dengue virus serotypes 1 (DEN-1), -2, and -4 were isolated each year, with DEN-2 predominating in 1995 and 1996, and DEN-4 in 1997. From 1995 through 1997 incidence was highest (0.61-0.77/1,000) in persons under 30 years of age; males and females were equally affected. Among positive cases, 28.3% to 37.9% were hospitalized; 28.9% to 35.2% had hemorrhagic manifestations; at least 1.1% to 1.6% fulfilled the criteria for dengue hemorrhagic fever/dengue shock syndrome; and 0.2% to 0.3% died. Neither hurricane preparations (1995) nor widespread floods (1996) seem to have affected dengue incidence. Most municipalities with the highest laboratory-diagnosed dengue rates in 1995 were in the eastern foothills of the central mountains, an area relatively spared by the 1994 epidemic. In the next two years, at least half of the municipalities with the highest laboratory-diagnosed dengue rates were in the west. The most intense municipal outbreak of this period (DEN-2, Villalba, 1995, rate of 11.67/1,000) is described to highlight the importance of local conditions and epidemiologic history in determining the risk of dengue.

A dengue outbreak among camp participants in a Caribbean island, 1995.

BACKGROUND: Dengue, a mosquito-transmitted viral disease, is a risk for visitors in tropical and subtropical areas. Several participants in a community-assistance program in Tortola, British Virgin Islands, in August, 1995, reported dengue-like symptoms either before or soon after leaving the island. METHODS: We conducted a retrospective cohort study to determine the extent of the outbreak, risk factors for illness, and the proportion of inapparent infections. Program participants were interviewed by telephone or mail, and asked to submit a serum sample for dengue diagnosis. A clinically-diagnosed case of dengue was defined as a person with fever and two or more of the following: headache, retro-orbital pain, myalgia, arthralgia, rash, or hemorrhagic manifestations. Serum specimens were tested for virus isolation, polymerase chain reaction (PCR), plaque-reduction neutralization (PRNT) or anti-dengue IgM and IgG antibody. RESULTS: Thirty-two (97%) of the 33 program participants responded; 21 of the 32 (66%) provided at least one serum sample for study. The median age was 17 years; 20 (62%) were women. Of 32 respondents, 22 (69%) met the clinical case definition for dengue: 15 of them (68%) had a positive IgM antibody response and 7 did not submit a serum sample. Dengue 1 virus (DEN-1) was identified by PCR in one case and all 11 positive PRNT results. No asymptomatic infections were identified. No respondent used effective mosquito repellent, and only 2 (6%) used bednets. CONCLUSIONS: A DEN-1 outbreak with a high attack rate (69%) occurred in a group of young short-term community aid workers. There were no asymptomatic infections documented. Participants' rare use of bednets or effective mosquito repellent highlights the importance of providing travelers to tropical areas with information about dengue fever and the recommended precautions to protect against infection.

Common occurrence of concurrent infections by multiple dengue virus serotypes.

The co-circulation of all 4 dengue virus serotypes in the same community, common since the 1950s in Southeast Asia, has now become a frequent occurrence in many Caribbean Islands, Mexico, and Central and South America in the past 20 years. As a consequence, the frequency of concurrent infections would be expected to increase in these areas. To assess this, using state of the art technology, we screened viremic serum samples and mosquitoes inoculated with serum samples collected during epidemics involving multiple dengue virus serotypes in Indonesia, Mexico, and Puerto Rico for virus isolation. Of 292 samples tested, 16 (5.5%) were found to contain 2 or more dengue viruses by an indirect immunofluorescence test and/or the reverse transcriptase-polymerase chain reaction.

Dengue and dengue haemorrhagic fever.

The incidence and geographical distribution of dengue have greatly increased in recent years. Dengue is an acute mosquito-transmitted viral disease characterised by fever, headache, muscle and joint pains, rash, nausea, and vomiting. Some infections result in dengue haemorrhagic fever (DHF), a syndrome that in its most severe form can threaten the patient's life, primarily through increased vascular permeability and shock. The case fatality rate in patients with dengue shock syndrome can be as high as 44%. For decades, two distinct hypotheses to explain the mechanism of DHF have been debated-secondary infection or viral virulence. However, a combination of both now seems to be the plausible explanation. The geographical expansion of DHF presents the need for well-documented clinical, epidemiological, and virological descriptions of the syndrome in the Americas. Biological and social research are essential to develop effective mosquito control, medications to reduce capillary leakage, and a safe tetravalent vaccine.

Dengue in Spanish travelers returning from the tropics.

Dengue is seldom recognized as an imported disease in Europe. A prospective clinical study was carried out in 37 travelers suspected to have dengue infection on their return from dengue-endemic areas. Anti-dengue antibodies were found in 24 of 37 patients (14 recent infections and 10 undetermined). The most common features among the recent infections were fever (100%), thrombocytopenia (61.5%), abnormal liver function tests (61.5%), and rash (53.8%). In one case, denguehemorrhagic fever grade III was confirmed and later followed by depression and suicide of the patient. The possibility of local transmission of dengue in Spain is discussed.

Dengue surveillance--United States, 1986-1992.

PROBLEM/CONDITION: Dengue is an acute, mosquito-transmitted viral disease characterized by fever, headache, arthralgia, myalgia, rash, nausea, and vomiting. The worldwide incidence of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) increased from the mid-1970s through 1992. Although dengue is not endemic to the 50 United States, it presents a risk to U.S. residents who visit dengue-endemic areas. REPORTING PERIOD COVERED: 1986-1992. DESCRIPTION OF SYSTEM: Dengue surveillance in the 50 United States and the U.S. Virgin Islands relies on provider-initiated reports to state health departments. State health departments then submit clinical information and serum samples to CDC for diagnostic confirmation of disease among U.S. residents who become ill during or after travel to dengue-endemic areas and among residents of the U.S. Virgin Islands. In Puerto Rico, an active, laboratory-based surveillance program receives serum specimens from ambulatory and hospitalized patients throughout the island, clinical reports on hospitalized cases, and copies of death certificates that list dengue as a cause of death. Laboratory diagnosis relies on virus isolation or serologic diagnosis of disease (i.e., IgM or IgG antibodies against dengue viruses). RESULTS: In 1986, the first indigenous transmission of dengue in the United States in 6 years occurred in Texas; from the time of that incident through 1992, however, no further endemic transmission was reported. During 1986-1992, CDC processed serum samples from 788 residents of 47 states and the District of Columbia. Among these 788 residents, 157 (20%) cases of dengue were diagnosed serologically or virologically. Of the 157 patients, 71 (45%) had visited Latin America or the Caribbean; 63 (40%), Asia and the Pacific; seven (4%), Africa; and nine (6%), several continents. All four dengue virus serotypes (DEN-1, DEN-2, DEN-3, and DEN-4) were isolated from travelers to Asia and the Pacific; however, travelers to the Americas acquired infections with only DEN-1, DEN-2, or DEN-4. Even though the number of laboratory-diagnosed dengue infections among travelers was small, severe and fatal disease was documented. In the U.S. Virgin Islands and Puerto Rico, three serotypes (DEN-1, DEN-2, and DEN-4) circulated during 1986-1992. In Puerto Rico, disease transmission was characterized by a cyclical pattern, with peaks in incidence occurring during months with higher temperatures and humidity (usually from September through November). The highest incidence of laboratory-diagnosed disease (1.2 cases per 1,000 population) occurred among persons < 30 years of age; rates were similar for males and females.(ABSTRACT TRUNCATED AT 400 WORDS)

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.

We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

Study of anti-dengue NS1 antibody by western blot.

The presence of anti-nonstructural protein (NS1) antibody in natural dengue infections has been suspected to be associated with development of dengue haemorrhagic fever (DHF). The Western blot technique was used to study the dynamics of the immune response to NS1 and to determine the frequency of anti-NS1 antibody among confirmed dengue patients in Indonesia, where DHF is common, and in Puerto Rico, where DHF is less frequently observed. Anti-NS1 antibody was rarely found in those with primary infections in either group. The antibody occurred at a significantly higher frequency in acute-phase serum samples from secondary infections in Indonesia than in those from Puerto Rico. No difference was observed, however, in Indonesian patients with secondary infection who had dengue fever or DHF.

Dengue 2 virus envelope protein expressed by a recombinant vaccinia virus fails to protect monkeys against dengue.

A cDNA copy of the dengue (DEN) 2 virus genome region encoding the virion capsid, membrane and envelope structural proteins has been inserted into vaccinia virus (VV) DNA under the control of its 11K late promoter. The DEN-2 envelope protein was expressed and processed in cells infected with the VV recombinant (VV/D2S). No DEN-2 virus antibody response was detected in mice, hamsters or monkeys vaccinated with VV/D2S. Furthermore, a viraemia was observed in recombinant-vaccinated monkeys after challenge with infectious DEN-2 virus.

Partial nucleotide sequence of St. Louis encephalitis virus RNA: structural proteins, NS1, ns2a, and ns2b.

cDNA clones of the St. Louis encephalitis (SLE) virus genome have been obtained and the nucleotide sequence of 4.7 kb corresponding to the 5' terminal half of the genome determined. The genome contains a 5' noncoding region of 98 nucleotides followed by a single continuous open reading frame that encodes three structural proteins in the order capsid (C), membrane precursor (prM)-membrane (M), and envelope (E). Immediately following the C-terminus of E are located nonstructural proteins NS1 through NS3. The SLE amino acid sequence homology with yellow fever (YF), Murray Valley encephalitis (MVE), West Nile (WN), and dengue-2 (DEN) viruses over the sequenced region is 39, 66, 64, and 43%, respectively. The start of each SLE protein has been assigned on the basis of N-terminal sequence data and potential proteolytic cleavage sites homologous with YF and MVE viruses. Flaviviruses have conserved glycosylation sites in prM and NS1 proteins, although only one of the two glycosylation sites in the SLE E protein is conserved in MVE and DEN viruses. An evolutionary tree showing relationships of SLE, MVE, WN, YF, and DEN-2 flaviviruses is proposed on the basis of the amino acid sequences of the C proteins.

Genetic and epidemiological studies of dengue type 2 viruses by hybridization using synthetic deoxyoligonucleotides as probes.

A rapid nucleic acid hybridization procedure was developed for examining the genotypic variation of dengue type 2 viruses (DEN 2) having distinct RNase T1 fingerprints and isolated from different geographical areas. Synthetic DNA hybridization probes were constructed complementary in nucleotide sequence to common and unique RNase T1 oligonucleotides of topotype viruses from Puerto Rico/South Pacific, Jamaica, the Seychelles, Thailand/Burma and Africa. Hybridization probes with both type- and topotype-specific reactivities were observed, as were probes specific for two or more of the DEN 2 topotypes. These results confirm geographical movement of topotype virus strains and suggest possible origins. Detection of DEN 2 RNA by hybridization is a rapid and reproducible method that can be modified and applied as a viable alternative to the laborious T1 oligonucleotide fingerprinting.

Genetic and epidemiological studies of dengue type 2 viruses by hybridization using synthetic deoxyoligonucleotides as probes

A rapid nucleic acid hybridization procedure was developed for examining the genotypic variation of dengue type 2 viruses (DEN 2) having distinct RNase T1 fingerprints and isolated from different geographical areas. Synthetic DNA hybridization probes were constructed complementary in nucleotide sequence to common and unique RNase T1 oligonecleotides of topotype viruses from Puerto Rico/South Pacific, Jamaica, the Seychelles, Thailand/Burma and Africa. Hybridization probes with both type- and topotype-specific reactivities were observed, as were probes specific for two or more of the DEN 2 topotypes. These results confirm geographical movement of topotype virus strains and suggest possible origins. Detection of DEN 2 RNA by hybridization is a rapid and reproducible method that can be modified and applied as a viable alternative to the labourious T1 oligonucleotide fingerprinting.(AU)

Identification and characterization of Bahia Grande, Reed Ranch and Muir Springs viruses, related members of the family Rhabdoviridae with widespread distribution in the United States.

Sixteen virus isolates with similar biological characteristics were obtained from salt-marsh mosquitoes collected in south Texas in 1974. When compared antigenically, these and 13 other isolates from mosquitoes collected between 1972 and 1979 in west Texas, New Mexico, Louisiana, Colorado and North Dakota were shown to be related but not identical. Three distinct serotypes were determined: Bahia Grande (prototype strain TB4-1054), Reed Ranch (TB4-222) and Muir Springs (76V-23524). When examined by electron microscopy, these three viruses were shown to be rhabdoviruses. Structural analysis of the prototype strain of Bahia Grande virus from Texas revealed five proteins. Comparative oligonucleotide fingerprint maps showed 51 to 86% sharing of the large oligonucleotides between Bahia Grande virus (strain TB4-1054) and 11 other antigenically related isolates but not with Muir Springs virus (strain 76V-23524), an antigenically distinct isolate from mosquitoes collected in Colorado. A serological survey for antibody to Bahia Grande virus showed that humans, cattle, sheep, reptiles and wild mammals from south Texas had neutralizing antibodies to this virus.

Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses.

Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains.

Purification of small oligonucleotides by polyacrylamide gel electrophoresis and transfer to diethylaminoethyl paper.

The application of polyacrylamide gel electrophoresis with subsequent electroelution onto DEAE paper for the purification of small oligonucleotides is described. We demonstrate that synthetic DNAs and hydrolyzed RNAs as small as three nucleotides in length can be purified by this technique. The product is undegraded and homogeneous in length.

Purification of the structural and nonstructural proteins of St. Louis encephalitis virus.

We have developed a procedure for purifying both the structural and nonstructural proteins of flaviviruses from lysates of infected cell cultures. The procedure involves: immunoprecipitation to concentrate viral proteins and eliminate most of the cellular proteins, preparative polyacrylamide gel electrophoresis to separate the viral proteins, and hydroxyapatite chromatography, which eliminates most of the unlabeled cellular protein. This procedure offers an improvement over previous purification schemes in that there is no loss of viral proteins after the immunoprecipitation step, any combination of labeling isotopes may be used, and it is not necessary to soak proteins out of gel slices.

Interferon-mediated persistent infection of Saint Louis encephalitis virus in a reptilian cell line.

A persistent infection with Saint Louis encephalitis (SLE) virus in a poikilothermic cell line TH-1 (turtle heart cells) was studied. Infected TH-1 cells were subcultured weekly at 31 degrees C for 1 year and continued to produce low levels (10(2) to 10(3) p.f.u./ml) of virus without obvious cytopathic effects or marked cyclic events. Indirect fluorescent antibody and infectious centre assays indicated that less than 1% of the cells were producing detectable virus proteins or infectious virus. Defective-interfering particles, temperature-sensitive mutants and DNA provirus were not detected. Interferon (IFN) mediation of the persistent infection as considered since the persistently infected cells (PIC) and normal TH-1 cells were resistant to heterologous virus challenge after treatment with virus-free culture fluid from PIC. A direct relationship was found between the m.o.i. and the amount of IFN produced, plateauing at an m.o.i. of approx. 10. The reptilian IFN was physically and chemically similar to mammalian and avian IFN. Certain biological markers of the SLE virus changed during the persistent infection. It was less virulent for mice, showed distinct differences in cell culture host range and had increased thermal lability.