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Wild populations of nonhuman primates live in regions of sylvatic arbovirus transmission. To assess the status of arbovirus transmission in Bornean forests and the susceptibility of wild orangutans to arboviral infection, blood samples of wild orangutans, semi-captive orangutans, and humans were examined. Samples were tested by plaque reduction neutralization test for antibodies to viruses representing three families (Flaviviridae, Alphaviridae, and Bunyaviridae), including dengue-2, Japanese encephalitis, Zika, Langat, Tembusu, Sindbis, Chikungunya, and Batai viruses. Both wild and semi-captive orangutan groups as well as local human populations showed serologic evidence of arbovirus infection. The presence of neutralizing antibodies among wild orangutans strongly suggests the existence of sylvatic cycles for dengue, Japanese encephalitis, and sindbis viruses in North Borneo. The present study demonstrates that orangutans are susceptible to arboviralinfections in the wild, although the impact of arboviral infections on this endangered ape remain unknown.

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.

A commercial IgM immunoblot kit was evaluated for dengue diagnosis with a panel of serum specimens collected from patients in a dengue endemic area. The kit is not recommended for use in its present form because of its undesirable rate of false-positive results. However, by substituting internal controls with the reference positive and negative controls that are more representative of those seen in endemic areas and by modifying the positive and negative scoring criteria, sensitivity and specificity of 80.3% and 94.5%, respectively, were obtained. These results are comparable with those obtained with the IgM ELISA on specimens, most of which were obtained from outpatient health care facilities. With further technical modifications, inclusion of a visual guide to ensure scoring standardization, and a more complete elaboration of the limitations of the test, wide application of the kit in diagnostic laboratories should be possible.

Outbreaks of yellow fever (YF) have never been recorded in Kenya. However, in September 1992, cases of hemorrhagic fever (HF) were reported in the Kerio Valley to the Kenya Ministry of Health. Early in 1993, the disease was confirmed as YF and a mass vaccination campaign was initiated. Cases of suspected YF were identified through medical record review and hospital-based disease surveillance by using a clinical case definition. Case-patients were confirmed serologically and virologically. We documented 55 persons with HF from three districts of the Rift Valley Province in the period of September 10, 1992 through March 11, 1993 (attack rate = 27.4/100,000 population). Twenty-six (47%) of the 55 persons had serologic evidence of recent YF infection, and three of these persons were also confirmed by YF virus isolation. No serum was available from the other 29 HF cases. In addition, YF virus was isolated from a person from the epidemic area who had a nonspecific febrile illness but did not meet the case definition. Five patients with confirmed cases of YF died, a case-fatality rate of 19%. Women with confirmed cases of YF were 10.9 times more likely to die than men (P = 0.010, by Fisher's exact test). Of the 26 patients with serologic or virologic evidence of YF, and for whom definite age was known, 21 (81%) were between 10 and 39 years of age, and 19 (73%) were males. All patients with confirmed YF infection lived in rural areas. There was only one instance of multiple cases within a single family, and this was associated with bush-clearing activity. This was the first documented outbreak of YF in Kenya, a classic example of a sylvatic transmission cycle. Surveillance in rural and urban areas outside the vaccination area should be intensified.

The first recorded outbreak of yellow fever in Kenya occurred from mid-1992 through March 1993 in the south Kerio Valley, Rift Valley Province. We conducted entomologic studies in February-March 1993 to identify the likely vectors and determine the potential for transmission in the surrounding rural and urban areas. Mosquitoes were collected by landing capture and processed for virus isolation. Container surveys were conducted around human habitation. Transmission was mainly in woodland of varying density, at altitudes of 1,300-1,800 m. The abundance of Aedes africanus in this biotope, and two isolations of virus from pools of this species, suggest that it was the principal vector in the main period of the outbreak. A third isolate was made from a pool of Ae. keniensis, a little-known species that was collected in the same biotope. Other known yellow fever vectors that were collected in the arid parts of the valley may have been involved at an earlier stage of the epidemic. Vervet monkeys and baboons were present in the outbreak area. Peridomestic mosquito species were absent but abundant at urban sites outside the outbreak area. The entomologic and epidemiologic evidence indicate that this was a sylvatic outbreak in which human cases were directly linked to the epizootic and were independent of other human cases. The region of the Kerio Valley is probably subject to recurrent wandering epizootics of yellow fever, although previous episodes of scattered human infection have gone unrecorded. The risk that the disease could emerge as an urban problem in Kenya should not be ignored.

This study presents the disability-adjusted life years (DALYs), a non-monetary economic measure of impact, lost to dengue in Puerto Rico for the period 1984-1994. Data on the number of reported cases, cases with hemorrhagic manifestations, hospitalizations, and deaths were obtained from a surveillance system maintained at the Dengue Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention (San Juan, PR). The reported cases were divided into two age groups (0-15 years old and >15 years old), and then multiplied by predetermined factors (10 for 0-15 years; 27 for >15 years) to allow for age-related under-reporting of cases. Severity of dengue was modeled by classifying cases into three groups: dengue fever, dengue with severe manifestations, and hospitalized cases. Each group was assigned a different number of days lost because of dengue-related disability. Dengue caused an average of 658 DALYs per year per million population (SE = 114, range = 145-1,519). A multivariate sensitivity analysis, which simultaneously altered the values of six input variables, produced a mean of 580 DALYs/year/million population, with a maximum average of 1,021 DALYs/year/million population, and a maximum, single-year estimate for 1994 of 2,153 DALYs/million population. The most important input was the number of days lost to classic dengue. The DALYs/year/million population lost to dengue in Puerto Rico are much greater than previous estimates concerning the impact of dengue hemorrhagic fever alone. The loss to dengue is similar to the losses per million population in the Latin American and Caribbean region attributed to any of the following diseases or disease clusters; the childhood cluster (polio, measles, pertussis, diphtheria, tetanus), meningitis, hepatitis, or malaria. The loss is also of the same order of magnitude as any one of the following: tuberculosis, sexually transmitted diseases (excluding human immunodeficiency virus), tropical cluster (e.g., Chagas' disease, leishmaniasis), or intestinal helminths. The results objectively suggest that when governments and international funding agencies allocate resources for research and control, dengue should be given a priority equal to many other infectious diseases that are generally considered more important.

A dengue fever/dengue hemorrhagic fever (DF/DHF) outbreak in Yap State caused by dengue-4 virus was confirmed serologically and by virus isolation from serum samples collected on each of three island groups. Most DF/DHF cases occurred during a three-month period between mid-May and early August 1995. Five fatal cases, three of which were in children between the ages of four and 11, occurred between June 20 and July 26. A serosurvey conducted in late August revealed anti-dengue IgM prevalence rates of 18% on Yap, 36% on Eauripik, and 6% on Woleai. The majority of residents (93-100%) on the three islands were positive for anti-dengue IgG antibodies, indicating widespread exposure to dengue viruses. The IgG titers indicative of secondary antibody response were noted on Eauripik (6.5%) and Woleai (17%), but were rare on Yap (0.7%). Entomologic investigations implicated the native mosquito species, Aedes hensilli, a member of the Scutellaris Group of Aedes (Stegomyia), as a previously unrecognized epidemic vector of dengue viruses. Aedes hensilli was the most abundant and widespread member of Ae. (Stegomyia) in Yap State, the only species of Ae. (Stegomyia) on Woleai, and the only mosquito species present on Eauripik. New distribution records for mosquito species are reported.

The etiologic spectrum of acute encephalitis syndrome (AES) has not been well defined in Vietnam. Cohort and case-control studies were performed on all adult and pediatric AES patients admitted to the Neurology Service of Bach Mai Hospital between June 5 and August 3, 1995. Among pediatric AES patients, 31 (67%) of 46 had acute Japanese encephalitis (JE), compared with only two (6%) of 33 adult AES patients (P < 0.0001). For confirmed JE cases, serum specimens obtained 15-21 days after symptom onset had the highest mean anti-JE IgM signal-to-noise (P/N) ratios (8.08 + 1.09 SE). A serosurvey of adult household members did not reveal any cases of recent subclinical JE infection, although 26% had evidence of past JE infection. The use of bed netting was nearly universal but did not appear to reduce the risk of AES or JE. Given the high incidence of JE, particularly among children, Vietnam seems well suited for the development of a targeted JE vaccination strategy.

Single-strand conformation polymorphism (SSCP) analysis was used to characterize genetic polymorphisms among 12 isolates of dengue-2 virus, which were previously genetically characterized by RNase T1 oligonucleotide mapping and by sequencing the viral envelope (E) gene. Specific cDNA fragments from the dengue-2 isolates were amplified by the reverse transcriptase-polymerase chain reaction. The viral E, premembrane (prM), and nonstructural 5 (NS5) gene cDNAs of 291 basepairs (bp), 291 bp, and 201 bp, respectively, were denatured, rapidly chilled to promote intrastrand reassociation, electrophoretically separated on nondenaturing polyacrylamide gels, and SSCP patterns were observed by silver staining. The SSCP analysis revealed polymorphisms among a number of dengue-2 isolates from the same topotype, and these were markedly different between isolates of different topotype (distinct genetic group). Comparison of nucleotide sequence and SSCP analyses of the 291-bp E cDNA demonstrated that virus isolates that produced identical SSCP patterns contained 0-7 nucleotide substitutions, whereas isolates that showed different SSCP patterns contained 4-25 nucleotide substitutions. Positive predictive value and negative predictive value as measures of certainty for predicting identical and different sequences were 26% and 100%, respectively. The SSCP patterns of the 12 dengue-2 isolates suggested greater genetic variation in the prM gene region than in either the E or NS5 gene regions. The SSCP analyses should allow easy, sensitive, and rapid screening of dengue viruses isolates and the assessment of variation at a number of sites in the virus genome. Additionally, SSCP screening of dengue-2 virus for genetic variability may reveal the introduction of new viral genotypes in a given geographic area. These genetic variants of the virus could serve as markers of the epidemic potential of the virus strain.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

During studies of arboviral epidemiology in Vietnam, five virus isolates were recovered from Culex tritaeniorhynchus mosquitoes. Three of the five isolates were identified as strains of Japanese encephalitis virus, but the others, collected at Me Tri village, Hanoi, were shown to represent an alphavirus, for which we propose the name Me Tri virus. This newly recognized virus is most closely related to Semliki Forest virus. The two isolates appear to be antigenic subtypes of a single virus, and each was associated with central nervous system illnesses in children. Serologic surveys indicate widespread distribution of these viruses in both humans and livestock in Vietnam. We suggest that Me Tri virus is an etiologic agent of human disease in southeast Asia.

Dengue viruses are maintained in endemic transmission cycles in tropical urban areas where epidemics periodically occur. Until about 30 years ago, there were long intervals (10-40 years) between epidemics but they are now occurring in many areas at 3-5-year intervals. These epidemics are most likely caused by virus strains with different epidemic potential. Accompanying this increased frequency in epidemic activity has been a change in the disease pattern with cases of the severe form of dengue (dengue hemorrhagic fever) becoming much more common. The occurrence of these factors and the expanding geographic distribution of dengue hemorrhagic fever in the past 15 years have made it necessary to re-evaluate currently recommended methods for prevention and control. The result has been increasing emphasis on the development of effective sustainable Aedes aegypti control programs based on source reduction using community participation. A brief overview of global programs using this approach is presented with emphasis on the Puerto Rican program, one of the earliest developed.

During 1990 and 1991, adult mosquitoes were collected along the Ob River and its tributaries in western Siberia from approximately 51 degrees 18'N to 66 degrees 4'N. Fifteen virus strains were isolated from 74,196 mosquitoes tested in 1,874 pools. These included Tahyna virus from Aedes cataphylla-punctor subgroup (one) and Ae. excrucians (one), and Inkoo (INK) virus from Ae. communis (one), Ae. communis subgroup (one), Ae. hexodontus (two), Ae. punctor subgroup (two), Ae. punctor complex (one), and unidentified Aedes species (three). In addition, a single Ae. euedes yielded a strain of snowshoe hare (SSH) virus and a strain of Getah, an alphavirus. A Bunyamwera serogroup virus was isolated from Ae. excrucians. With the exception of the two isolates from a single mosquito, minimum infection rates among mosquito taxa ranged from 0.4 to 16.7 per 1,000. The INK virus isolates were widely distributed geographically; however, seven of the 10 isolates were from two sites north of the Arctic Circle. During 1991, sera from two mouse species, five vole species, and four shrew species were collected along the upper Ob River for serologic tests. The prevalence of neutralizing antibody to SSH virus in these sera was 80%. Prevalence rates in the four most abundant species were Apodemus agrarius, 73%; Clethrionomys rutilus, 71%; Microtus arvalis, 80%; and Sorex araneus, 91%. This is the first attempt to clarify the vector and vertebrate host relationships of California serogroup viruses in western Siberia.

Dengue 4 (DEN-4) virus strain 341750 Carib was modified by serial passage in primary canine kidney (PCK) cell cultures. By the 15th PCK passage, this virus was less infectious for monkeys and resulted in a significantly reduced viremia as compared to the parent DEN-4 virus. The 30th PCK passage of DEN-4 341750 Carib was non-infectious for monkeys. A vaccine prepared at the 20th PCK passage in DBS-FRhL-2 cells stimulated the production of both neutralizing and hemagglutination inhibition antibodies in monkeys; these animals were also protected against challenge with the homologous strain as well as a heterologous strain of DEN-4. An ID50 titration in monkeys resulted in a titer of greater than 10(4) plaque-forming units (PFU) for the vaccine virus and 0.5 PFU for the parent virus. Reduced monkey infectivity of this magnitude has been correlated with human attenuation in previous dengue vaccine candidates. The DEN-4 strain 341750 Carib PCK-20/FRhL-4 vaccine has been characterized and sufficiently tested to be considered for safety and immunogenicity trials in humans.

During the fall of 1981, a new method for the routine isolation and identification of dengue viruses in Puerto Rico was implemented utilizing C6/36 cell cultures and serotype specific antidengue monoclonal antibodies. A blind comparison of the monoclonal antibody indirect fluorescent antibody test (IFAT) and the complement fixation (CF) test for identification of 89 newly isolated dengue viruses of all four serotypes from the Caribbean, Asia and Africa showed 100% agreement. Although virus isolation rates were slightly lower than with the mosquito inoculation technique, use of the C6/36 cell culture system was much less time-consuming and allowed the processing of larger numbers of sera. Beginning in November 1981, a new virologic surveillance system was begun in Puerto Rico. Acute sera from persons with suspected dengue were selected for virus isolation attempts on the basis of geographic area of residence on the island, day after onset the blood was taken and clinical signs and symptoms. These sera were processed for virus isolation in C6/36 cell cultures, and virus isolates were identified by the IFAT using the monoclonal antibodies. Using this system, 2,702 sera were tested from November 1981 through August 1982. Dengue virus was isolated from 518, for an isolation rate of 19.2%. Dengue 1 was the predominant virus until December 1981, when dengue 4 became dominant. The changing patterns of dengue 1 and 4 distribution by time and geographic location on Puerto Rico were followed. This system allows the dengue viruses being transmitted in an area to be monitored with a minimal amount of effort and provides the early warning capability necessary to predict epidemic dengue.

The first documented case of concurrent human infection with 2 dengue viruses is reported. Dengue 1 and 4 viruses were isolated from the serum of a 16-year-old male during the 1982 outbreak in Puerto Rico. The illness was mild and does not support the hypothesis that double infection with dengue viruses leads to more severe hemorrhagic disease.

Clinical observations were made on 95 serologically or virologically confirmed dengue fever cases during an epidemic in a rural area of Indonesia in December 1976. The age distribution was similar to that observed in patients with dengue hemorrhagic fever in Jakarta, a highly endemic urban area. The observed disease ranged in severity from undifferentiated fever to shock and death. The majority of patients had acute onset of fever with nausea, vomiting, headache, and abdominal pain. Hepatomegaly was observed in only 19% of the patients. A positive tourniquet test was the most frequently observed hemorrhagic manifestation, but epistaxis was observed in 20% and hematemesis in 6% of the patients. Dengue shock syndrome was observed in 37% of the patients. There were four deaths, three of which were confirmed as due to dengue infection by virus isolation. The data suggest that one, and possibly two, of the fatal cases with virus isolation were primary infections, based on the results of hemagglutination-inhibition test using all four dengue antigens.

Laboratory studies were carried out with two geographic strains of Aedes polynesiensis and one strain of Aedes aegypti to determine whether they could transmit Ross River virus (RRV). Both species were shown to be good vectors of RRV, but Ae. polynesiensis was the most susceptible. Ae. polynesiensis represents a new vector for this virus and the epidemiologic implications of RRV spread by both mosquito species are discussed.

Serum specimens from Puerto Rican residents were tested for antibodies to human T lymphotropic virus type I (HTLV-I) using an enzyme immunoassay, Western immunoblot, and radioimmunoprecipitation assays. Of 1,279 specimens obtained during a dengue virus surveillance program in 1986 and 1987, 3 (0.2%) tested positive; an additional 11 were indeterminate. Of 602 specimens obtained from blood donors in Ponce in 1987, 1 (0.2%) was positive; an additional specimen was indeterminate. Of 21 persons hospitalized for problems related to intravenous drug use in 1986 and 1987, 1 (5%) tested positive for HTLV-I antibodies.

An epidemic of Ross River virus infection occurred in the Cook Islands early in 1980 and affected the majority of the inhabitants of Rarotonga, the most populated island in the group. This represents the easternmost extension of the virus which, until 1979, was believed limited to Australia, New Guinea, and the Solomon Islands. The clinical manifestations of Ross River disease, predominantly polyarthritis, did not differ significantly from those observed previously in Australia. However, unlike the experience in Australia, where Ross River virus has never been isolated from a patient with polyarthritis, the agent was recovered from the serum of one-half of approximately 100 such patients with serologically proven infections. It is not known if this latter observation is the result of a change in the virus, the different virus isolation technique employed, or other factors. It was found that the incubation period of the disease could be as short as 3 days--much less than previously suspected. Ross River virus was isolated from six pools of Aedes polynesiensis mosquitoes collected in nature and it appeared that this species was the most probable vector on Rarotonga. In view of the widespread distribution of Ae. polynesiensis on islands, in the eastern Pacific it would not be surprising if Ross River virus occurs in other previously unaffected areas in the future.

Transovarial transmission of La Crosse virus by experimentally infected Aedes albopictus females to 2.7% of their F1 generation offspring was demonstrated. Progeny of both sexes were infected. Mean virus titers in parent mosquitoes and infected F1 generation adults were 10(4.6) and 10(3.4) plaque forming units/insect, respectively. The La Crosse-infected offspring were randomly distributed among the female parents. After two serial passages in A. albopictus, a marked change occurred in the plaque morphology of the virus but this had no apparent effect on the subsequent vertical transmission rate. In contrast, transovarial transmission did not occur in La Crosse-infected Culex fatigans or in A. albopictus and C. fatigans infected with vesicular stomatitis-Indiana, Cache Valley, Batai, Arumowot, and Itaporanga viruses. Results of this experiment suggest that the La Crosse model might be useful in studying the mechanism of transovarial transmission in additional mosquito species.

Prior laboratory studies and field observations suggested that it might be possible to reduce the size of the population of, or eliminate, Aedes polynesiensis by the introduction of Aedes albopictus. The former mosquito is the principal vector of nonperiodic filariasis caused by Wuchereria bancrofti and the latter is a closely related species refractory to the development of human filariae. The practicability of such competitive displacement was studied by a field trial on a remote coral atoll where there was an established population of A. polynesiensis. Three strains of Al albopictus were liberated at separate localities on the atoll and their fate was followed for 4 years. One strain disappeared within 12 months after release and the other two disappeared within 48 months. It was not clear whether A. albopicuts failed to become established because the strains were unsuitable, the general environment was inappropriate, or A. polynesiensis was present in such numbers that A. albopictus rarely succeded in mating with its own species.

An outbreak of dengue type 2 infection occurred in the Pacific island Kingdom of Tonga in 1974 and an outbreak of dengue type 1 occurred there in 1975. The 1974 outbreak was characterized by relatively mild clinical disease with few hemorrhagic manifestations, a low attack rate, and relatively low viremia levels. The 1975 outbreak was characterized by relatively severe disease with frequent hemorrhagic manifestations and a high attack rate. The differences between the outbreaks could not be attributed to differences in abudance of, or susceptibility to infection of, mosquito vectors or to the prior immune status or other characteristics of the human population. It appeared that a difference in viral virulence was the most likely explanation.

Virological studies were carried out during an epidemic of dengue hemorrhagic fever in Central Java, Indonesia in 1976. Dengue virus was isolated from the acute sera of 45 of 69 patients (65%). The isolation rate was higher in primary than secondary cases. Dengue 3 was the predominant serotype being transmitted (27 isolates), but both dengue 1 (8 isolates) and dengue 4 (10 isolates) were also being transmitted. A composite picture of magnitude and duration of viremia showed that many patients were circulating over 10(8) MID50 per milliliter dengue 3 virus for the first 3 days of illness and that viremia persisted for 5-6 days in some persons. If all shock cases were considered, there was no relationship between dengue serotype and severity of disease. All three confirmed fatal cases, however, were associated with dengue type 3 infections.

A variety of methods have been employed to detect viral immune complexes (IC) in clinical specimens. However, most techniques used were not antigen-specific. We developed a simple, specific double antibody sandwich technique to detect artificial anti-dengue (DEN) IgM immune complex (IgM-IC). Positive reactivity with IgM-ICs prepared with live DEN-1, -2, and -3 viruses was found to be related to IgM titers exceeding 1:20 and to the titer of the viruses. Most IgM-ICs prepared with live DEN-4 virus did not react. In contrast, IgM-ICs prepared with hemagglutination antigens, representing all 4 serotypes, reacted positively with amounts of antigens ranging from 2 to 8 units. These IgM-ICs were not type-specific.

From January to April 1984, 63 Rattus rattus and 40 R. norvegicus were trapped in northeastern Puerto Rico and examined for Angiostrongylus cantonensis adults. Nineteen (47.5%) of the R. norvegicus and 10 (15.9%) of the R. rattus were infected, giving an overall infection rate of 28.2%. Four species of terrestrial snails and one species of brown slug were examined for A. cantonensis larvae. Two snail species, Subulina octona and Aquebana belutina, were found infected with third stage larvae of A. cantonensis. These larvae were harvested and inoculated per os into adult white mice. Immature adult worms were found in the brain tissue of all mice inoculated. This is the first report in the rat and snail populations of Puerto Rico.

An outbreak of dengue type 3 was studied in Central Java, Indonesia, in 1978. In contrast to previous dengue 3 epidemics in Central and East Java, this outbreak was less explosive, associated with mild illness, and low viremia. The dengue virus isolation rate from serologically confirmed patients was only 32% compared to 65% for an epidemic in Bantul a year earlier. Neither dengue hemagglutination-inhibition antibody titers nor day of illness on which specimens were collected accounted for this difference. These data suggest that some naturally occurring strains of dengue virus (endemic strains) are associated with low viremia and generally cause only mild illness in man.

The comparative susceptibility of 13 geographic strains of Aedes aegypti to oral infection with dengue viruses was studied by feeding the mosquitoes on a virus-erythrocyte-sugar suspension. Significant variation in susceptibility to four dengue serotypes was observed among the geographic strains tested. Mosquito strains which were more susceptible to one serotype were also more susceptible to the other serotypes, suggesting that the factors controlling susceptibility were the same for all types. The amount of virus required to infect mosquitoes orally varied inversely with the susceptibility of the geographic strain. Thresholds of infection were not the same for dengue types 1, 2, 3 and 4. There was no apparent difference in infectivity between prototype and recently isolated strains of dengue types 1 and 3. Crossing experimentibility as the resistant parent. No difference was observed between resistant and susceptible mosquito strains in the rate or the amount of viral replication after infection by the parenteral route, or in their ability to transmit dengue 2 virus after infection by the oral route.

Two types of variation were observed when the susceptibility of 16 different geographic strains of Aedes albopictus to oral infection with chikungunya (CHIK) virus was studied. One was differential susceptibility to infection with the virus. The other was variation in the quantity of virus present in infected mosquitoes after a standard incubation period. Mean virus titers of infected mosquitoes of different geographic strains varied almost 1,000-fold. Attempts to develop increasingly resistant or susceptible mosquito lines through genetic selection were unsuccessful. Infection rates did not change significantly despite 3 to 6 generations of selective inbreeding. In contrast, crosses between strains of high and low CHIK susceptibility yielded hybrid mosquitoes with infection rates and mean virus titers intermediate between those of the parent colonies. These data suggest that at least one factor controlling the susceptibility of A. albopictus to CHIK infection is genetic. Two lines of A. albopictus with a marked difference in susceptibility to oral infection with dengue viruses were equally susceptible to oral infection with chikungunya virus.

Interviews and physical examinations for filarial disease were conducted in Howrah near Calcutta, West Bengal, India, in a study area where long term quantitative entomological studies have disclosed high intensity, year round exposure to Wuchereria bancrofti. Little elephantiasis or other serious filariasis-related disease was noted, but half the males over 20 years of age had genital lesions of various types. Ninety-two percent of households had at least one resident with microfilaremia or presumptive clinical evidence of filarial disease.

We evaluated direct fluorescent antibody (FA) testing of peripheral blood leukocytes (PBL) from patients in Puerto Rico with serologically and/or virologically confirmed dengue fever as a possible rapid diagnostic test and compared rates of dengue virus isolation from PBL with the rates from plasma or serum using the mosquito inoculation technique. Dengue antigen was detected in the PBL of only 1 of 19 patients with confirmed dengue. Virus was isolated from 3 of 19 PBL specimens and from 6 of 19 acute-phase serum or plasma samples. Four viruses were obtained from serum or plasma only and 1 isolate came from PBL only. We conclude that FA testing of PBL from dengue fever patients has little promise as a rapid diagnostic technique. Despite small numbers, our data suggest that virus isolation from PBL is less sensitive than that from serum or plasma. Our results differ considerably from those of previous studies of dengue hemorrhagic fever patients conducted in Thailand.

Entomological studies were carried out during a dengue hemorrhagic fever epidemic in Central Java in December 1976. Both Aedes aegypti and Aedes albopictus were widely distributed in Bantul, but the latter species was more abundant. Comparative studies on the vector competence of the two species showed that Ae. albopictus had a higher susceptibility than Ae. aegypti to oral infection with all four dengue serotypes. The two species were equally compentent in transmitting the Bantul strain of dengue 3 virus after parenteral infection. The data suggest that Ae. albopictus could have been an important vector in this epidemic, but no direct observations were obtained to define the respective contribution of either Ae. albopictus or Ae. aegypti.

A previously reported case of childhood dengue shock syndrome in an American traveler to India was investigated serologically. The original studies neither indicated the infecting serotype nor proved primary or secondary infection. However, BHK suspension PRNT of 6-year convalescent serum now indicates that the child had primary dengue type 3 infection. Dengue, dengue hemorrhagic fever, and dengue shock syndrome are potential hazards for American travelers and American residents of dengue-receptive areas.

The first known transmission of dengue 3 virus in Africa was documented by virus isolation during an epidemic of dengue-like illness in Pemba, Mozambique, in late 1984 and early 1985. Dengue 3 virus was the only serotype isolated. Most patients appeared to be experiencing secondary flavivirus infections, but whether this was the result of previous dengue, yellow fever, or other flavivirus infection is not known. Two cases of hemorrhagic disease with shock and death were associated with the epidemic.

The comparative susceptibility to dengue virus infection of 13 geographic strains of Aedes albopictus was studied by feeding the mosquitoes on a virus-erythrocyte-sugar suspension. Significant variation in susceptibility for each of the four dengue serotypes was observed among the geographic strains. Mosquito strains which were more susceptible to infection with one dengue serotype also were more susceptible to the other dengue serotypes. There was a direct relationship between the amount of virus ingested and the infection rate in a given mosquito strain. A 100-fold difference in oral ID50 was noted between the most and the least susceptible strains. Crossing experiments between susceptible and resistant mosquito strains produced hybrid progeny with intermediate susceptibility. Susceptibility to infection by dengue 2 virus was decreased by selective inbreeding in one strain from 74% to 13% in two generations. Further selection, however, failed to produce a completely resistant line. It appeared that the actual "barrier" to infection was in the mosquito midgut.

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.

Aedes albopictus mosquitoes infected with dengue type 2 virus transmitted virus to measured small amounts of fluid which could be titrated readily for virus content. It was found that the percentage of mosquitoes transmitting was related to the extent of salivary gland infection. It was not uncommon for mosquitoes to transmit as much as 10(4) mosquito infectious doses50 of virus and transmission of significant amounts of virus was observed even though an insect only probed the test suspension without feeding to repletion. Transmission of virus was demonstrated as early as 10 days after oral infection when mosquitoes were held at 32 degrees C.

Thirty-four strains of Asian and Pacific mosquitoes belonging to 22 species of 7 genera were compared for oral and/or parenteral susceptibility to infection with 1 or more strains of each of the 4 dengue serotypes. Surprisingly, several species of common man-biting Aedes were much more susceptible to oral infection with each of the 4 dengue serotypes than was Aedes aegypti. These species included Aedes albopictus and members of the scutellaris group of the subgenus Stegomyia found on South Pacific islands. Mosquito strains and species relatively susceptible to 1 dengue serotype usually were relatively susceptible to the others also. Almost all species of Aedes tested were uniformly susceptible to parenteral infection with the dengue viruses but, with the exception of a species of Tripteroides, species of all other genera were comparatively resistant to that mode of infection. Dengue viruses usually replicated to about the same extent in orally-infected mosquitoes as they did in parenterally-infected specimens of the same species. Seventeen species of mosquitoes of 7 genera also were tested for parenteral susceptibility to infection with Japanese encephalitis virus. With the possible exception of 2 species of Anopheles, the virus replicated to about the same degree in all species tested and achieved levels considerably higher than did any of the dengue viruses in the same mosquito strain and species held under the same conditions.