Dengue virus type 1

Dengue fever and dengue hemorrhagic fever (DF/DHF) are caused by the dengue viruses, which belong to the genus Flavivirus, family Flaviviridae. There are four antigenically related, but distinct, dengue virus serotypes (DEN-1, DEN-2, DEN-3 and DEN-4), all of which can cause DF/DHF.

All analyses undertaken to date show that the four serotypes of dengue virus are phylogenetically distinct, and often to the same degree as different "species" of flaviviruses.

The first dengue viruses were isolated from soldiers who became ill in Calcutta, India, New Guinea, and Hawaii. The viruses from India, Hawaii, and one strain from New Guinea were antigenically similar, whereas three other strains from New Guinea appeared to be different. They were called dengue 1 (DEN-1) and dengue 2 (DEN-2) and designated as prototype viruses (DEN-1, Hawaii and DEN-2, New Guinea-C).

Dengue virus type 1 (strain 836-1)

Philippine 836-1 strain isolated in 1984.

Dengue virus type 1 (strain 924-1)

Four DEN-1 virus strains were isolated from humans with classical DF: AHF82-80 (Thailand 1980), 836-1 (Philippines 1984, strain 162, AP2), 924-1 (Mexico 1983, strain 1378) and CV1636/77 (Jamaica 1977).

Dengue virus type 1 (strain AHF 82-80)

Thai AHF 82-80 strain isolated in 1980.

Dengue virus type 1 (strain CV1636/77)

Caribbean CV1636/77 strain isolated in 1977.

CV 1636/77 was isolated in 1977 in Jamaica.

Dengue virus type 1 (strain Singapore S275/90)

Singapore S275/90 strain, isolated from a patient with DHF in 1990.

Dengue virus type 1 (strain TH-SMAN)

TH-36 was isolated in 1958 by Hammon and co-workers from a patient with DHF in Bangkok. TH-Sman was isolated from a similar patient by Dr. Sman Vardhanabhuti.

Dengue virus type 1 (strain Western Pacific)

Western Pacific strain (West Pac) of DEN-1, isolated from a patient with a mild case of DF in 1974.

The parent DEN-1 strain used in these studies, 45AZ5 PDK-0, was derived from the human isolate, West Pac 74, made from a mild case of dengue fever during an outbreak on Nauru Island in the Western Pacific in 1974.

Dengue virus type 2

Dengue virus type 2 (isolate Malaysia M1)

Dengue-2 (DEN-2) viruses, MI, M2 and M3, isolated in Malaysia from patients with dengue haemorrhagic fever, dengue shock syndrome and dengue fever, respectively.

Dengue virus type 2 (isolate Malaysia M2)

Dengue-2 (DEN-2) viruses, MI, M2 and M3, isolated in Malaysia from patients with dengue haemorrhagic fever, dengue shock syndrome and dengue fever, respectively.

Dengue virus type 2 (isolate Malaysia M3)

Dengue-2 (DEN-2) viruses, MI, M2 and M3, isolated in Malaysia from patients with dengue haemorrhagic fever, dengue shock syndrome and dengue fever, respectively.

Dengue virus type 2 (NGC-prototype)

Isolated in 1944 from a DF patient in New Guinea.

Synonyms: Dengue virus prototype strain New Guinea C (NGC), Dengue virus type 2 (strain New Guinea C).

Dengue virus type 2 (strain 16681)

Isolated in 1964 from a DHF patient in Bangkok, Thailand.

Dengue virus type 2 (strain 16681-PDK53)

The Mahidol D2 vaccine virus, the PDK-53 strain, was derived by passage of the wild-type D2 16681 virus 53 times in PDK cells.

Dengue virus type 2 (strain D2-04)

Strain 04 isolated in 1985 from a human in China.

Dengue virus type 2 (strain Jamaica)

DEN 2 virus strain 1409 isolated in 1983 from a human in Jamaica.

Dengue virus type 2 (strain PR159/S1)

Strain PR159S1 isolated in 1969 from a human in Puerto Rico.

Dengue virus type 2 (strain PUO-218)

Strain PUO-218 isolated in 1990 from a human in Thailand.

Dengue virus type 2 (strain TH-36)

TH-36 was isolated in 1958 by Hammon and co-workers from a patient with DHF in Bangkok.

In 1958 an epidemic of hemorrhagic fever occurred in and near Bangkok, Thailand during the rainy season. The disease there was called Thai hemorrhagic fever. Over 2500 patients were hospitalized with about 10% case fatality rate. During the epidemic, Hammon et al. isolated several viruses both from human sera and from Aedes aegypti. Among these isolates a prototype strain named TH-36 (representing a number of apparently identical isolates) was found to be antigenically closely related to dengue type 2.

Dengue virus type 2 (strain Tonga 1974)

Dengue-2 virus isolated during an epidemic of benign dengue fever in Tonga in 1974.

Dengue virus type 3

Dengue virus type 4

40 to 60 nm in diameter, containing an electron dense core (about 30 nm in diameter) surrounded by a lipid bilayer.

Dengue viruses are spherical, lipid-enveloped viruses.

Flavivirus virions consist of a spherical ribonucleoprotein core surrounded by a lipoprotein envelope with small surface projections. The projections seen in electron micrographs are clarified by x-ray crystallography and represent molecules of envelope glycoprotein, which form rodlike structures anchored to the viral membrane at their basal ends. Envelope lipids constitute about 17% of the virion dry weight and are derived from the host cell lipids.

Dengue virus is transmitted in a cycle involving humans and mosquitoes, Aedes aegypti being the most important vector.

Two distinct transmission cycles have been described for DENV: Endemic and epidemic cycles that occur in urban/periurban environments and involve human reservoir and amplification hosts. The peridomestic mosquito Ae. aegypti is the principal DENV vector, with Ae. albopictus and other anthropophilic Aedes mosquitoes serving as secondary vectors. Ecologically distinct, sylvatic, enzootic cycles of DENV occur in west Africa and Malaysia, probably involving non-human primate reservoir hosts and sylvatic Aedes spp. mosquito vectors. The two kinds of DENV cycles are also evolutionarily distinct, and all four serotypes of endemic/epidemic DENV are believed to have evolved independently from sylvatic progenitors during the past few 1,000 years.

The four serotypes of dengue (DEN) virus belong to the genus Flavivirus in the family Flaviviridae. These are single-stranded positive-sense RNA viruses with a genome of about 11000 bases that codes for three structural proteins, C-prM-E; seven nonstructural proteins, NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5; and short non-coding regions on both the 5' and 3' ends.

10735 bp ss-RNA.

The genome of flaviviurses consists of a single-stranded RNA about 11 kilobases (kb) in length. This RNA contains a 5' cap [m(7)G51ppp5'A) at the 51 end and lacks a polyadenylate tail. Genomic RNA is the messenger RNA for translation of a single long open reading frame (ORF) as a large polyprotein.

The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-O and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-O was derived. Twenty-five (0.23%) nucleotide and 10 (0.29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-O, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine. These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-O and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0.23%) nucleotide and 11 (0.32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-O strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-O and 27.

10703 bp ss-RNA

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.

10,696 bp ss-RNA.

The complete nucleotide sequence of the genome of the dengue virus type 3 was determined. Sequence analyses of the genomic RNA and cloned cDNA revealed that the genomic RNA contains 10,696 nucleotides and encodes a single open reading frame of 10,170 nucleotides corresponding to 3390 amino acid residues. The N-terminal amino acid sequences of three structural proteins (C, M, and E proteins) and the preM protein were also determined from the purified virion. When the deduced amino acid sequence and N-terminal amino acid sequence determined from purified proteins were compared with those of other flaviviruses, the genome organization was found to be the same as that of other flaviviruses.

10,649 bp ss-RNA.

Unpublished sequence.

The first reported epidemics of dengue-like disease occurred on three separate continents almost simultaneously in 1779 and 1780. Although there is some disagreement as to whether all of these epidemics were caused by dengue viruses, it is clear that dengue and other arboviruses with similar ecology had widespread distribution in the tropics as long as 200 years ago. For the next 175 years major pandemics of dengue-like illness, occurred in Asia and the Americas at variable intervals ranging from 10 to 30 years. With the advent of modern diagnostic virology, and the isolation and identification of the four dengue virus serotypes, their distribution became better known. Asia historically has been the area of highest endemicity, with all four dengue serotypes circulation in the large urban centers of most countries. During and shortly after World War II, Ae. aegypti became more widespread in Asia, and with the subsequent urbanization that occurred in most countries, the incidence of dengue infection increased dramatically. This increase coincided with the emergence of epidemic DHF in the 1950s.

The factors responsible for this global resurgence of DF and the emergence of DHF include unprecedented population growth, unplanned and uncontrolled urbanisation, increased air travel, the lack of effective mosquito control, and the deterioration, during the past 30 years, of public health infrastructure.

The average number of DF/DHF cases reported to WHO per year has risen from 908 between 1950 and 1959 to 514,139 between 1990 and 1999. The real figure is estimated to be closer to 50 million cases a year causing 24,000 deaths. Of an estimated 500,000 cases of DHF/DSS requiring hospitalisation each year, roughly 5% die according to WHO statistics.

Asia. Epidemic DF was a common occurrence in Asia in the first 50 years of the 20th century. Epidemic waves would move through the region every 10 to 40 years, depending on when a new virus was introduced. DEN viruses were endemic in many cities of Asia during this time as documented by the numerous accounts of expatriates arriving in a tropical Asian city only to become ill with a severe dengue-like illness within weeks to months of arrival.

Maldives. Maldives has experienced an outbreak of dengue since January 2006, with 602 suspected cases until 5 March 2006 (including 64 cases of dengue haemorrhagic fever and 9 cases of dengue shock syndrome).

India. The new dengue paradigm viz, the burst of sudden disease activity, persistence and diffusion of the disease in different areas has secured a foot-hold in Southern India and has emerged as a serious public health problem. In Tamil Nadu, annual reports of dengue cases and deaths due to dengue were ranging from 128 to 264 and 2 to 21 respectively up to the year 2000. Recently, between October 2001 and January 2002, an epidemic of dengue emerged in Chennai, Tamil Nadu, affecting adults and children; majority of the victims were children less than 15 yrs of age.

Between October 2001 and January 2002, there was an epidemic of dengue in Chennai, with a peak in October. The case occurrence was reported to be high among pediatric group. The number of cases confirmed during the study period was considered as the representative of the cases reported (about 700 cases) to the health surveillance system in Chennai.

In India, dengue virus activity has been reported in many parts of the country with sudden epidemics over the last few years. Seasonal and cyclic epidemic pattern of dengue is a recent phenomenon in Northern India. The DF, DHF and DSS have spread dramatically in many parts of the country. Though all age groups were affected in these epidemics, the occurrence was high among children more than 6 yr; and few infants also presented symptoms of DHF.

Timor-Leste. As of 28 February 2005, WHO has received reports of 336 hospitalized cases of dengue infection and 22 deaths.Of the 336 cases, 263 had clinical features compatible with dengue haemorrhagic fever (DHF) and the remaining 73 cases were diagnosed as having suspected dengue fever (DF)using WHO standard case definitions. Districts reporting DF/DHF cases are Baucau, Dili, Ermera, Liquica, Maliana, Manatuto and Viqueque, with 76% of the cases reported from Dili. Preliminary laboratory results have identified Dengue 3 as the main circulating strain in this outbreak.

Cuba. During the past three decades there have been four major dengue epidemics in Cuba. The first which was widespread throughout Cuba occurred in 1977 and only dengue fever was observed. This epidemic was caused by an American genotype DENV-1 virus. Subsequently, two independent DHF epidemics caused by DENV-2 of Asiatic origin occurred in 1981 throughout Cuba and 1997 in Santiago de Cuba, four and twenty years respectively, after the epidemic caused by DENV-1

Brazil. The following study was intended to evaluate the occurrence of typical signs and symptoms in the cases of classic dengue and hemorrhagic dengue fever, during the 2001-2002 epidemic in the city of Rio de Janeiro. The authors reviewed 155,242 cases notified to the Information System of Notification Diseases, from January/2001 to June/2002: 81,327 cases were classified as classic dengue and 958 as hemorrhagic dengue fever, with a total of 60 deaths.

In early 2004, an outbreak of dengue began to spread throughout Indonesia. On 16 February 2004, the Indonesian Ministry of Health declared a national DF/DHF epidemic. Jakarta, the capital city with approximately 16 million inhabitants, was the most affected area

The Indonesian Ministry of Health reported cases of DF in 30 of 32 providences within the archipelago. In the capital city of Jakarta, a total of 20 503 cases were recorded, with an epidemic peak between March and April.

Palau. An epidemic of dengue 4 virus occurred in the island nation of Palau between January and July 1995. The last known epidemic of dengue in the Palau Islands occurred in 1988 when dengue type 2 was introduced. Before that, dengue transmission had not been reported since 1944. In 1995, higher than expected rainfall for January and February may have contributed to a large mosquito population and increased transmission of the virus. Increased rainfall has been reported previously to be associated with epidemic dengue fever.

During January and February 1995, 145 patients (an unusually high number) with viral syndrome were reported to the Palau Ministry of Health. On April 3, 1995, a 38-year-old man died at the Palau National Hospital soon after presentation with viral syndrome. He was noted to have had neutropenia and thrombocytopenia. Initially, an outbreak of leptospirosis was suspected (and later this patient was confirmed to have leptospirosis by immunohistochemical analysis). However, the majority of the initial serum samples from patients with febrile illness tested at the Centers for Disease Control and Prevention (CDC) Dengue Laboratory in San Juan, Puerto Rico were consistent with dengue virus infection, suggesting an outbreak of dengue fever.

Mosquito Human

After the mosquito becomes infective, it may transmit dengue by taking a blood meal, or by simply probing the skin of a susceptible person

Human Mosquito

The mosquito becomes infected by a blood meal from a viraemic person and becomes infective after an obligatory extrinsic incubation period of 10-12 days.

Human Human

Vertical transmission of dengue virus has been recorded in a small number of cases, leading to neonatal DF or even DSS. One case of nosocomial transmission from a needlestick injury has been reported.

The vertical transmission of dengue has been infrequently described world-wide, although there are reports from Cuba, Brazil, Malaysia, and Thailand which have occurred during outbreaks. These report variable neonatal outcomes, from asymptomatic infection to death.

Mosquito Mosquito

It was originally thought that all vertical transmission of arboviruses in mosquitoes was transovarial in nature because viral antigen has been demonstrated in developing eggs for bunyaviruses, and both bunyaviruses and flaviviruses have been recovered from progeny reared from surface-sterilized eggs. It was eventually discovered, however, that vertical transmission of dengue viruses, and at least certain other flaviviruses, takes place in the genital chamber of the female as mature eggs are fertilized during oviposition. This explains how vertical transmission of dengue viruses can occur without virus in developing eggs.

Progeny of Aedes aegypti mosquitoes infected intrathoracically with dengue-3 virus was reared to subsequent generations. In each generation, blood-fed females were confined individually and the eggs obtained from the transovarially infected females were pooled. The seventh generation obtained from the infected parental mosquitoes showed that virus could persist in mosquitoes in successive generations through transovarial passage. The rate of vertical transmission initially increased in the few generations (F1-F2), but in subsequent generations it was found to be steady.

These observations, which have great epidemiologic importance, suggest that vector mosquitoes may play an important role in the maintenance of virus in nature, and that mosquitoes may act as reservoirs of these viruses.

Male Ae. albopictus can transmit dengue virus sexually in the course of mating, and females can transmit it vertically more efficiently than can Ae. aegypti females. These two mechanisms could explain the maintenance of the virus in nature between epidemics in non-endemic areas where susceptible human or primate populations are not always present.

Human dengue viruses are mostly active in urban areas where the virus is maintained through a cycle in which humans are the principal reservoir host and Aedes aegypti is the principal mosquito vector.

The evidence that some Aedes (Stegomyia) species transmit dengue virus vertically suggests that they also could serve as reservoirs of the virus during dry season. their role in the epidemiology of dengue is not known in detail.

Dengue viruses multiply in the midgut epithelium, brain, fat body, and salivary glands of mosquitoes. No detectable pathologic changes result from infection, and mosquitoes remain infectious for life.

One or more dengue serotypes, transmitted by Aedes of the niveus group circulate in the forest canopy in primeval cycle among certain species of monkeys (Macaca sp. and Presbytis sp.-which have asymptomatic infections) in a silent cycle. Man is only occasionally involved in this cycle. Such a zoonotic reservoir of infection could exist in all the primary forests of tropical Asia: in Malaysia, in thailand, in Vietnam, in Cambodia, in Indonesia etc.

There is considerable field evidence from both Malaysia and Africa that lower primates are involved in forest maintenance cycles of dengue viruses. Moreover, experimental laboratory data show that chimpanzees, gibbons, and macaque are susceptible to infection with dengue viruses. All species develop detectable viremia in the absence of clinical illness. The experimental infection data suggest that dengue viruses have become well adapted to lower primates which, therefore, are not useful as laboratory animal models for the study of human disease.

Human

Homo sapiens

Humans are the major host of dengue virus.

There is no consensus on when dengue first appeared in human populations, largely because its symptoms are often not diagnostic. The earliest record suggested is from a Chinese medical encyclopaedia dating to 992 A.D.. However, it is generally agreed that by the late 18th century a disease bearing a strong resemblance to dengue was causing intermittent epidemics in Asia and the Americas, and that by the late 19th and early 20th centuries the virus was probably widespread in the tropics and subtropics. Shortly after World War II, a new dengue-associated disease was reported in endemically infected areas of Southeast Asia. This had a far more pronounced impact than DF, since the primary targets were children. The first well documented outbreak of what came to be known as dengue haemorrhagic fever took place in Manila in 1953/54, and was followed by a larger outbreak in Bangkok in 1958. Since this time DHF/DSS have become endemic in all countries in Southeast Asia, with dramatic increases in case numbers, so much so that dengue is considered an archetypal "emerging" disease.

Dengue viruses are efficiently transmitted in an urban cycle which involves man and Aedes aegypti, a day-biting species which often breeds in the clean water stored in houses. Infection with one type results in life-long immunity to that type but, after a short period of cross protection, individuals may become clinically ill during and infection with a second type.

Naturalistic methods involve changes to the natural environment designed to suppress the abundance of immature stages of vector mosquitoes. These measures may be either long-term, which are based on filling or draining of potential aquatic breeding sites for the vector, or short-term. On occasion, A. aegypti may breed in newly-constructed or abandoned septic tanks or latrines, and this may be prevented by draining or filling. More temporary naturalistic measures may include landscaping efforts to remove the vegetation that provides shade, food, or water collection that might contribute to the abundance of these vector mosquitoes; vegetation near the home may influence the abundance of A. aegypti. Where possible, brush should be cut back or removed from the immediate vicinity of homes. Treeholes and other natural rainwater receptacles should be filled with concrete, sand, packed earth, gravel, or other suitable materials. Stumps and other vegetation near houses that could become foci should be removed, or at least cut back annually.

One of the keys to the control of urban Aedes vectors, particularly A. aegypti, is improved domestic water supply.

Potable water must be delivered in sufficient quantity, quality, and consistency year-round in order to reduce the use of major breeding sites, duchas, drums, overhead tanks, and jars. Individual household piped water supplies are the preferred alternative to the use of wells, communal standpipes, rooftop catchments, and other water delivery systems.

We investigated the hypothesis that a deficient supply of piped water was causing a high prevalence of water storage containers, which in turn, become important aquatic habitats of Aedes aegypti in a small town in Venezuela. The House (71.2%) and Breteau indices (229) were considerably elevated. Prevalent positive containers were: metal drums, small disposable containers (bottles, tins, etc.), tires, house plants (flowers in vases and plants in pots with earth) and tanks. Most people reported frequent interruptions in the supply of piped water and considered it to be unreliable. The frequency of interruptions in the supply of water was positively correlated with the House and Container indices, and with the number of positive containers, water-storage devices and positive water-storage devices per house. Even people who considered that they had an adequate supply of water kept numerous water storage containers. Most people (60%) said they would not stop storing water even in the event of the establishment of a reliable supply of piped water.

Vector control efforts employing solid waste management protect public health and conserve natural resources. Proper storage, collection, and disposal of solid wastes protects the public health, while the reduction of the generation of wastes, reuse, and recycling conserve natural resources. Both approaches require community education and participation. Solid waste management for successful vector control consists of three aspects: waste reduction, recycling and re-use; collection; and appropriate disposal.

We established a waste recycling system and promoted a breeding site reduction campaign for waste management, including the application of Temephos in containers to kill larvae. For the drinking water management, fish were released in water containers to prevent larval breeding. It should be mentioned that with the integrated pest control and regular inspections of Aedes larvae in Taiwan the density figures 1, 2-5, and 6 or above for Aedes aegypti were 38.7%, 42.9%, and 18.4%, respectively, in 1988, and in 1993 were 90.8%, 9.2% and 0%. The incidence of dengue fever cases has 98% decreased since 1988. In 1990 and 1993, there was no indigenous cases. We have concluded that integrated pest control is the best and most effective method for dengue fever control, including solid waste and drinking water management.

Surveys were conducted in some townships along the national highways and trunk roads of northeast India to detect breeding of Aedes mosquitoes in used/waste tire dumps piled outdoors by the tire repairing shops during summer season of 1996-1997. The breeding of both the potential vectors of dengue, viz. Aedes aegypti and Ae. albopictus were detected, prevalence rate being in the range of 30.0-88.0 (CI = container index value). The preponderance of Ae. aegypti was considerably much higher than that of Ae. albopictus and all the urban and semiurban areas coming up along the side of the roads were observed to be infested with Ae. aegypti. With respect to transmission of dengue, this study clearly indicates that waste tire dumps in every urban agglomeration should receive primary attention in view of their relative contribution to the abundance and dispersal of these vector mosquitoes

Waste tire stockpiles are a significant public health (vectors) and safety (fire hazard) concern in cities, and imported used tires are believed responsible for the introduction of A. albopictus into the United States. Tires may be treated with insecticides, salt, or soap for chemical control of immature mosquitoes. New technologies for tire reuse and disposal are continually coming into use, but most of them have proved to be of limited application or cost-effectiveness.

VectoLex (22.6 kg) was used to treat approximately 6,000 car and truck tires; some of the tires were in direct sunlight whereas others were shaded. Aedes triseriatus was the dominant species in these tires. Tires treated with VectoLex contained significantly fewer mosquitoes than control tires, and even 65 days after application, control tires were 16.7 times more likely to contain larvae.

Larviciding of "focal" control of A. aegypti is usually limited to domestic-use containers that cannot be destroyed, eliminated, or otherwise managed. There are three insecticides that can be used for treating containers that hold drinking water: One percent temephos (Abate) sand granules applied to the containers using a calibrated plastic spoon to administer a dosage of 1 ppm. This dosage has been found to be effective for 8-12 weeks; The insect growth regulator methoprene (Altosid) is used in the form of briquettes; BTI (Bacillus thuringiensis H-14).

Trial tests and container observations were conducted in households to verify the residual effect of temephos in Manaus, Amazonas State, Brazil. Three plastic buckets, three tin cans, and three tires filled with water from an artesian well and larvicide were used in the experiment, with twenty-five third-instar larvae, which remained exposed for 24h, followed by mortality readings. The same types of containers were selected from common households. Collection and counts followed by chemical treatment were carried out on the larvae that were found. Follow-up was performed weekly to verify recolonization by Aedes aegypti. The experiment showed 100% mortality in the plastic buckets until day 90, and 80% in the tin cans until day 30, decreasing from day 45 onwards. Mortality in the tires decreased to 35% in the first month. Household results showed 100% mortality for all containers after 24h and differentiated values in the subsequent readings. Larvae were observed on day 35 in a tin can and on day 21 in a gallon can. There was a large diversity of results in the tires, with recolonization observed from day 7 onwards.

This dosage has been found to be effective for 8-12 weeks.

Methoprene-derived mortality occurs mainly at the pupa stage and pupa development is inversely proportional to methoprene concentration.

The tank bromeliad Billbergia pyramidalis was treated with 2 doses (0.5 and 2 g) of ALTOSID Granules or Pellets for the control of Aedes aegypti L. Emergence inhibition (EI) for all mosquito pupae (including natural populations) in the center wells and leaf axils was greater than 90% for at least 6 and 12 months for both doses of granules and pellets, respectively. No significant difference in %EI was found between center wells and leaf axils.

Space spraying involves the application of small droplets of insecticide into the air in an attempt to kill adult mosquitoes.

Pant and Yasuno demonstrated that 95% of A. aegypti rest indoors, and of these, greater than 90% do so on surfaces that could not be sprayed with residual compounds. Therefore the intervention commonly used during epidemics was and still is, the ground application of small quantities of an aerosol insecticide in gas oil or kerosene as carrier (ultra low volume (ULV).

Unfortunately many of the campaigns to reduce vector populations have not been successful due to practical problems associated with the treatments of densely populated areas, lack of funds and failure to sustain implementation of control programs as well as irritation problems in the inhabitants associated with the way compounds were applied.

Various fish can be used to eliminate mosquitoes from larger containers used to store potable water. These include Gambusia affinis and Poecilia spp. These and other fish may be potentially used where their introduction can be maintained and where the human population does not object to the presence of such obvious organisms in their domestic water-storage containers. Local, endemic larvivorous fish species also may be evaluated for efficacy against A. aegypti larvae.

Although various larval mosquitoes feed on other mosquitoes, Toxorhynchites mosquitoes have two advantages as predators: they develop in the same kinds of containers as A. aegypti and they do not feed on blood. Field trials have produced mixed results. On Union Island in Saint Vincent and the Grednadines, adult emergence was reduced when larval Toxorhynchites were introduced by hand, but the effect on adult abundance was not recorded. In Indonesia, sustained release of first instar predatory larvae into virtually all household water storage basins failed to reduce the abundance of adult A. aegypti. This lack of effect may have been due to an inability of first instar Toxorhynchites larvae to survive in the absence of small prey.

Each week, fifty Ae. aegypti first instar larvae were introduced to each of five water-filled drums (220 litres) of the type commonly used for domestic water storage in Caribbean dwellings. At the beginning of the fourth week, a certain number (0, 1, 2, 5 or 10) of first instar Tx. moctezuma larvae were introduced to each drum and the daily yield of Ae. aegypti adults from each drum was monitored thereafter. The experiment was repeated three times. With only one or two Tx. moctezuma larvae, predation on Ae. aegypti larvae stopped the output of Ae. aegypti adults for 1 week. Five or ten Tx. moctezuma prevented any Ae. aegypti emergence for up to 16 weeks.

The juvenile hormone (JH) analog methoprene is a larvicide that can be substituted for temephos in localities where Ae. aegypti exhibits resistance to this organophosphate.

Methoprene now promises to be the most environmentally acceptable larvicidal chemical usable against mosquitoes. The apparent absence of mammalian toxicity of this synthetic juvenile hormone permits its use in portable water.

Susceptibility of the Brazilian Ae. aegypti populations to methoprene alone suggests this insect growth regulator could substitute for temephos in the control of the dengue vector in the country.

Certain ubiquitous cyclopoid copepods ("water fleas") prey on newly hatched larvae. If the mosquitoes develop to the third instar, however, they are too large to be attacked by these minute predators.

Recently, copepods have emerged as a new tool for controlling mosquito vectors, particularly those inhabiting artificial containers. In Tahiti, Mesocyclops leuckarti (Claus) and Mesocyclops aspericornis (Daday) were successfully used against the dengue vector Aedes aegypti (L.) and in Honduras this mosquito was suppressed in peridomestic containers by Mesocyclops longisetus (Thiebaud). In New Orleans, Macrocyclops albidus (Jurine) eliminated Aedes albopictus (Skuse) from tire piles in a wooded area. More recently, in Vietnam, Mesocyclops sp. succeeded in eliminating Ae. aegypti

When copepods were present, the immature stages of Ae. albopictus were nearly eliminated. Macrocyclops and the mixture of three genera were the most effective in reducing the older instars of Ae. albopictus. Although predation pressure was high, a few Ae. albopictus pupated in all the treated containers.

Although these copepods suppressed Ae. albopictus in the 500 mL containers, this mosquito uses a wide variety of artificial containers as larval habitats and prefers to colonize cryptic microhabitats. Such behaviour makes it difficult to monitor all the productive sites. However, treatment of most of the artificial containers with predatory copepods such as those studied here could possibly reduce adult Ae. albopictus populations in our immediate environment.

Bacillus thuringiensis israelensis (BTI), which was discovered in the 1970s, is a proven, environmentally nonintrusive mosquito larvicide that appears to be entirely safe for people. This fermentation product of Bacillus thruingiensis H-14 is the most acceptable material currently on hand for use against mosquitoes. It has become commercially available under such trade names as Teknar, Vectobac, and Bactimos, and can be purchased in lots of up to one-quarter of a million pounds.

VectoBac DT, a tablet formulation of Bacillus thuringiensis israelensis (Bti) was evaluated for the potential control of dengue vectors in various types of potable water containers. On introduction to containers, the tablet sinks to the bottom and the Bti toxins are found concentrated at the sides and the base, while the treated water column is free of Bti toxins within 24 hours after tablet introduction. In a simulated study, earthen, HDPE and plastic containers were kept covered and laboratory-bred larvae were introduced to determine the control by the tablet.

The efficacy and persistence of the tablet, with a control of greater than 90%, was significantly longer in earthen containers in comparison to the HDPE and plastic containers. Efficacy and persistence were observed in earthen containers for a minimum period of 5.5 months (166 days) both without water replenishment and with weekly, 50% water volume, replenishment, and for a maximum period of 2.2 months (66 days) with daily, 50% water volume, replenishment. In plastic and HDPE containers, the tablet activity had a persistence of 2.1 months (63 days) without water replenishment and 1.8 months (54 days) with weekly water replenishment. The efficacy and persistence of the VectoBac DT was significantly longer in the earthen containers, with or without regularly treated water exchange, due to the Bti toxins being embedded in the porous earthen container surfaces, which protects them from rapid degradation. Lesser toxin amounts are removed from the water column during water exchange. The efficacy of VectoBac DT was also evaluated for the control of natural infestation of Aedes larvae which were resistant to temephos at the WHO diagnostic dosage of 0.012 mg/l. The tablet significantly reduced the pupal density by 8 fold in earthen containers for 67 days and 5 fold in HDPE containers for 55 days in comparison to untreated containers (p less than 0.05). However, the tablet was effective for a shorter period of 25 days post-tablet-introduction due to fungal infestation in the treated plastic containers.

Both syndromes, DF and DHF/DSS, are caused by any of the four dengue serotypes that belong to the family Flaviviridae.

The pathogenesis of DHF/DSS is not very well understood nor are the host conditions that favor the severe disease; however, children, females, individuals with chronic diseases such as asthma and diabetes, and whites appear to be at greater risk. Finally, recent reports argue the risk of DHF/DSS is higher if the interval is longer between primary and secondary dengue infection.

Although most dengue infections cause only mild clinical disease, understanding the basis of severe dengue disease is an important clinical and scientific goal. Dengue viruses are capable of replicating in many cell types and can be detrimental to cell function. However, the major target for dengue virus infection in vivo appears to be cells of the monocyte/macrophage lineage, in which dengue virus causes little cytopathic effect. It is thought that capillary leakage in DHF results from the release of circulating factors by dengue virus-infected monocytes, activated T cells and other cells. Hemorrhagic manifestations, on the other hand, may be multifactorial due to the direct and indirect effects of dengue virus infection on platelets and the coagulation system. Explanations for the occurrence of severe dengue disease have focused on possible viral and host factors. The available evidence supports the suggestion that severe dengue disease can be more frequently observed with some viral strains than others and that it can occur in the absence of 'enabling' host factors. However, the molecular basis for such an association, if any exists, remains unknown. There is also substantial evidence that the risk of DHF is increased during secondary dengue infections and this immunologic mechanism may predominate in the pathogenesis of capillary leakage. In vitro studies have identified both antibody and T-cell-dependent mechanisms that could exacerbate disease, and clinical studies have correlated the presence of enhancing antibodies and higher levels of T-cell activation with DHF. Thus, both viral and host factors are probably relevant to determining the risk of severe dengue disease, but the interactions and relative importance of all these factors in influencing the expression of clinical disease have not been established.

The incubation period for dengue is four to six days.

The prognosis in DHF/DSS depends on prevention or early recognition and treatment of shock. In hospitals with long experience of DSS the case fatality rate in DHF can be as low as 0.2%. Once shock has set in the fatality rate may be high (12% to 44%).

Dengue infection can cause a spectrum of illness ranging from mild, undifferentiated fever to illness up to 7 days' duration with high fever, severe headache, retro-orbital pain, arthralgia and rash, but rarely causing death. Dengue Haemorrhagic Fever (DHF), a deadly complication, includes haemorrhagic tendencies, thrombocytopenia and plasma leakage. Dengue Shock Syndrome (DSS) includes all the above criteria plus circulatory failure, hypotension for age and low pulse pressure. DHF and DSS are potentially deadly but patients with early diagnosis and appropriate therapy can recover with no sequelae.

The vast majority of infections, especially in children under age 15 years, are asymptomatic or minimally symptomatic. Population-based studies have shown increasing severity in the clinical features of DF with increasing age of the patient and with repeated infections. Infants and young children may have an undifferentiated febrile disease with a maculopapular rash. Older children and adults may have either a mild febrile syndrome or the classical and even incapacitating disease. Skin eruptions are reported in over 50% of laboratory-confirmed dengue cases in Puerto Rico, more commonly in children and adults with primary infections. There may be a flushing of the face, neck, and chest initially in the febrile period; or a centrifugal maculopapular rash arising on the third or fourth day; or a later confluent petechial rash with round pale areas of normal skin; or a combination of these manifestations.

Infants and young children usually develop an undifferentiated febrile disease that can be accompanied by a maculopapular rash.

The clinical features of dengue vary frequently, according to the age of the patient. Infants and young children may have an undifferentiated febrile disease with a maculopapular rash. Older children and adults may have either a mild febrile syndrome or the classical incapacitating disease with abrupt onset and high fever, severe headache, pain behind the eyes, muscle and joint-pains, and rash. Skin hemorrhages (with positive tourniquet test and or/petechiae) may be present. Leukopenia is usually found and thrombocytopenia may be observed. The case fatality rate is exceedingly low.

Clinical description: An acute febrile illness characterized by frontal headache, retro-ocular pain, muscle and joint pain, and rash.

Dengue virus infections may be asymptomatic or lead to a range of clinical presentations, even death. The incubation period is 4-7 days (range 3-14). Typically, DF is an acute febrile illness characterised by frontal headache, retroocular pain, muscle and joint pain, nausea, vomiting, and rash. The febrile, painful period of DF lasts 5-7 days, and may leave the patient feeling tired for several more days. A biphasic or "saddleback" fever curve is not the norm. Dengue virus disappears from the blood after an average of 5 days, closely correlated with the disappearance of fever, and no carrier state ensues.

Dengue is the most prevalent mosquito-borne viral infection worldwide, with 100 million cases of dengue fever (DF) and half a million cases of dengue haemorrhagic fever (DHF) annually.

Today, DF and DHF/DSS are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. More than 2.5 billion people are at risk of infection and more than 100 countries have endemic dengue transmission. DHF has been reported in 60 of them. The burden of DF and DHF disease is not very well documented; however in 1998 alone, more than 1.2 million cases were reported to the World Health Organization, with south-east Asia, the western Pacific and more recently the Americas being the most affected regions.

9.1% in patients with dengue fever had abdominal pain.

15.2% in patients with dengue fever had bleeding manifestations.

21.2% in patients with dengue fever had diarrhoea.

The most severe cases of dengue fever are usually seen in older children and are characterised by a rapidly rising temperature (greater than or equal to 39 C) that lasts 5-6 days.

76-100% in Thai adults with classical dengue fever.

36.4% in patients with dengue fever had a flushed appearance.

Minor haemorrhagic manifestations like petechiae, epistaxis, and gingival bleeding do occur.

3% in patients with dengue fever had gum bleeding.

The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting.

78.8% in patients with dengue fever had headaches.

30.3% in patients with dengue fever had an enlarged liver.

76-100% in Thai adults with classical dengue fever.

26-50% in Thai adults with classical dengue fever.

Over half of infected people report a rash during the febrile period that is initially macular or maculopapular and becomes diffusely erythematous, sparing small areas of normal skin ("islands of white in a sea of red").

26-50% in Thai adults with classical dengue fever.

The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting.

51-75% in Thai adults with classical dengue fever.

78.8% in patients with dengue fever had myalgia.

57.6% in patients with dengue fever had arthralgia.

Minor haemorrhagic manifestations like petechiae, epistaxis, and gingival bleeding do occur.

1-25% in Thai adults with classical dengue fever.

15.2% in patients with dengue fever had petechiae.

1.0% in patients with dengue fever had ecchymoses.

A positive tourniquet test (more than 20 petechiae in a square patch of skin 2.5 x 2.5 cm [greater than 20/in(2)]) may be found in over one-third of patients with DF.

The tourniquet test is another stumbling block, because there is confusion in the definition of a positive result (either ten or 20 petechiae per square inch [6.45 cm2]), it is a long test (5 min) for a doctor's visit, and it feels even longer for the patients, since it is very uncomfortable.The omission of the use of the tourniquet test has considerable impact on the detection of dengue haemorrhagic fever. Grade I dengue haemorrhagic fever, which might represent 15-20% of all dengue haemorrhagic fever cases, depends on tourniquet test positivity.

26-50% in Thai adults with classical dengue fever.

The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting.

54.5% in patients with dengue fever had vomiting.

Today, secondary infection by a different dengue serotype is considered the most significant individual risk factor for DHF/DSS. The presence of circulating non-neutralizing, cross-reactive antibodies in a previously immune individual allows for enhancement of infection, favoring the increased entrance of the virus into the target cell through the cell Fc receptor.

Clinical Case Definition for Dengue Hemorrhagic Fever. The following must all be present: 1. Fever or recent history of acute fever. 2. Hemorrhagic tendencies, as evidenced by at least one of the following: positive tourniquet test, petechiae, ecchymoses, or purpura; and bleeding from mucosa, gastrointestinal tract, injection sites, or others. 3. Thrombocytopenis [100,000 mm(3) or less]. 4. Plasma leakage due to increased capillary permeability as mainifested by at least one of the following: hematocrit on presentation that is greater than or equal to 20% above average for that age, sex, and population; greater than or equal to 20% drop in hematocrit following treatment; or commonly associated signs of plasma leakage-pleural effusion, ascites, and hypoproteinemia.

DHF commonly begins with a sudden rise in temperature and other symptoms resembling DF. The temperature is typically high (38-40 C) and continues for 2-7 days. DHF and dengue shock usually develop around the third to seventh day of illness. The most common haemorrhagic feature is a positive tourniquet test (over 50% of patients). Petechiae, easily bruised skin, and subcutaneous bleeding at venepuncture sites are present in most cases. Transudate due to excessive capillary permeability collects at the pleural and abdominal cavities.

Dengue is the most prevalent mosquito-borne viral infection worldwide, with 100 million cases of dengue fever (DF) and half a million cases of dengue haemorrhagic fever (DHF) annually.

Today an estimated 50-100 million cases of dengue fever and 500,000 cases of DHF, resulting in around 24,000 deaths, occur annually, depending on the epidemic activity.

Several symptoms and signs occur before defervescence and may serve as warning signs that DHF and DSS are impending: generalised abdominal pain, persistent vomiting, change in the level of consciousness, a sudden drop in the platelet count, and a rapid rise in the hematocrit.

14% in patients with dengue hemorrhagic fever had abdominal pain.

Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums.

49.3% in patients with dengue hemorrhagic fever had bleeding manifestations.

Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums.

1-25% in Thai children with classical dengue hemorrhagic fever.

32% in patients with dengue hemorrhagic fever had diarrhoea.

The temperature is typically high (38-40 C) and continues for 2-7 days.

76-100% in Thai children with classical dengue hemorrhagic fever.

100% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory.

44% in patients with dengue hemorrhagic fever had a flushed appearance.

Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums.

1-25% in Thai children with classical dengue hemorrhagic fever.

Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums.

6% in patients with dengue hemorrhagic fever had gum bleeding.

The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting.

60% in patients with dengue hemorrhagic fever had headaches.

6% in patients with dengue hemorrhagic fever had haematemesis.

38% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory.

76-100% in Thai children with classical dengue hemorrhagic fever.

52% in patients with dengue hemorrhagic fever had an enlarged liver.

45% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory.

17.3% in patients with dengue hemorrhagic fever had hypotension.

26-50% in Thai children with classical dengue hemorrhagic fever.

10.3% in patients with dengue hemorrhagic fever had melena.

17% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory.

The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting.

1-25% in Thai children with classical dengue hemorrhagic fever.

74.7% in patients with dengue hemorrhagic fever had myalgia.

57.3% in patients with dengue hemorrhagic fever had arthralgia.

Minor haemorrhagic manifestations like petechiae, epistaxis, and gingival bleeding do occur.

26-50% in Thai children with classical dengue hemorrhagic fever.

16% in patients with dengue hemorrhagic fever had petechiae.

9.3% in patients with dengue hemorrhagic fever had ecchymoses.

48% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory.

A prospective study recorded pleural effusions in 84% (22/26) of DHF cases and the mean pleural effusion index (the proportion of the width of the right hemithorax occupied by a pleural effusion in the right lateral decubitus chest radiograph) was 14.1%.

14.7% in patients with dengue hemorrhagic fever had pleural effusions or ascites.

A positive tourniquet test (more than 20 petechiae in a square patch of skin 2.5 x 2.5 cm [greater than 20/in(2)]) may be found in over one-third of patients with DF.

76-100% in Thai children with classical dengue hemorrhagic fever.

2.7% in patients with dengue hemorrhagic fever had splenomegaly.

76-100% in Thai children with classical dengue hemorrhagic fever.

100% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory.

9.3% in patients with dengue hemorrhagic fever had vaginal bleeding.

68% in patients with dengue hemorrhagic fever had vomiting.

62% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory had vomiting.

The World Health Organisation defines DSS as DHF with circulatory failure as manifested by a rapid, weak pulse with narrowing of the pulse pressure (less than or equal to 20 mmHg, regardless of pressure levels, e.g. 100/90 mmHg) or hypotension with cold, clammy skin and restlessness. In Asia, DHF and DSS mainly affect children under 15 years of age in hyperendemic areas. The age distribution is different in the Americas, where these syndromes occur in all age groups. However, the majority of fatalities during epidemics in the Americas occur in children.

In severe cases, the patient's condition suddenly deteriorates after a few days of fever. At the time of or shortly after the temperature drop, between 3 and 7 days after onset, there are signs of circulatory failure: the skin becomes cool, blotchy, and congested; circumoral cyanosis is frequently observed, and the pulse becomes weak and rapid. Although some patients may appear lethargic, they become restless and then rapidly go into a critical stage of shock. Acute abdominal pain is a frequent complaint shortly before the onset of shock.

The liver may be palpable and tender; and liver enzymes are usually mildly abnormal but jaundice is rare. The four warning signs for impending shock are intense, sustained abdominal pain; persistent vomiting; restlessness or lethargy; and a sudden change from fever to hypothermia with sweating and prostration. The development of any of these signs or any suggestion of hypotension are indications for hospital admission and management to prevent shock. The patient may recover rapidly after volume replacement but shock may recur during the period of excessive capillary permeability. The prognosis in DHF/DSS depends on prevention or early recognition and treatment of shock. In hospitals with long experience of DSS the case fatality rate in DHF can be as low as 0.2%. Once shock has set in the fatality rate may be high (12% to 44%).

26-50% in Thai children with classical dengue hemorrhagic fever developed shock.

18.7% in patients with dengue hemorrhagic fever developed shock.

An increasing number of dengue infections have been related to other unusual manifestations. These include dengue fever with severe haemorrhage, fulminant liver failure, cardiomyopathy, and neurological phenomena such as altered consciousness, convulsions, and coma resulting from enchephalitis and encephalopathy. Previously, neurological manifestations were ascribed to nonspecific complications secondary to DHF and DSS. Possible causes of dengue encephalopathy include hypotension, cerebral oedema, focal haemorrhage, hyponatraemia, and fulminant hepatic failure. However, a recently documented possibility is the invasion of the central nervous system. Other unusual presentations include ocular manifestations.

Dengue diagnosis can be performed through virus isolation, genome and antigen detection and serological studies. Serology is currently the most widely applied in routine diagnosis. Of course, clinical, geographical, and epidemiological data associated with the patient remain critical considerations when evaluating a laboratory result.

The definitive diagnosis of dengue virus infection can only be made in the laboratory, and it depends on the isolation of these viruses, the detection of viral antigens or RNA in serum or tissues, or the detection of specific antibodies in the patients' serum.

Five serological tests have been used for the diagnosis of dengue infection: hemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), immunoglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA) and indirect immunoglobulin G ELISA. The limitations of these techniques are the high cross-reactivity observed with these tests, requiring a comprehensive pool of antigens, including all four serotypes, another flavivirus (yellow fever virus, Japanese encephalitis virus, or St. Louis encephalitis virus), and in some areas, another virus that causes similar clinical manifestations but that is not flavivirus, such as Oropouche, Mayaro or Chikungunya viruses. Furthermore, the dengue antibodies are better detected around the fifth day of disease onset, making this technique unfeasible for rapid diagnosis

Treatment is supportive and includes bed rest, anitpyretics, and analgesics. In case of dehydration, fluid and electrolyte replacement are used in addition.

Ribavirin has marginal value.

Suckling albino mice.

Litters of 1- to 2-day-old suckling albino mice were intracerebrally inoculated with 0.02 ml of dengue-2 virus suspension.

The encephalitic model in suckling mouse is very well known and has been extensively used for many years. It shows direct cytopathology of the viruses to the neurons of suckling mice. This, however, is not a model of dengue fever of DHF in humans.

The infected mice showed definite paralysis on day 4 postinoculation with dengue-2 virus, and they were severely paralyzed on day 5. None of the infected mice survived to day 6.

Monkeys and chimpanzees.

Chimpanzees, gibbons, and macaques.

Monkeys and chimpanzees infected with dengue viruses develop viremia and specific antibodies against dengue viruses, but they show no clinical disease.

Experimental laboratory evidence shows that several species of lower primates (chimpanzees, gibbons, and macaques)become infected and develop viremia titers sufficient to infect mosquitoes. Onset of viremia after infection in these animals is similar to that in humans, but the magnitude and duration of viremia generally are lower and shorter, respectively.

Female SCID (C.B.-17/Icr Tac-scid) mice.

To develop a useful animal model for DEN virus infection, HepG2 cells, which support DEN viral replication, were transplanted into SCID mice.

HepG2-grafted SCID mice were infected ip with 10(6) PFU/mouse of DEN-2 virus at 7 to 8 weeks after transplantation.

An elegant mouse model for dengue virus infection has now been described, which may be useful in studying various aspects of the pathogenesis of DHF. A human hepatocarcinoma cell line, HepG2, known to support dengue virus replication was transplanted into severe combined immunodeficient (SCID) mice. Such HepG2-grafted mice were infected with DEN-2 virus, and the authors showed very convincingly that the mice developed many of the features of DHF, including thrombocytopenia and increased haematocrit. Virus inoculated intraperitoneally was detected in the serum and in the liver, and also induced paralysis, at which point virus was also detected in the brain.

Bonnet macaque

Macaca radiata

To investigate the ecology of dengue and Japanese encephalitis (JE) viruses in the forest in Asia, a seroepidemiological survey was carried out on 358 Southeast Asian cynomologus (Macaca iris), 33 Indian bonnet (Macaca radiata) and 37 Japanese (Macaca fuscata) monkey sera by a plaque reduction neutralization test. The results indicated that Southeast Asian monkeys were naturally infected with these viruses but the frequency of antibody to them varied considerably according to the geographical origin of the monkeys.

Crab-eating macaque

Macaca fascicularis

Yuwono et al. reported that out of 74 cynomolgus monkeys (Macaca iris) in the Philippines, 14.9% were positive for DEN and 2.7% were positive for JE, as evidenced by a plaque reduction neutralization test.

Synonyms: Macaca irus, Macaca cynomolgus.

Japanese macaque

Macaca fuscata

Mitred leaf monkey

Presbytis melalophos

Of 114 monkeys tested by hemagglutinatin inhibition (HI), 82.5% had dengue antibody and only 25.9% had Japanese encephalitis (JE) antibody at a serum titer of 1:20 or higher. Of 233 monkeys, including the same 114 tested by HI, 62.8% had dengue neutralizing antibody of 2 logs or greater, while only 2 of 46 tested had JE neutralizing antibody (4.3%).

It appears reasonable to consider that most of the high-titered dengue neutralizing antibody (3.0 logs or greater) in monkeys is actually a result of dengue infection.

P. melalohos-HI for Dengue, 3 positives out of 4 total tested, 66.7% positive. P. melalohos-NI for Dengue, 2 positives out of 2 total tested, 100% positive.

Orangutan

Pongo pygmaeus

Between 1996 and 1998, 84 free-ranging orangutans captured for translocation, underwent a complete health evaluation. Analogous data were gathered from 60 semi-captive orangutans in Malaysia. Baseline hematology and serology; vitamin, mineral and pesticide levels; and results of health evaluations, including physical examination, provide a baseline for future monitoring. Free-ranging and semi-captive orangutans shared exposure to 11 of 47 viruses.

There was evidence of exposure to respiratory syncytial virus, coxsackie virus, dengue virus, and zika virus in both groups.

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.

Pig-tailed macaque

Macaca nemestrina

It appears reasonable to consider that most of the high-titered dengue neutralizing antibody (3.0 logs or greater) in monkeys is actually a result of dengue infection.

M. nemestrina-HI for Dengue, 1 positives out of 2 total tested,50% positive. P. melalohos-NI for Dengue, 1 positives out of 2 total tested, 50% positive.

Presbytis obscura

The studies by Rudnick's group, which had as their objective the demonstration of a sylvatic cycle of dengue, were carried out in forests of various ecologic types, but all were characterized by abundant monkey populations and relative isolation from human populations. Some were characterized by a small human population at the forest edge in the form of a sentinel village and, especially by the complete absence of Aedes aegypti and the presence of Aedes albopictus. These studies led to the isolation of several strains of dengue (types 1, 2 and 4) from sentinel monkeys [Prebytis obscura and Macaca fascicularis (=irus)] placed in the forest canopy.

Silvered leaf monkey

Trachypithecus cristatus

Of 238 monkey sera tested by HI, the majority represented Macaca irus, the long-tailed macaque, and Presbytis cristatus, the silvered leaf monkey, two of the most common species in Malaya. For all species, the percentage of positives for the four dengue types ranged from 52 to 62%.

Toque macaque

Macaca sinica

The population of toque macaques used in the current study inhabit the natural dry evergreen forest within the Nature and Archaeological Reserve at Polonnaruwa. The behavior, ecology, demography, and genetics of these wild monkeys have been intensively studied since 1968 by Dittus and others. In 1995, the population comprised nearly 1,000 monkeys distributed among some 28 social groups. All the macaques in the population have been individually identified and the dates of birth and life histories of nearly all the animals are known. The ages of animals whose births were not observed, such as immigrants into the study population, were estimated based on known relationships between morphologic development and age.

Two hundred forty-four serum samples collected between July and October 1995 were screened for the presence of antibodies against dengue-2 virus. Twenty-one percent (52 of 244) of the animals tested positive for dengue virus antibody.

Armadillo

Dasypus spp.

Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer. The role of free-ranging species in the maintenance of DENV in the wild in South America is disputed. Nevertheless, it is unlikely that another flavivirus induced the seroneutralization reaction seen, since DENV makes its own antigenic complex within the Flaviviridae. After Platt et al. reported neutralizing antibodies to DENV in bats, we found evidence for infection of a very large number of diverse forest neotropical mammals, indicating that wild animals could be exposed to DENV, once it has been introduced into a pristine area, possibly consecutive to human activities, such as tourism, hunting, logging, and gold mining. The exact role of accidentally exposed species is not known, but they may act as temporary reservoirs, with transmission by either forest populations of A. aegypti

60 Dasypus spp tested, 3 positive for Dengue 2.

Brown four-eyed opossum

Metachirus nudicaudatus

Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer.

19 Metachirus nudicaudatus tested, 1 positive for Dengue 2.

Brazilian agouti

Dasyprocta leporina

Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer.

29 Dasyprocta leporina tested, 1 positive for Dengue 2.

Porcupines

Coendou spp.

Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer.

42 Coendou spp tested, 2 positive for Dengue 2.

Brocket deer

Mazama spp.

Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer.

10 Mazama spp tested, 1 positive for Dengue 2.

Yellow fever mosquito

Aedes aegypti

Both Yellow Fever (YF) and Dengue (DEN) viruses can be transmitted in an urban cycle between humans by the highly domesticated Aedes aegypti mosquito. This species is a very efficient epidemic vector of both viruses because of its close association with humans in urban settings, and its blood-feeding behavior of taking blood from multiple human hosts during a single gonotrophic cycle. The DEN viruses are unique in that they are the only known arboviruses that have fully adapted to humans and are maintained in large urban centers of the tropics in an Ae. aegypti-human-Ae. aegypti cycle without apparent input from the enzootic cycles

Stegomyia aegypti

Aedes africanus

While the vectors involved in transmission from monkey to monkey, and possibly, from monkey to man are not known with certainty, certain species are suspected in view of the isolation of dengue viruses from them and of their biology (i.e. their contact with both monkeys and man, at least in certain ecosystems). In Africa, these species have already been implicated in the transmission of yellow fever, and include the subgenera Stegomyia (Ae. luteocephalus, Ae. africanus and Ae. opok) and Diceromyia (Ae. taylori and Ae. furcifer).

Asian tiger mosquito

Aedes albopictus

The mosquito Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae), originally indigenous to South-east Asia, islands of the Western Pacific and Indian Ocean, has spread during recent decades to Africa, the mid-east, Europe and the Americas (north and south) after extending its range eastwards across Pacific islands during the early 20th century. The majority of introductions are apparently due to transportation of dormant eggs in tyres. Among public health authorities in the newly infested countries and those threatened with the introduction, there has been much concern that Ae. albopictus would lead to serious outbreaks of arbovirus diseases (Ae. albopictus is a competent vector for at least 22 arboviruses), notably dengue (all four serotypes) more commonly transmitted by Aedes (Stegomyia) aegypti (L.). Results of many laboratory studies have shown that many arboviruses are readily transmitted by Ae. albopictus to laboratory animals and birds, and have frequently been isolated from wild-caught mosquitoes of this species, particularly in the Americas.

Ae. albopictus can be unequivocally incriminated as a vector of dengue only where transmission occurs in the absence of Ae. aegypti or any other potential vector. Such transmission in the absence of Ae. aegypti or other species of Stegomyia, has been seen to occur in parts of China, at one time in Japan and the Seychelles, most recently in Hawaii and possibly La Reunion Island in the Indian Ocean. In other areas, particularly in South-east Asia, it appears that Ae. albopictus serves primarily as a maintenance vector of dengue in rural areas.

Aedes cooki

Usually, a species has come under suspicion because it was the only plausible vector present during a dengue epidemic when Ae. aegypit was either absent or so localized that it could not have been responsible for all transmission. Thus, Ae. polynesiensis (in French Polynesia, the Cook Islands, and Futuna) and Ae. scutellaris (in new Guinea) have been shown to be vectors both by epidemiologic and transmission studies to man or monkeys. Others are suspect based on epidemiologic studies and laboratory infection (Ae. cooki on Niue) or epidemiologic studies alone (Ae. hebrideus on Espiritu Santo in Vanuatu, or Ae. rotumae on Rotuma Island).

Aedes furcifer

In west Africa, many mosquitoes in the Aedes subgenera Stegomyia and Diceromyia, most notably Ae. furcifer, Ae. luteocephalus, and Ae. aegypti, are suspected of DENV-2 transmission. Although susceptibility and ability to transmit are not the only important factors determining vectorial capacity, our results improve understanding of the role of each species in DENV-2 transmission cycles and emergence potential. The vectorial role of Ae. furcifer and Ae. luteocephalus, hitherto suspected based on frequent DENV-2 isolations in nature, is supported by their high susceptibility to infection. In the light of our data and their bionomics, Ae. furcifer and Ae. aegypti are good candidate vector species for domestic DENV-2 transmission. As indicated in previous studies, only Ae. furcifer is a strong candidate for virus exchange between the forest and human habitations. Of the susceptible forest mosquitoes, it is the most common in villages and the only one found infected in a domestic environment. In contrast, Ae. luteocephalus appears to be confined to the forest habitat

During 1990, Dengue-2 (DEN-2) virus was isolated fro the first time from mosquitoes (Aedes furcifer, six isolates; Ae. taylori, six isolates; Ae. luteocephalus, seven isolates) during an epidemic in which DEN-2 virus also was isolated from humans.

Aedes hebrideus

Aedes hensilli

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

Aedes luteocephalus

Aedes mediovittatus

We have circumstantial evidence that Ae. mediovittatus was responsible for transmission of the dengue 5 epidemic in at least 1 rural community of Puerto Rico (San Juan laboratories, unpubl data). Collectively, the data lead us to believe that Ae. mediovittatus may be playing an important role in the maintenance of dengue viruses in Puerto Rico during interepidemic periods. This could explain how viruses like dengue 2 and possibly dengue 3 persisted in a limited island population for over 10 years.

Aedes (Gymnometopa) mediovittatus is a forest mosquito that has become adapted to the rural peri-domestic environment in the Caribbean region (Puerto Rico, Cuba and other islands). It can be considered a potential vector of dengue in view of its anthropophilia and high degree of susceptibility to infection in the laboratory.

Aedes niveus

The sylvatic transmission cycle of DEN viruses among forest monkeys by Ae. niveus is indicated, based on epidemiological studies in the Peninsula Malaya.

Aedes opok

Aedes polynesiensis

Epidemiologic observations have led to the incrimination of several species of Aedes of the subgenus Stegomyia (Ae. aegypti, Ae. albopictus and Ae. polynesiensis) from which virus has been isolated in nature, whose abundance coincides with the incidence of epidemic or endemic dengue, and that are at least partially domestic and anthropophilic.

During an outbreak occurring in Futuna (Horne Islands) from October 1976 to January 1977, II strains quite similar to dengue virus type I were isolated from blood of patients in acute phase. Immunitary [sic] responses were noted on 8/12 paired sera submitted to IH test; 4/17 serum samples showed antibody titer presumptive of a recent infection. Entomological survey gave evidence that virus was transmitted by Aedes polynesiensis and confirmed that Futuna is free of A.E. aegypti; other species found were: Culex annulirostris, C. pipiens fatigans, C. sitiens. A viral strain was isolated from Ae. polynesiensis only.

Aedes rotumae

An explosive epidemic of dengue occurred in Fiji between January and July 1975. All laboratory evidence indicated that type 1 dengue was the only prevalent dengue virus. This type had probably not been in Fiji for 30 years and over 70% of the population was susceptible. Aedes aegypti appeared to be the major vector in urban areas, but circumstantial evidence indicated that Aedes rotumae was a vector in at least one remote area

Aedes scutellaris

Aedes taylori

During 1990, Dengue-2 (DEN-2) virus was isolated for the first time from mosquitoes (Aedes furcifer, six isolates; Ae. taylori, six isolates; Ae. luteocephalus, seven isolates) collected during an epidemic in which DEN-2 virus also was isolated from humans.

Ochlerotatus triseriatus

Another possible vector is Ae. triseriatus, of the subgenus Protomacleaya, which has been shown to be capable of transmitting dengue virus in the laboratory. There is, as yet, no evidence that it does so in nature.

Synonym: Aedes triseriatus.

Biosafety Level 2 is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; (2) access to the laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

Mosquito cell culture is the most recent methodology developed for dengue virus isolation. Three cell lines of comparable sensitivity are frequently used, but the most widely used is the C6/36 clone of A. albopictus cells. The use of this cell lineage has provided a rapid, sensitive and economical method for dengue virus isolation. Dengue antigens can be detected in infected cell culture by IFA. This technique is less sensitive than the intrathoracic inoculation of adult mosquitoes, but due to its ability to process several samples at the same time, it has become the standard technique for dengue virus isolation. Compared to the other techniques, the advantages of the mosquito cells are: 1) higher sensitivity than the vertebrate cell lines for the recovery of dengue viruses, 2) they are relatively easy to maintain and grow at room temperature, and 3) it is possible to maintain cultures for up to 14 days without changing the medium. Although some reports describe a cytopathic effect (syncytium formation, presence of multinucleated giant cells) induced by all four serotypes of dengue virus, the cytophatic effect produced in mosquito cell culture by many dengue viruses is difficult to detect, and it can be variable. The cytophatic effect is usually seen when these cells are cultured in tubes.

Singh and Paul first succeeded in the maintainence of the four dengue virus serotypes in a mosquito cell line established from larvae of Aedes albopictus. Since then, several other mosquito cell lines have also been used or recommended for dengue virus isolation, such as the AP61 (Aedes pseudoscutellaris), Tra-284 (Toxorynchites amboinensis), C636 (A. albopictus), AP64 (clone of an A. pseudoscutellaris cell line), and CLA-1 (clone of an A. pseudoscutellaris cell line) cell lines.

Although all the four serotypes were initially isolated by intracerebral inoculation of suckling mice, this technique has several disadvantages, including high cost, long time for isolation, and low sensitivity. These problems have prevented further recommendation of this methodology for vi