Dengue: an update

Review

Dengue

Dengue: an update María G Guzmán and Gustavo Kourí

Lancet Infectious Diseases 2001 2: 33–42

At the end of the last century, the world faced the resurgence of many infectious diseases, dengue being one of the most important in terms of morbidity and mortality. The dengue virus is transmitted to man by the bite of a domestic mosquito, Aedes aegypti being the principal vector although some other species such as Aedes albopictus are of importance. The disease has been described since 1779–1780; however, there is evidences that a similar disease occurred earlier on several continents.1,2 Four viruses, dengue 1 to 4, classified in an antigenic complex of the flavivirus genus, family flaviviridae, are the aetiological agents of this entity.3 These spherical agents of 40–50 nm in diameter have a lipid envelope and a positive single stranded RNA. The viral genome of approximately 11 kb in length encodes three structural proteins (capsid, C, membrane protein, M, envelope glycoprotein, E) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The 5´and 3´non-coding regions are important for regulating viral replication.4 The main biological properties of the viruses are located in the E protein, including receptor binding, haemagglutination of erythrocytes, neutralising antibody induction, and protective immune response.4 The spectrum of illness ranges from inapparent, mild disease to a severe and occasionally fatal haemorrhagic clinical picture. THE LANCET Infectious Diseases Vol 2 January 2002

800 000 WPRO SEARO Americas

700 000 600 000

Cases

This review is an update of dengue and dengue haemorrhagic fever (DHF) based on international and Cuban experience. We describe the virus characteristics and risk factors for dengue and DHF, and compare incidence and the case fatality rates in endemic regions (southeast Asia, western Pacific, and the Americas). The clinical picture and the pathogenesis of the severe disease are explained. We also discuss the viral, individual, and environmental factors that determine severe disease. Much more research is necessary to clarify these mechanisms. Also reviewed are methods for viral isolation and the serological, immunohistochemical, and molecular methods applied in the diagnosis of the disease. We describe the status of vaccine development and emphasise that the only alternative that we have today to control the disease is through control of its vector Aedes aegypti.

500 000 400 000 300 000 200 000 100 000 0 92

19

93

19

94

19

95

19

96

19

97

19

98

19

99

19

00

20

Figure 1. World DF/ DHF reported cases 1992–2000 by WHO region. SEARO=South East Asia, WPRO=Western Pacific.

Disease burden and global situation Dengue fever (DF) and dengue haemorrhagic fever (DHF) are increasingly important public health problems in the tropics and subtropics. Dengue has been recognised in over 100 countries and 2·5 billion people live in areas where dengue is endemic. Yearly, an estimated of 50–100 million cases of DF and several hundred thousand cases of DHF occur, depending on epidemic activity. About 250 000–500 000 cases of DHF are officially notified annually; however, the true incidence is not very well known.5–8 In 1998, 1·2 million cases of dengue and DHF were reported to WHO, including 3442 deaths.7,8 Case fatality rates vary from 0·5% to 3·5% in Asian countries.9 The disease is endemic in the Americas, southeast Asia (SEAR), western Pacific (WPR), Africa, and the eastern Mediterranean, with the major disease burden in the three first regions. Figure 1 shows the DF and DHF reported cases by geographical regions from 1992 to 2000 (data kindly provided by C Prasittisuk, WHO southeast Asian regional office, K Palmer, WHO western Pacific regional office, and J Arias, WHO American regional office). During the past 8 years incidence of dengue has grown in the endemic areas, particularly the American region (AMR); however, in the past 3 years, the case fatality rate was higher in southeast Asia and western Pacific regions (table 1). Much Mar a G Guzm n is head of the Virology Department and director the PAHO/WHO Collaborating Center for Viral Diseases, Pedro Kour Tropical Medicine Institute, Ciudad Habana, Cuba; and Gustavo Kour is director of the Pedro Kour Tropical Medicine Institute. Correspondence: Professor Mar a G Guzm n, Virology Department and PAHO/WHO Collaborating Center for Viral Diseases, Pedro Kour Tropical Medicine Institute, Autopista Novia del Mediod a, Km 6, PO Box Marianao 13, Ciudad Habana, Cuba. Tel +53 7 220450; fax +53 7 246051; email [email protected]

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Review

Dengue

Table 1. Case fatality rate (per 1000 dengue cases) by geographical region, 1998–2000 SEAR

1998 14·14

1999 9·64

2000 3·37

WPR

4·14

1·83

NR

AMR

NR

0·32

0·17

NR=not reported.

more research on DF and DHF risk factors is needed to explain this observation. Why the recent recrudescence of dengue and DHF?

Two main factors are directly responsible for the emergence and re-emergence of DF and DHF: increases in the density and geographic distribution of the vector, and in the rate and geographic range of virus transmission. The first factor is widely influenced by major global demographic changes, particularly unprecedented global population growth and unplanned urbanisation resulting in substandard housing, and inadequate water supply and waste management systems. The epidemiological situation is worsening by deterioration of the health systems and of mosquito-control programmes in most dengue-endemic countries.5,10,11 The American region is perhaps the best example: an eradication campaign started at the end of the 1940s and most countries became free of the vector; however, during the 1960s and the 1970s A aegypti reinfested Central and South America.5 Today, only a few countries are free of the vector. Figure 2 shows the current and potential distribution of A aegypti in the world according to WHO. The increase in air travel allows the movement of the different serotypes, strains, and even genotypes of virus from one region to another. Individuals in viraemic phase are able to introduce a new virus into a susceptible

population. 17 years after the last report, dengue 3 was reintroduced in Central America and in less than 7 years it had spread to Caribbean and South American countries producing DF and DHF epidemics.12 In general those factors that enhance the contact between vector and host favour an increase in dengue transmission.13 However, it is not possible to exclude other factors that could influence emergence of this disease such as climate change and virus evolution. WHO has reported that a temperature rise of 1–20C could result in an increase of the risk population by several hundred million, with 20 000– 30 000 more fatal cases annually.14 Dengue transmission is a complex phenomena where the factors mentioned above are all involved; however, living conditions15 and specifically poverty, social inequalities, and illiteracy constitute the general background.15,16

Clinical picture Most dengue infections are symptomless or very mild characterised by undifferentiated fever with or without rash mainly in infants and young children. Older children and adults may develop a mild febrile syndrome or typical DF consisting of high fever, severe headache, myalgia, arthralgia, retro-orbital pain, and maculopapular rash. Signs of skin bleeding such as positive tourniquet test, petechiae, or ecchymosis are observed in some patients. DF cases with bleeding complications such as epistaxis, gingival bleeding, gastrointestinal bleeding, haematuria, and hypermenorrhea can be observed during some epidemics. Thrombocytopenia has been also reported in some cases.17,18 DF is a very incapacitating disease; however, its prognosis is favourable. Plasma leakage is the major pathophysiological feature observed in DHF and differentiates this from typical DF. High fever, bleedings, moderate to marked

Figure 2. Current and potential distribution of Aedes aegypti, WHO 1998.

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Review

Dengue

thrombocytopenia (below 100 000/mL) and haemoconcentration (haematocrit increase by 20%) characterise severe disease. Hepatomegaly has been an important sign in different settings.17–20 Both DF and DHF begin with a sudden rise in temperature; after 3–4 days, signs of haemorrhage such as petechiae, ecchymosis, epistaxis, or gingival or gastrointestinal bleeding are observed in DHF cases. Plasma leakage such as pleural effusion, ascites, and hypoproteinaemia are common. Some patients deteriorate to circulatory failure, dengue shock syndrome (DSS), presenting as rapid and weak pulse, narrow pulse pressure or hypotension, cold clammy skin, and altered mental status. Disease severity is classified as mild (grades I and II) or severe (grades III and IV), the presence of shock being the main difference.17,18 There is not a specific antiviral treatment but patients usually recover after fluid and electrolyte supportive therapy, particularly if early measures are applied. Early recognition of the warning signs of DHF (intense continuous abdominal pain, persistent vomiting, and restlessness or lethargy) and early treatment are of utmost importance to reduce case fatality rate.17,18,20 An iceberg characterises dengue virus infections. Most cases are symptomless, followed, in increasing rarity, by undifferentiated fever, DF, and DHF. Studies of the 1997 DHF Cuban epidemic21 illustrate this fact (figure 3). DHF has been primarily a disease of children.17,18 Reports from 1975 to 1978 from areas where DHF is endemic revealed only eight of 629 patients in Indonesia and 18 of 694 in Thailand to be older than 15 years.19,22 However, the age distribution of DHF cases has changed progressively, and is different in the Americas to that observed in Asia. In the outbreaks in Cuba and Venezuela the disease occurred in all age groups, although about two-thirds of the fatalities were among children.5,23,24 Similar observations have been made in Brazil and Puerto Rico.25,26 In general, an increase in the number of DHF adult cases has been observed during the 1980s and the 1990s in countries such as Philippines and Malaysia. Table 2 illustrates some of the main signs and symptoms observed in adult patients.24

12 fatalities

205 DHF/DSS cases

5208 DF/DHF cases

17926 Dengue infections

Table 2. Main signs and symptoms observed in DHF fatal cases, Cuba 1981 and 1997 Clinical manifestation 1981 adults

1997 adults

Fever

88

100

Vomiting

81

100

Hepatomegaly

35

66·6

Abdominal pain

58

83·3

Ascites

8

91·6

Pleural effusion

8

58·3

Shock

100

100

Haemorrhagic manifestations

65

100

Petechiae

38

41·6

Haematemesis

35

58·3

Melaena

4

25

Vaginal bleeding

44

42·8

Haemoconcentration

92

91·6

Thrombocytopenia

71·8

83·3

Numbers given as %.

One of the unusual manifestations of dengue infection is the involvement of the central nervous system. Gubler et al27 concluded that neurologic disorders can occur in both DF and DHF. In DF, neurological symptoms range from irritability and depression through mononeural palsies to encephalitis with seizures and death. Encephalopathy in DHF could result from cerebral anoxia, oedema, intracanial haemorrhage, and vessel occlusion. In general, encephalitic symptoms in DHF are attributable to liver failure and oedema associated with leakage through the cerebral vasculature; however, in DF, the pathogenesis of encephalopathy is less clear. There is controversy over whether dengue viruses produce neurological disease as a non-specific complication or because of direct invasion of the brain in the manner of other flaviviruses such as Japanese encephalitis and St Louis encephalitis. In a study of 378 Vietnamese patients with suspected central nervous system infections, 4·2% were infected with dengue viruses.28 Viruses were isolated or detected by PCR in cerebrospinal fluid in some cases. With the development of molecular diagnosis, dengue detection from cerebrospinal fluid or brain has increased; however, the question of contamination with blood has been raised. It is possible that haemorrhage or leakage through the bloodbrain barrier enables antibody and virus present in the blood to move into the cerebrospinal fluid, or dengue virus could cross the blood-brain barrier and infect the cerebrospinal fluid. Finally, dengue antigen has been detected by immunoperoxidase stain in the brain of fatal cases.29 In general, the pathological findings observed in the brain are associated with cerebral oedema and haemorrhage without evidences of encephalitis. More pathological studies are needed.

Dengue diagnosis—still a need Figure 3. Reported and estimated DF/ DHF and dengue-2 infections during the 1997 DHF Cuban epidemic.21

THE LANCET Infectious Diseases Vol 2 January 2002

Three factors have been fundamental in dengue diagnosis: development of ELISAs for dengue-specific IgM detection; mosquito cell lines and monoclonal antibody development 35

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Review for viral isolation and identification; and, most recently, the introduction of reverse transcriptase PCR for molecular diagnosis and strain characterisation. These three methods cover the serological, virological, and molecular diagnosis of dengue.30 Once an individual is infected, an incubation period of 7–10 days occurs. 2 days before the onset of disease to 5–6 days after, virus could be recovered from blood. Serum inoculation in mosquito cell lines such as A albopictus (C6-36) and Aedes pseudoscutellaris (AP61) are the most common method for virus isolation.30–32 However, inoculation of samples directly into mosquitoes, specifically adult or larval inoculation of Toxorhynchites spp mosquitoes (Tx amboinensis, Tx splendens) is the best isolation system in terms of sensitivity.33,34 Unfortunately this last method is not available in most endemic countries. In routine diagnosis, the C6-36 cell line has become most widely used. 7–10 days postinoculation in cell lines or 14 days post-inoculation in mosquitoes, virus identification is done by immunofluorescence assay with serotype-specific monoclonal antibodies.30,35,36 Viral isolation rates are up to 36% with C6-36 cell lines or up to 80% by direct mosquito inoculation.32,36 A rapid centrifugation assay for dengue virus improved the isolation rate, even in tissue samples.37 Several PCR protocols for dengue detection have been described that vary in the RNA extraction methods, genomic location of primers, specificity, sensitivity, and the methods to detect PCR products and to determine the serotype.38–41 Reverse transcriptase PCR has provided one of the most important steps in the molecular diagnosis of dengue virus. A rapid assay was developed by Lanciotti et al,38 which allows the detection of virus in viraemic sera with consensus primers located in the C and prM genes. A second PCR with specific primers allows serotype identification (DNA products of different sizes according to the dengue serotype are obtained). PCR has been applied to dengue diagnosis (with sera, tissue from fatal cases, mosquito pool, infected cell cultures, and mosquito larvae), molecular surveillance, and genetic strain characterisation. PCR combined with the nucleotide sequencing and restriction enzyme analysis has become a powerful tool for dengue strain characterisation.42–47 Nucleotide sequencing studies have allowed classification of dengue viruses into different genotypes according to their nucleotide sequence. Rico-Hesse42 studying the E/NS1 gene junction demonstrated the presence of five genotypes for both dengue 1 and dengue 2 viruses. Others have obtained similar results by studying a fragment encoding aminoacids 29–94 in the E protein of 28 dengue 2 and aminoacids 28–87 in 35 dengue 1 isolates.43,44 Dengue 3 has been classified into four subtypes by sequencing the M and E structural genes of 23 geographically and temporally distinct strains, and dengue 4 has been classified into two genotypes by studying the complete E gene of 19 strains.44,45 A direct correlation between viraemia, disease severity, and explosiveness of epidemic transmission has been reported previously. Illness severity seems to correlate with the level of circulating virus. Viraemia levels should also correlate with mosquito infection rates and thus epidemic transmission rates.48 The 36

Dengue

recent development of protocols for dengue RNA quantification will allow improved study of the role of level of viraemia in severity of the disease.49 Primary, secondary, and even tertiary dengue infections can be observed taking into account the existence of four serotypes. During a primary infection, individuals develops IgM after 5–6 days and IgG antibodies after 7–10 days. During a secondary infection high levels of IgG are detectable even during the acute phase and they rise considerably over the next 2 weeks. IgM levels are lower and in some cases absent during secondary infection. IgM antibodies suggest a recent infection although they are still present after 2–3 months. High titres of IgG are a criterion of secondary infection.17,18,50 There are different methods to detect both IgM and IgG immunoglobulins; however, ELISA is the most widely used in routine practice.50–52 The sensitivity of IgM ELISA ranges from 90 to 97% compared with the gold standard haemagglutination-inhibition test. Some false positive reactions can be observed in less than 2% of cases and also a low or negative IgM reaction in secondary infections.50,53 IgM false positive reactions are illustrated with the data from Cuban dengue surveillance in the period 1998–1999 where no dengue circulation was detected (table 3). Commercial kits are available for serological diagnosis, but they still need careful evaluation.53–55 The capture ELISA for IgM detection is the most useful serologic procedure currently available and it is widely recommended for serological surveillance allowing health authorities to be alerted before there is an increase in number of cases and severity of illness.17,56 One example of this occurred in Havana, a city of two millions inhabitants, in September 2000. The national dengue surveillance system detected a positive IgM sample from a dengue suspected case. Five more related cases were detected in the following days. Rapid surveillance and established control actions allowed this outbreak to be controlled in less than 3 months. About 135 DF confirmed cases were reported, dengue 3 and dengue 4 viruses being the aetiological agents. No DHF cases were observed. This epidemic was located in three health areas of Havana City. No cases were reported in the rest of the country and no DHF cases were observed (MG Guzman, unpublished observation). PAHO/WHO guidelines for prevention and control of dengue in the Americas commend the usefulness of epidemiological surveillance with laboratory support (including dengue IgM detection and viral isolation) for rapid detection of dengue epidemics.17 Antigen detection in tissues and serum samples complete the dengue diagnosis. Immunohistochemistry with specific antibodies has allowed detection of dengue antigen in liver, spleen, lung, and lymph nodes from fatal Table 3. IgM false positive reaction, dengue surveillance, Cuba, 1998–1999*. Year

False positive reactions/total (%)

1998

26/4794 (0·54)

1999

9/10012 (0·08)

Total

35/14806 (0·23)

*Samples tested by IgM antibody capture ELISA

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Dengue

cases.57 There are scarce reports of antigen detection in serum, probably because of the low sensitivity of the employed system in the context of a high number of secondary infections and the presence of immune complexes of virus and antibodies.58,59 Although dengue diagnosis has improved, we still need better tools for an early, rapid, specific, sensitive, and non-expensive diagnosis.

•Age •Sex •Race •Nutritional Status •Secondary infection •Host response

What we know about DHF and what we don’t

Epidemiological

Individual risk

risk

factors

factors

•Number of susceptible •Vector high density •Wide viral circulation •Hyperendemicity

Viral

factors For years, DHF pathogenesis has been a controversial matter. Some workers argued that secondary infection was the main factor in the severity of this disease, whereas others pointed to •Strain virulence viral virulence.60–65 Today, the majority •Serotype view is that secondary infection is the main risk factor for DHF; however, Figure 4. Risk factors for DHF/DSS: an integral hypothesis. other factor such as viral virulence and host characteristics are also of utmost importance. other hand, when no neutralising antibodies are present, DHF occurs as a consequence of a very complex heterotypic antibodies form complexes with dengue mechanism where virus, host, and host immune response viruses, which infect mononuclear phagocytes with interact to give the severe disease in 2–4% of individuals enhanced efficiency and as a consequence a higher number with secondary infection.21 An integral hypothesis for the of cells are infected. This phenomenon has been called development of DHF epidemics was published in 198765 antibody dependent enhancement (ADE).66,67 In an elegant taking into account the international and Cuban experiences study, Kliks and colleagues68 demonstrated that preon DHF (figure 4). The intersection of three groups of infection sera of children experiencing non-severe dengue factors (host, viral, and epidemiological factors) determine 2 secondary infection had cross-reactive denguethe occurrence of a DHF epidemic. A high vector density, a neutralising antibodies, whereas sera from children with high virus circulation, and a susceptible population (at risk severe dengue 2 secondary infection did not. Furthermore, of secondary infection) are necessary to get a high number the sera of children with severe infection exhibited of DHF cases.65 In general the epidemiological and viral significant enhancement activity, supporting the fact that factors are the determinants for an epidemic of disease. heterologous antibodies are an important controlling Individual risk factors such as sex, race, and chronic diseases determinant of infection enhancement. It is understood are predisposing factors that make the disease more frequent today that dengue virions and IgG antibody to dengue virus in a certain race or age group. However, the pre-existence of form virus-antibody complexes, whose binding to Fc␥R via antibodies is the main individual risk factor, but not the only the Fc portion of IgG results in augmentation of dengue one, for the occurrence of severe disease. Individual risk virus infection.66,69 Epidemiological and serological studies done both in factors are the ones that determine the appearance of the disease in a particular person in a given population. The Thailand and Cuba are good examples of the importance of presence or absence of these individual risk factors, in the secondary infections as risk factor of DHF. Since the first matrix of epidemiological and viral factors, determines observations done by Halstead et al in 1970, DHF has been whether or not all persons with a secondary type of infection present in situations where more than one serotype circulates.17,21,62,69,70 present the clinical picture of DHF. Children are in higher risk of DHF than adults, as have Antibody levels may have a dominant role in driving dengue infection to more or fewer infected cells. Epitopes been reported in Thailand and some others southeast Asian present on E protein are able to induce neutralising countries.19,71 These studies showed that age-specific DHF antibodies, both homologous and heterologous. People incidence was bimodal, with severe cases peaking at 7 infected with one serotype maintain a life-long protective months of age and again at 3–5 years of age (figure 5).61,72 immunity to infection by the homologous virus, although DHF or DSS occurred in infants who acquired maternal protective immunity to infection with heterologous dengue antibody and subsequently experienced a dengue serotypes is short-lived. It has been proposed that infection. In general, children less that 1 year of age were neutralising antibodies down regulate the severity of the hospitalised almost exclusively during primary dengue disease.66 During a secondary infection with a different infections. These infants were born to dengue immune serotype, the presence of low amount of heterotypic mothers.73 On the other hand, children 3–5 years old have neutralising antibodies could prevent severe disease; on the DHF during a secondary infection. THE LANCET Infectious Diseases Vol 2 January 2002

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18 16 14 Cases

12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Age Figure 5. Age distribution in children with DHF/DSS, Cuba 1981.23

It has been suggested that baseline microvascular permeability in children is considerably greater than that of adults and this could explain, at least partly, why DHF is more frequently observed in children.74 There seems to be no time limit to sensitisation after a primary dengue infection. The 1997 Cuban epidemic clearly demonstrated that dengue 2 DHF still occurs 16–20 years after the primary dengue 1 infection.21,24 How the homotypic and heterotypic antibodies raised after the primary dengue infection change in time in terms of neutralising titre, immune enhancement ability, and avidity are important matters that deserve careful study. Besides secondary infection, chronic diseases such as bronchial asthma and diabetes have been suggested as risk factors for DHF. Finally, whites have higher risk of developing DHF than blacks. Dengue 2 virus is known to replicate to higher concentration in the peripheral blood cells of whites compared with those of blacks.24 Neutralising antibodies are key factors in the aetiopathogenesis of the disease; however, the cellular immune response is also of importance. It has been demonstrated that memory dengue T lymphocyte response after a primary infection includes both serotype-specific and serotype-cross-reactive T lymphocytes.75 NS3 protein seems to be the major target for CD4+ and CD8+ T cells, although some T cell epitopes have been recognised in other proteins such as envelope and capsid.76,77 The magnitude of proliferation to heterologous dengue serotypes is variable depending on different factors such as the serotype causing the primary infection and the ethnicity of the individual.78 These findings support the possibility that during a secondary infection T cells become activated due to interactions with infected monocytes. Recent observations suggest a massive T-cell activation during DHF, which could explain partly if not totally the mechanism of plasma leakage through cytokine production and infected cell lysis by CD4+ and CD8+ dengue-specific T lymphocyte. Cytokines could be released either directly from monocytes/macrophages as a result of infection or after interactions between infected and immune cells, or both.78,79 Cytokines that may induce plasma leakage such as interferon ␥, interleukin (IL) 2, and tumour necrosis factor (TNF) ␣ are increased in DHF cases.79,80 Also, interferon ␥ 38

enhances uptake of dengue particles by target cells through increasing Fc cell receptors.81 Others cytokines such as IL-6, IL-8, and IL-10 are also increased. A protein of 22–25 kDa has been associated with the pathogenesis of DHF. This cytotoxic factor is able to induce increased capillary permeability in mice, is capable of reproducing in mice all the pathological lesions that are seen in human beings, and has been detected in sera of DHF patients.82 Dengue virus through an indirect more than a direct mechanism could mediate endothelial cell activation. It has been demonstrated that dengue infected peripheral blood monocytes in ADE conditions generate soluble mediators that activate endothelial cells through the enhanced expression of adhesion molecules such as VCAM-1 and ICAM-1.83 At the same time, high levels of TNF␣ (induced by infected monocytes and by T-cell activation or both) could be responsible in part for transient vascular damage. The role of TNF␣ in the pathogenesis of the disease is critical, and it probably initiates several processes relating to plasma leakage and haemorrhage.83 Avirutnan and colleagues84 have shown that infection of human endothelial cells with dengue virus induces the secretion of RANTES and IL-8, and the formation of nonlytic complement complexes. King et al demonstrated the potential role of mast cells/basophils in the pathogenesis of the disease.85 They reported that mast cell/basophil KU812 cells are permissive to dengue virus infection with the production of viral particles and vasoactive cytokine production. Furthermore, they demonstrated that virus-antibody complexes are much more potent that virus alone in inducing cells activation. Finally, a high release of IL-6 and IL-1␤ was also observed. Both cytokines could activate endothelial cells modulating the expression of the adhesion molecules as well as altering endothelial cell morphology.85 Complement activation as a result of immune complexes (virus-antibody) or immune activation and cytokine production could be also involved in the mechanism of plasma leakage. Haemorrhage appears to be due to several factors such as vasculopathy, thrombocytopenia, platelet dysfunction, and prothrombin-complex deficiency.86 However, the mechanisms that trigger events leading to haemorrhage have not been well studied. Development of antibodies potentially cross-reactive to plasminogen (due to a similarity in 20 aminoacid sequence of dengue E glycoprotein and a family of clotting factors) could have a role in causing haemorrhage in DHF.86 The increased destruction or decreased production of platelets could result in thrombocytopenia.86 Virus-antibody complexes have been detected on the platelet surface of DHF patients suggesting a role for immune-mediated destruction of platelets.87 The release of high levels of platelet-activating factor by monocytes with heterologous secondary infection may explain the haemorrhage, given that platelet-activating factor may induce platelet consumption and augment adhesiveness of vascular endothelial cells resulting in thrombocytopenia.88 The presence of IgM antibodies in the sera of DHF cases that cross-reacted with platelets has been demonstrated.89 These autoantibodies were able to cause platelet lysis and could be involved in the pathogenesis of the disease. THE LANCET Infectious Diseases Vol 2 January 2002

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In spite of this knowledge, it is still uncertain what kind of host and virus-specified factors determine why certain individuals have only mild DF while others develop DHF. Nucleotide and deduced aminoacid sequences of the different serotypes have been studied to define possible molecular markers of attenuation and virulence.90–92 Sequence comparison of virulent and attenuated strains has demonstrated that mutations could be important for attenuation.90 Two important genotypes for dengue 2 virus have been identified, one, from southeast Asian origin, related to most of the DHF epidemics in southeast Asia and the Americas; and the other, the American genotype, only related to DF epidemics in the American region.91,93,94 The 1981 Cuban epidemic has been considered one of the most severe DHF epidemics to date in the Americas.23 The genomic study of Cuban dengue 2 strains and the RNA from a liver sample of a fatal case demonstrated their similarity with southeast Asian strains, at least at the studied fragment.93,95 A similar epidemiological situation to that in Cuba was reported in Iquitos, Peru. A dengue 1 epidemic occurred in 1990 followed 5 years later by a dengue 2 epidemic.94 No DHF cases were observed. The dengue 1 viruses that affected Cuba in 1977 and Iquitos in 1990 were similar; by contrast, the 1981 Cuban dengue 2 strain belonged to an Asian genotype and the Peruvian dengue 2 virus belonged to the American genotype.93,94 These results suggest that the Asian genotype possess a virulence determinant that is absent from viruses originating in the Americas. At least two branches of the Asian genotype are circulating in the Americas, one related to old dengue 2 strains that have been isolated in Cuba in 1981, Venezuela in 1994, and Mexico in 1995, and the other isolated in Jamaica in 1981, Cuba in 1997, and many other Latin-American countries and related to the most recent Thailand dengue 2 strains. Both branches are linked to DHF cases.91,93,95 Recently, some aminoacid changes on M and E proteins of dengue 2 strains have been related to DHF epidemics.91 These aminoacid changes are present in both 1981 and 1997 dengue 2 Cuban strains (MG Guzman, unpublished observation). In a recent study, dengue 2 Thailand strains were classified in three subtypes, according the non-synonymous aminoacid replacements and the authors proposed that clinical severity depend both on the molecular structure of the viruses and the serological response of patients.92 Subtype I included one strain isolated from a DSS case with a secondary serological response, subtype II included strains from DHF with secondary serological response and DF cases with a primary serological response, and subtype III included only DF cases with both primary or secondary serological response. The relation of specific genotypes with severe disease has also being observed for dengue 3 virus.12,96 The significant genetic variation between genotypes could be responsible of differences in virus interactions with macrophages and suggest that certain strains are more virulent than others are. Morens and Halstead97 reported that virulence differences between dengue 2 strains could be associated with subtle antigenic differences that affect the degree to which strains form immune complexes THE LANCET Infectious Diseases Vol 2 January 2002

with heterotypic antibodies. Recently, intraserotype recombination was demonstrated.98,99 The implications of these genetic changes for epidemics, severity of disease outcome, and virus emergence in new areas remain to be determined In addition to the effect of strain and serotype of virus in determining the magnitude of an epidemic and severity of the disease, the serotype that produces the secondary infection and particularly the serotype sequence of infection are important. Although the four dengue viruses are able to produce DHF cases, dengue 2 and dengue 3 are the most frequently associated with the severe disease. Dengue 1 infection followed by dengue 2 has been associated with DHF epidemics, although in hyperendemic areas it is not easy to define the virus producing the primary infection.100 Finally, escape mutants could be another factor that adds to the complexity of the DHF phenomenon.101 Still a matter for discussion is the target cell for dengue virus replication. Cells of the monocyte/macrophage lineage have long been viewed as the primary target cells for dengue viruses. Dengue antigen has been detected in Kuffer cells, alveolar macrophages, mononuclear phagocytes in the skin, and circulating monocytes.102 The viruses have been isolated or detected in organs such as lymphoid organs, liver, spleen, kidney, and brain.24,102 Immune effector cells such as monocytes and T lymphocytes, and non-immune cells such as hepatocytes, endothelial cells, and brain cells have been reported as potential hosts. Langerhans and dendritic cells could be targets for dengue viruses and could play an important part in the pathogenesis of dengue infection through an increase in virus load and cell activation.102,103 Dendritic cells may be required for establishing a primary immune response, producing virus particles, and TNF␣ and interferon ␣ production. They could be the early, primary target of dengue virus in natural infection and the vigour of cell-mediated immunity could be modulated by the relative presence or absence of interferon ␥ in the microenvironment surrounding the virus-infected dendritic cells.104

Dengue control—a challenge? Today we are closer to getting a dengue vaccine, although problems remain to be solved. For decades, scientists have considered that a dengue vaccine should provide protective immunity to the four serotypes to avoid the ADE phenomenon.105 However, we are facing a new challenge. The report of DHF 20 years after the primary infection and the higher severity observed when secondary infection occurs after long interval compared with shorter interval give a new dimension to this disease and reinforce the need to get a long-lasting immunity to the four viruses.70 The absence of an animal model, poor understanding of

Strategies for a dengue vaccine Conventional (inactivated and attenuated vaccines) Non recombinant (structural and non-structural purified proteins, synthetic peptides) Recombinant subunit (Escherichia coli, baculovirus, yeast) Recombinant vector Infectious cDNA clone DNA vaccines 39

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Review Search strategy and selection criteria The references covered in this review come mostly from a search of journals and books published in English and Spanish in the past decades. The review includes the most advance knowledge on dengue today.

the pathogenesis of the disease, and poor financial support are other main problems. The panel shows some of the strategies used to produce a dengue vaccine.106–110 The first human studies have been conducted for the attenuated tetravalent vaccine and have demonstrated that it is safe and immunogenic in both children and adults. Results obtained with chimeric viruses in mice and non-human primates suggest their possible usefulness in people. Independently of the applied strategy, careful human studies must be conducted to demonstrate the usefulness of the vaccine candidates and its innocuousness. Until a dengue vaccine is available, vector control is the only way to diminish dengue transmission. But how can the mosquito be controlled when control programmes deteriorate, the economical situation in most of endemic countries is unfavourable, and the population does not recognise dengue as a priority? Control of dengue transmission is harder today than ever before, but some principles remain fundamental if control is to be achieved: political will (financial support, human resources), improvement of public health infrastructure and vector control programmes, intersectorial coordination (partnerships among donors, the public sector, civil society, non-governmental organisations, and the private and commercial sectors), active community participation, and reinforcement of health legislation. Ministries of health must direct control, and must establish epidemiological and entomological surveillance and education campaigns for the community. It is fundamental that the community recognises its responsibility in dengue control. References

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9 Halstead SB. Is there an inapparent dengue explosion? Lancet 1999; 353: 1100–01 10 Guzman MG, Kouri G, Bravo J. La emergencia de la fiebre hemorragica del dengue en las Americas: re-emergencia del dengue. Rev Cub Med Trop 1999; 51: 5–13 11 Guzman MG, Kouri G, Pelegrino JL. Enfermedades virales emergentes. Rev Cub Med Trop 2001; 53: 5–15 12 Figueroa R, Ramos C. Dengue virus (serotype 3) circulation in endemic countries and its reappearance in America. Arch Med Res 2000; 31: 429–30 13 Monath TP. Epidemiology of yellow fever: current status and speculations on future trends. In Saluzzo JF, Dodet B, eds. Factors in the emergence of arboviruses diseases. Paris: Elsevier, 1997: 143–56 14 WHO. The world wealth report. Life in the 21st century. A vision for all. Geneva: WHO, 1998. 15 Reiter P. Global warming and mosquito-borne disease in USA. Lancet 1996; 348: 622 16 Farmer P. Social inequalities and emerging infectious diseases. Emerg Infect Dis 1996; 2: 259–69 17 Pan American Health Organization. Dengue and dengue hemorrhagic fever in the Americas: guidelines for prevention and control. Scientific publication no. 548. Washington: PAHO, 1994 18 WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. Geneva: WHO, 1997. 19 Nimmannitya S. Clinical spectrum and management of dengue haemorrhagic fever. Southeast Asian J Trop Med Pub Health 1987; 20: 325–30 20 Martinez E. Dengue hemorragico en criancas. La Habana: Editorial Jose Marti, 1992: 1–180 21 Guzman, MG, Kouri G, Valdes L, et al. Epidemiological studies on dengue in Santiago de Cuba, 1997. Am J Epidemiol 2000; 152: 793–99. 22 Sumarmo, Wulur H, Jahja E, Gubler DJ, Suharyono W, Sorensen K. Clinical observations on virologically confirmed fatal dengue infections in Jakarta, Indonesia. Bull World Health Organ 1983; 61: 693–701 23 Kouri GP, Guzman MG, Bravo JR, Triana C. Dengue hemorrhagic fever/dengue shock syndrome: lessons from the Cuban epidemic, 1981. Bull World Health Organ 1989; 67: 375–80. 24 Guzman MG, Alvarez M, Rodriguez R, et al. Fatal dengue haemorrhagic fever in Cuba, 1997. Int J Infect Dis 1999; 3: 130–35. 25 Zagne SMO, Alves VGF, Nogueira RMR, Miagostovich MP, Lampe E, Tavares W. Dengue hemorrhagic fever in the state of Rio de Janeiro, Brazil: a study of 56 confirmed cases. Trans R Soc Trop Med Hyg 1994; 88: 677–79. 26 Rigau-Perez, the Puerto Rico Association of Epidemiologists. Clinical manifestations of dengue hemorrhagic fever in Puerto Rico, 1990–1991. Rev Panam Salud Publica 1997; 1: 381–88. 27 Gubler DJ, Kuno G, Waterman SH. Neurologic disorders associated with dengue infection. Proceedings of the International Conference on Dengue/Dengue Hemorrhagic Fever; Kuala Lumpur, Malaysia; 1983: 290–306 28 Solomon T, Dung NM, Vaughn DW, et al. Neurological manifestations of dengue infection. Lancet 2000; 355: 1053–59. 29 Nogueira RMR, Miagostovich MP, Cunha RV, et al. Fatal primary dengue infections in Brazil. Trans R Soc Trop Med Hyg 1999; 93: 418 30 Guzmán MG, Kourí G. Advances in dengue diagnosis. Clin Diagnostic Immunol 1996; 3: 621–27. 31 Race MW, Williams MC, Agostini CFM. Dengue in the Caribbean: virus isolation in a mosquito (Aedes pseudoscutellaris) cell line. Trans R Soc Trop Med Hyg 1979; 73: 18–22 32 Kuno G, Gubler DJ, Velez M, Oliver A. Comparative sensitivity of three mosquito cell lines of dengue viruses. Bull World Health Organ 1985; 63: 279–86. 33 Gubler D, Rosen L. A simple technique for demonstrating transmission of dengue virus by mosquitoes without the use of vertebrate hosts. Am J Trop Med Hyg 1976; 25: 146–50. 34 Rosen L, Shroyer DA. Comparative susceptibility of five species of Toxorhynchities mosquitoes to parenteral infection with dengue and other flaviviruses. Am J Trop Med Hyg 1985; 34: 805–09. 35 Henchal EA, McCown JM, Seguin MC, Gentry MK, Brandt WE. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. Am J Trop Med Hyg 1983; 32: 164–69. 36 Vorndam V, Kuno G. Laboratory diagnosis of dengue virus infections. In: Gubler DJ, Kuno G, eds. Dengue and dengue haemorrhagic fever. New York: CAB International, 1997: 313–33. THE LANCET Infectious Diseases Vol 2 January 2002

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37 Rodriguez R, Alvarez M, Guzman MG, Morier L, Kouri G. Isolation of dengue 2 in C636/HT cells by rapid centrifugation/shell vial assay. Comparison with conventional virus isolation method. J Clin Microbiol 2000; 38: 3508–10. 38 Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992; 30: 545–51. 39 Morita K, Meomoto T, Honda D, et al. Rapid detection of virus genome from imported dengue fever and dengue hemorrhagic fever patients by direct polymerase chain reaction. J Med Virol 1994; 44: 54–58. 40 Harris E, Roberts G, Smith L, et al. Typing of dengue virus in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR. J Clin Microbiol 1998; 36: 2634–39. 41 Rosario D, Alvarez M, Díaz J, et al. Rapid detection and typing of dengue viruses from clinical samples using reverse transcriptase-polymerase chain reaction. Pan Am J Public Health 1998; 4: 1–5. 42 Rico-Hesse R. Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology 1990; 147: 479–93. 43 Deubel V, Nogueira R, Drouet MT, Zeller H, Reynes JM, Ha DQ. Direct sequencing of genomic cDNA fragments amplified by the polymerase chain reaction for molecular epidemiology of dengue-2 viruses. Arch Virol 1993; 129: 197–210. 44 Chungue E, Cassar O, Drouet MT, et al. Molecular epidemiology of dengue-1 and dengue-4 viruses. J Gen Virol 1995; 76: 1877–84. 45 Lanciotti RS, Gubler DJ, Trent DW. Molecular evolution and phylogeny of dengue-4 viruses. J Gen Virol 1997; 78: 2279–86. 46 Balmaseda A, Sandoval E, Perez L, Gutierrez CM, Harris E. Application of molecular typing techniques in the 1998 dengue epidemic in Nicaragua. Am J Trop Med Hyg 1999; 61: 893–97. 47 Tolou H, Coussinier-Paris P, Mercier V, et al. Complete genomic sequence of a dengue type 2 virus from the French West Indies. Biochem Bioph Res Comm 2000; 277: 89–92. 48 Gubler DJ, Suharyono W, Tan R, Abidin M, Sie A. Viraemia in patients with naturally acquired dengue infection. Bull World Health Organ 1981; 59: 623–30. 49 Sudiro TM, Zivny J, Ishiko H, et al. Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction. J Med Virol 2001; 63: 29–34. 50 Kuno G, Gomez I, Gubler DJ. An ELISA procedure for the diagnosis of dengue infections. J Virol Methods 1991; 33: 101–13. 51 Miagostovich MP, Nogueira RMR, Santos FB, Schatzmayr HG, Araujo ESM, Vorndam V. Evaluation of an IgG enzyme-linked immunosorbent assay for dengue diagnosis. J Clin Virol 1999; 14: 183–89. 52 Nawa M, Takasaki T, Yamada KI, Akatsuka T, Kurane I. Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody. J Virol Methods 2001; 92: 65–72. 53 Jelinek T, Wastlhuber J, Proll S, Schattenkirchner M, Loscher T. Influence of rheumatoid factor on the specificity of a rapid immunochromatographic test for diagnosing dengue infection. Eur J Clin Microbiol Infect Dis 2000; 19: 555–56. 54 Cussubo AJ, Vaughn DW, Nisalak A, et al. Comparison of PanBio dengue Duo IgM and IgG capture ELISA and Venture technologies Dengue IgM and IgG Dot Blot. J Clin Virol 2000; 16: 135–144. 55 Chakravarti A, Gur R, Berry N, Mathur MD. Evaluation of three commercially available kits for serological diagnosis of dengue haemorrhagic fever. Diagnostic Microbiol Infect Dis 2000; 36: 273–74. 56 Gubler DJ. Surveillance for dengue and dengue hemorrhagic fever. Bull PAHO 1989; 23: 397–404. 57 Pelegrino JL, Arteaga E, Rodriguez AJ, Gonzalez E, Frontela MC, Guzman MG. Normalizacion de tecnicas inmunohistoquimicas para la detección de antigenos del virus dengue en tejidos embebidos en parafina. Rev Cubana Med Trop 1997; 49: 100–07. 58 Malergue F, Chungue E. Rapid and sensitive streptavidin-biotin amplified fluorogenic enzyme-linked immunosorbent-assay for direct detection and identification of dengue viral antigens in serum. J Med Virol 1995; 47: 43–47. 59 Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. J Clin Microbiol 2000; 38: 1053–57. THE LANCET Infectious Diseases Vol 2 January 2002

Review 60 Hammon WM. Dengue hemorrhagic fever—do we know its cause? Am J Trop Med Hyg 1973; 22: 82–90. 61 Halstead SB. Observations related to pathogenesis of dengue hemorrhagic fever.VI: hypotheses and discussion. Yale J Biol Med 1970; 42: 350–62. 62 Sangkawibha N, Rojanasuphot S, Ahandrik S, et al. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. Am J Epidemiol 1984; 120: 653–69. 63 Rosen L. Disease exacerbation caused by sequential dengue infections: myth or reality? Rev Infect Dis 1989; 11: S840–42. 64 Halstead SB. Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenic cascade. Rev Infect Dis 1989; 11(suppl 4): S830–39. 65 Kouri GP, Guzman MG, Bravo JR. Why dengue haemorrhagic fever in Cuba? 2: an integral analysis. Trans R Soc Trop Med Hyg 1987; 81: 821–23. 66 Halstead S. Pathophysiology and pathogenesis of dengue hemorrhagic fever. In Thongcharoen P, ed. Monograph on dengue/dengue haemorrhagic fever. WHO Regional Publication, SEARO no 22, 1993: 80–103. 67 Halstead SB, Nimmannitya S, Yamarat C, Russell PK. Haemorrhagic fever in Thailand: newer knowledge regarding etiology. J Med Sci Biol 1967; 17: 96–103. 68 Kliks SC, Nisalak A, Brandt WE, Wahl L, Burke DS. Antibodydependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg 1989; 40: 444–51. 69 Morens DM. Antibody-dependent enhancement of infection and the pathogenesis of viral disease. Clin Inf Dis 1994; 19: 500–12. 70 Guzman MG, Kouri, Bravo J, Soler M, Vazquez S, Morier L. Dengue hemorrhagic in Cuba, 1981: a retrospective seroepidemiologic study. Am J Trop Med Hyg 1990; 42: 179–84. 71 Jatanasen S, Thongcharoen P. Dengue haemorrhagic fever in southeast Asian countries. In Thongcharoen P, ed. Monograph on dengue/dengue haemorrhagic fever. WHO Regional Publication, SEARO no 22, 1993: 23–30. 72 Guzman MG, Kouri G, Morier L, Fernandez A. A Study of fatal haemorrhagic dengue cases in Cuba 1981. Bull PAHO 1984; 18: 213–20. 73 Kliks SC, Nimmanitya S, Nisalak A, Burke DS.Evidence that maternal dengue antibodies are important in the development of dengue haemorrhagic fever in infants. Am J Trop Med Hyg 1988; 38: 411–19. 74 Gamble J, Bethell D, Day NPJ, et al. Age-related changes in microvascular permeability: a significant factor in the susceptibility of children to shock? Clinical Science 2000; 98: 211–16. 75 Kurane I, Ennis FA. Cytokines in dengue virus infections: role of cytokines in the pathogenesis of dengue hemorrhagic fever. Semin Virology 1994; 5: 443–48. 76 Kurane I, Zeng L, Brinton MA, Ennis FA. Definition of an epitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clones cross-reactive for dengue types 2, 3 and 4. Virology 1998; 240: 169–74. 77 Spaulding AC, Kurane I, Ennis FA, Rothman AL. Analysis of murine CD8+ T-cell clones specific for the dengue virus NS3 protein: flavivirus cross-reactivity and influence of infecting serotype. J Virol 1999; 73: 398–403. 78 Rothman AL, Ennis FA. Toga/flaviviruses: immunopathology. In: Cunningham MW, Fujinami RS, eds. Effects of microbes on the immune system. Philadelphia: Lippincott Williams & Wilkins, 1999: 473–87. 79 Hober D, Nguyen TL, Shen L, et al. Tumor necrosis factor alpha levels in plasma and whole-blood culture in dengue infected patients: relationship between virus detection and pre-existing specific antibodies. J Med Virol 1998, 54: 210–18. 80 Green S, Pichyangkul S, Vaughn DW, et al. Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever. J Infect Dis 1999; 180: 1429–35. 81 Kontny U, Kurane I, Ennis FA. Gamma interferon augments FC_ receptor-mediated dengue virus infection of human monocytic cells. J Virol 1988; 62: 3928–33. 82 Mukerjee R, Chatuverdi UC, Dhawan R. Dengue virus-induced human cytotoxic factor: production by peripheral blood leucocytes in vitro. Clin Exp Immunol 1995; 102: 262–67. 83 Anderson R, Wang S, Osiowy C, Issekutz AC. Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes. J Virol 1997; 71: 4226–32. 84 Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M. Dengue

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virus infection of human endothelial cells leads to chemokine production, complement activation and apoptosis. J Immunol 1998; 161: 6338–46. King CA, Marshall JS, Alshurafa H, Anderson R. Release of vasoactive cytokines by antibody-enhanced dengue virus infection of a human mast cell/basophil line. J Virol 2000; 74: 7146–50. Chungue E, Poli L, Roche C, Gestas P, Glaziou P, Markoff LJ. Correlation between detection of plasminogen cross-reactive antibodies and hemorrhage in dengue virus infection. J Infect Dis 1994; 170: 1304–07. Wang S, He R, Patarapotikul J, Innis BL, Anderson R. Antibodyenhanced binding of dengue-2 virus to human platelets. Virology 1995; 213: 254–57. Yang KD, Wang CL, Shaio MF. Production of cytokines and platelet activating factor in secondary dengue virus infections. J Infect Dis 1995; 172: 604. Lin CF, Lei HY, Liu CC, et al. Generation of IgM anti-platelet autoantibody in dengue patients. J Med Virol. 2001 ;63: 143–49. Puri B, Nelson WM, Henchal EA, et al. Molecular analysis of dengue virus attenuation after serial passage in primary dog kidney. J Gen Virol 1997; 78: 2287–29. Leitmeyer KC, Vaughn DW, Watts DM, et al. Dengue virus structural differences that correlate with pathogenesis. J Virol 1999; 73: 4738–47. Pandey BD, Igarashi A. Severity-related molecular differences among nineteen strains of dengue type 2 viruses. Microbiol Immunol 2000; 44: 179–88. Guzman MG, Deubel V, Pelegrino JL, et al. Partial nucleotide and amino acid sequences of the envelope and the envelope/nonstructural protein-1 gene junction of four dengue-2 virus strains isolated during the 1981 Cuban epidemic. Am J Trop Med Hyg 1995; 52: 241–46. Watts DM, Porter KR, Putvatana P, et al. Failure of secondary infection with American genotype dengue 2 to cause dengue haemorrhagic fever. Lancet 1999; 354: 1431–33. Sariol C, Pelegrino JL, Martinez A, Arteaga E, Kouri G, Guzman MG .Detection and genetic relationship of dengue virus sequences in seventeen-year old paraffin embedded samples from Cuba. Am J Trop Med Hyg 1999; 61: 994–1000. Briseño B, Gomez H, Argott E, et al. Potential risk for dengue hemorrhagic fever: the isolation of serotype dengue-3 in Mexico. Emerg Infect Dis 1996; 2: 133–35.

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97 Morens DM, Halstead SB. Disease severity-related antigenic differences in dengue 2 strains detected by dengue 4 monoclonal antibodies. J Med Virol 1987; 22: 169–74. 98 Worobey M, Rambaut A, Holmes EC. Widespread intra-serotype recombination in natural populations of dengue virus. Proc Natl Acad Sci USA 1999; 96: 7352–57 99 Holmes E, Burch SS. The causes and consequences of genetic variation in dengue virus. Trends Microbiol 2000; 8: 74–77. 100 Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239: 476–81. 100 Guzman MG, Kouri G, Halstead S. Do escape mutants explain rapid increases in dengue case-fatality rates within epidemics? Lancet 2000; 355: 1902–03 102 Bhamarapravati N. Pathology in dengue infections. In: Gubler DJ, Kuno G, eds. Dengue and dengue haemorrhagic fever. New York: CAB International, 1997: 115–132. 103 Wu SJ, Grouard-Vogel G, Sun W, et al. Human skin Langerhans cells are target of dengue virus infection. Nat Med 2000; 6: 816–820. 104 Libraty DH, Pichyangkul S, Ajariyakhajorn C, Endy TP, Ennis FA. Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis. J Virol 2001; 75: 3501–08. 105 Chambers TJ, Tsai TF, Pervikov Y, Monath TP. Vaccine development against dengue and Japanese encephalitis: report of a World Health Organization meeting. Vaccine 1997; 15: 1494–1502. 106 Bhamarapravati N, Sutee Y. Live attenuated tetravalent dengue vaccine. Vaccine 2000; 18: 44–47. 107 Alvarez M, Guzman MG, Pupo M, Morier L, Bravo J, Rodriguez R. Study of biologic attributes of Cuban dengue 2 virus after serial passage in primary dog kidney cells. Int J infect Dis 2001; 5: 35–39. 108 Guirakhoo F, Weltzin R, Chambers TJ, et al. Recombinant chimeric yellow fever–dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 2000; 74: 5477–85. 109 Kochel TJ, Raviprakash K, Hayes CG, et al. A dengue virus serotype 1 DNA vaccine induces virus neutralizing anibodies and provides protection from viral challenge in Aotus monkeys. Vaccine 2000; 18: 3166–73. 110 Raviprakash K, Porter KR, Kochel TJ, et al. Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques. J Gen Virol 2000; 81: 1659–67.

A list of references for further reading is available on The Lancet Infectious Diseases website, http://infection.thelancet.com

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