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Department of Microbiology and Molecular Genetics, Center for Vaccine Research, University of Pittsburgh, 9047 BST3, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA
Dengue fever (DF), the most prevalent arthropod-borne viral illness in humans, is caused by the dengue virus (DENV). The 4 serotypes of DENV (DENV 1-4) are transmitted to humans primarily by the Aedes aegypti mosquito (Fig. 1).
Fig. 1The A aegypti mosquito is the most common epidemic vector for spread of dengue virus. It can be identified by the white bands or scale patterns on its legs and thorax.
DENV is a member of the Flaviviridae family and is related to the viruses that cause yellow fever and the Japanese, St. Louis, and West Nile encephalitides.
Infection by DENV causes a spectrum of clinical diseases that range from an acute debilitating, self-limited febrile illness, DF, to a life-threatening hemorrhagic and capillary leak syndrome of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DENV causes an estimated 25 to 100 million cases of DF and 250,000 cases of DHF per year worldwide, with 2.5 billion people at risk of infection.
Epidemic DHF was first recognized in the 1950s in Southeast Asia, and by 1975 it had become a leading cause of hospitalization and death among children in many countries in that region. In the 1980s, DHF began a second expansion into Asia, and in countries where DHF is endemic, the epidemics have become progressively larger over the last 15 years (Box 1). In 1980, the first indigenous transmission of dengue in the United States in more than 40 years occurred. Later, infections also occurred in Texas. In 2001 to 2002, a dengue outbreak occurred in Hawaii spread by Aedes albopictus mosquitoes.
Recent dengue virus infections in the United States
Texas:
1980: 23 cases, first locally acquired since 1945
1986: 9 cases
1995: 7 cases
1997: 3 cases
1998: 1 case
1999: 18 cases
2005: 25 cases
Hawaii:
2001 to 2002: 122 cases (first since 1944)
The Americas have seen the most dramatic rise in the emergence of dengue cases (Fig. 3). The mosquito vector for dengue was eradicated in most of the region as part of the Pan American Health Organization's yellow fever eradication campaign in the 1950s and 1960s. The A aegypti eradication program was officially discontinued in the United States and other Western Hemisphere regions, leading to reinfestation of the mosquito vector in most countries during the 1980s and 1990s. By 1997, the geographic distribution of A aegypti was wider than its distribution before the eradication program. Dengue is now endemic in much of the Western Hemisphere. Hyperendemicity, the presence of multiple circulating serotypes, is widespread in most countries and epidemics caused by multiple serotypes are more frequent.
Fig. 3Reinfestation of A aegypti in the Americas Unfortunately, the success of the eradication campaign was not sustained. Beginning in the early 1970s, it began to be disbanded, and many countries channeled their limited resources into other areas. Consequently, A aegypti began to reinfest the countries from which it had been eradicated. Comparing the 1970 and 2006 maps, the mosquito is seen reestablishing itself throughout Central America and most of South America. As the mosquito has spread, the number and frequency of dengue epidemics have increased, as has dengue hemorrhagic fever activity in the Americas.
which is translated as a single polyprotein and then cleaved into 3 structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and 7 nonstructural (NS) proteins by virus- and host-encoded proteases. The 3 structural components are required for capsid formation (C) and assembly into viral particles (prM and E). The NS proteins contain a serine protease and ATP-dependent helicase (NS3), which is required for virus polyprotein processing, a methyltransferase and RNA-dependent RNA polymerase (NS5), and a cofactor for the NS3 protease (NS2B). NS4B has been implicated in blocking the interferon (IFN) response. NS1, NS2A, and NS4A have either unknown or incompletely understood functions. All the NS proteins appear to be necessary for efficient replication.
In primary DENV infection, the virus enters target cells after the E protein adheres to cell surface receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on dendritic cells.
Viral uptake occurs by receptor-mediated endocytosis. Endosomal acidification induces a conformational change in the E protein, resulting in fusion of the viral and endosomal membranes and nucleocapsid release into the cytoplasm.
Virus genome replication occurs in discrete domains within the endoplasmic reticulum (ER). Virus assembly occurs at the ER, and virions are exocytosed via Golgi-derived secretory vesicles.
Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells.
DENV can cause a range of mild-to-severe illnesses. The mosquito eradication program, which was officially discontinued in the United States in 1970, gradually weakened elsewhere, and the mosquito began to reinfest countries from which it had been eradicated. Consequently, the geographic distribution of A aegypti in 2002 was much wider than that before the eradication program and there was a corresponding increase in dengue infections. There are 4 distinct serotypes of DENV. Primary infection with one DENV serotype provides lifelong immunity to that specific serotype. However, when an individual is infected with a different serotype of DENV, there is an increased risk of severe dengue disease.
This can occur with all 4 serotypes; therefore, in regions with multiple endemic serotypes, the risk of severe disease is higher.
Pathogenesis
The pathogenesis of DHF/DSS, the most severe form of DENV infection, reflects a complex interplay of the host immune response and the viral determinants of virulence.
Epidemiologic studies have suggested an immune system linkage, because there is an increased risk of DHF with secondary DENV infection and in children within the first year of life born to DENV-immune mothers.
Observations related to the pathogenesis of dengue hemorrhagic fever. II. Antigenic and biologic properties of dengue viruses and their association with disease response in the host.
From these observations, the hypothesis of antibody-dependent immune enhancement (ADE) of infection emerged. In support of the ADE pathogenesis concept, antibody enhancement of DENV infection in monocytes in vitro correlated with increased risk of DHF,
Replication of dengue virus type 2 in human monocyte-derived macrophages: comparisons of isolates and recombinant viruses with substitutions at amino acid 390 in the envelope glycoprotein.
Finally, a pathologic cytokine response that occurs after extensive T-cell activation may contribute to the capillary leak syndrome associated with DHF.
Tumor necrosis factor alpha levels in plasma and whole-blood culture in dengue-infected patients: relationship between virus detection and pre- existing specific antibodies.
; and disease severity has been associated with activation of CD8+ T cells and the expansion of serotype-reactive low-affinity DENV-specific T cells that produce high levels of vasoactive cytokines.
Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes.
Dengue fever may present in many forms: as an undifferentiated febrile illness with a maculopapular rash, particularly in children, as flulike symptoms, or as classic Dengue with 2 or more symptoms, such as fever, headache, bone or joint pain, muscular pain, rash, pain behind the eyes, and petechial hemorrhaging. Often, there is prolonged fatigue and depression. During dengue epidemics, hemorrhagic complications may also appear, such as bleeding from the gums, nosebleeds, and bruising. Case fatalities due to DF are low, whereas DHF mortality is fairly high. There is no specific treatment for dengue fever except for symptomatic treatment, rest, and rehydration. Recognizing the warning signs and symptoms of dengue infection are critical for appropriate diagnosis and treatment (Fig. 4).
DHF is characterized by spontaneous bleeding, plasma leakage, fever, and thrombocytopenia. Four clinical manifestations need to be observed to be classified as DHF. These include (1) fever; (2) hemorrhagic episodes with the presence of at least one of the following: a positive tourniquet test result (also called a capillary fragility test: a clinical diagnostic method to determine a patient's hemorrhagic tendency and assess fragility of capillary walls); petechiae, ecchymoses, or purpura; or bleeding from mucosa, gastrointestinal tract, injection sites, or others; (3) plasma leakage due to increased capillary permeability; and (4) thrombocytopenia (100,000/mm3 or less). Moderate-to-marked thrombocytopenia with concurrent hemoconcentration is a distinctive clinical laboratory finding of DHF. However, to distinguish DHF from DF, an observation of plasma leakage manifested by a rising hematocrit value (ie, hemoconcentration) must be observed (Fig. 5).
Fig. 5Petechial hemorrhages from a dengue infected patient.
The normal course of DHF lasts between 7 to 10 days, and with appropriate intensive maintenance of the circulating fluid volume, mortality may be reduced to less than 1%. Only severe DF and DHF cases should be hospitalized. Serologic tests are necessary to confirm cases of dengue. However, these tests may take several days.
Developing countries may not have the resources to perform these expensive confirmatory assays, and therefore, many suspected cases of dengue are not fully diagnosed. In severe cases of DHF, the patient's condition may suddenly deteriorate after a few days of fever; the temperature drops, followed by signs of circulatory failure; and the patient may rapidly go into a critical state of shock (dengue shock syndrome), dying within 12 to 24 hours or quickly recovering following appropriate volume replacement therapy Box 2.
Grade 1: Fever and nonspecific constitutional symptoms and positive tourniquet test result
Grade 2: Grade 1 manifestations plus spontaneous bleeding.
Grade 3a: Incipient shock with signs of circulatory failure.
Grade 4a: Profound shock with undetectable pulse and blood pressure.
a Grades 3 and 4 are Dengue Shock Syndrome.
DSS is the most severe form of DHF and is characterized by the presence of all 4 DHF clinical manifestations and circulatory failure. All 3 manifestations of circulatory failure must be present: rapid and weak pulse; narrow pulse pressure or hypotension for the patient's age; and cold, clammy skin and altered mental state.
Diagnosis
Establishing a laboratory diagnosis of dengue infection is critical for diagnosis of dengue. A major challenge for disease surveillance and case diagnosis is that the dengue viruses produce asymptomatic infections and a spectrum of clinical illness ranging from a mild, nonspecific febrile illness to fatal hemorrhagic disease. Important risk factors of DHF include the strain and serotype of the infecting virus and the age, immune status, and genetic predisposition of the patient. The most common method of detecting the virus is to propagate virus from serum in cell culture or detect antidengue antibodies by serology. Virus can be cultured in vitro or by detection of viral RNA and specific dengue virus antigens. Countries that do not have access to sophisticated laboratory tests rely on identification of early clinical or simple laboratory indicators that can provide a reliable diagnosis of dengue before hospitalization. Early distinction between dengue and other febrile illnesses could help identify patients that should be monitored for signs of DHF.
Differential diagnosis
Febrile illnesses, such as measles, typhoid fever, leptospirosis, and severe acute respiratory syndrome (SARS), can produce symptoms similar to DF.
There are no specific antivirals that can eliminate the virus from an infected individual. However, supportive care and treatment can be effective in treating DF. Paracetamol and other antipyretics can be used to treat fever. Bone pain should be treated by analgesics or painkilling tablets. During episodes of DHF/DSS, the mortality rate in the absence of hospitalization can be as high as 50%. With proper treatment, such as intravenous fluid replacement, the mortality rate is greatly reduced.
Vaccines and immunity
Multiple correlates of protection have been described for dengue. However, the primary correlate seems to be long-term homotypic protection.
Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis.
Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis.
Currently, no DENV vaccine is approved by the US Food and Drug Administration (FDA). Four related but serologically distinct DENVs can cause disease. Non-neutralizing, cross-reactive antibodies may contribute to DHF pathogenesis via antibody-dependent enhancement. Therefore, an effective vaccine must induce high-titer neutralizing antibodies against all 4 strains
; failure to do so could increase the risk of severe disease on natural challenge. To circumvent this problem, tetravalent live-attenuated candidate vaccines are in varying stages of development.
In clinical trials, tetravalent serologic responses were observed in some individuals, but many do not develop high titer neutralizing antibodies despite multiple immunizations.
Additionally, each part of the tetravalent vaccine does not elicit high titer immune response leading to immunodominance. Subunit-based vaccines, as purified proteins or DNA plasmid, are alternative vaccine strategies. Repeated immunization of purified recombinant DENV domain III of the E protein (DIII) or DIII-encoding plasmids induced protective antibodies in mice, albeit at fairly low neutralizing titers.
Induction of neutralizing antibodies specific to dengue virus serotypes 2 and 4 by a bivalent antigen composed of linked envelope domains III of these two serotypes.
A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice.
Live attenuated vaccines and nonreplicating vaccines, such as inactivated virus vaccines, viruslike particles, and DNA vaccines, have been developed for dengue (Table 1). These vaccines elicit protective neutralizing antibodies. These vaccines can elicit long-lasting immunity against the specific serotype of DENV. However, they are poorly cross-reactive against infection with another subtype of DENV.
Table 1Experimental dengue virus vaccines
Type
Sponsor
Stage of Development
Live attenuated
Tetravalent
Mahidol University/Sanofi Pasteur
Phase I
Tetravalent
WRAIR/GSK
Phase II
Chimeric
ChimeriVax (17D YF)
Acambis/Sanofi Pasteur
Phase I
DENV-2/4d30 (all serotypes)
NIAID, NIH
Phase I/II
DENV-1
US FDA
Phase I
DENV-2 (16,681, PDK53)
CDC/Inviragen
Preclinical
DNA
Several approaches
Various
(ie, Domain III, prM/E, NS1)
NMRC/University of Pittsburgh
Phase I/Preclinical
Inactivated
Several approaches
WRAIR
Preclinical
Subvirion particles/viruslike particles
Drosophila cells
Hawaii Biotech
Phase I
Baculovirus (E, NS1)
Various
Preclinical
Replication-defective AV (E)
RepliVax-UTMB/Acambis
Preclinical
Yeast (C/prM/E, E-IIBsAg)
Various
Preclinical
Escherichia coli (E, E-NS1)
Various
Preclinical
DNA
University of Pittsburgh
Preclinical
Subunit/recombinant
Various
Preclinical
Abbreviations: AV, adenovirus; CDC, Centers for Disease Control and Prevention; GSK, GlaxoSmithKline; NIAID, National Institute of Allergy and Infectious Diseases; NIH, National Institutes of Health; UTMB, University of Texas Medical Branch; WRAIR, Walter Reed Army Institute of Research; YF, yellow fever.
Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells.
Observations related to the pathogenesis of dengue hemorrhagic fever. II. Antigenic and biologic properties of dengue viruses and their association with disease response in the host.
Replication of dengue virus type 2 in human monocyte-derived macrophages: comparisons of isolates and recombinant viruses with substitutions at amino acid 390 in the envelope glycoprotein.
Tumor necrosis factor alpha levels in plasma and whole-blood culture in dengue-infected patients: relationship between virus detection and pre- existing specific antibodies.
Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes.
Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis.
Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis.
Induction of neutralizing antibodies specific to dengue virus serotypes 2 and 4 by a bivalent antigen composed of linked envelope domains III of these two serotypes.
A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice.
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