Dengue is caused by Dengue virus (DENV), a mosquito-borne flavivirus. DENV is an single stranded RNA positive-strand virus of the family Flaviviridae, genus Flavivirus. This genus includes also the West Nile virus, Tick-borne Encephalitis Virus, Yellow Fever Virus, and several other viruses which may cause encephalitis. DENV causes a wide range of diseases in humans, from a self limited Dengue Fever (DF) to a life-threatening syndrome called Dengue Hemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS).

There are four antigenically different serotypes of the virus:

	DENV-1
	DENV-2
	DENV-3
	DENV-4

Here, a serotype is a group of viruses classified together based on their antigens on the surface of the virus. These four subtypes are different strains of dengue virus that have 60-80% homology between each other. The major difference for humans lies in subtle differences in the surface proteins of the different dengue subtypes. Infection induces long-life protection against the infecting serotype, but it gives only a short time cross protective immunity against the other types. The first infection cause mostly minor disease, but secondary infections has been reported to cause severe diseases (DHF or DSS) in both children and adults. This fenomenon is called Antibody-Dependent Enhancement.

Figure 1. Dengue virus particle and microscopic picture of dengue viruses

DENV is a 50-nm virus enveloped with a lipid membrane (see figure 1). There are 180 identical copies of the envelope (E) protein attached to the surface of the viral membrane by a short transmembrane segment. The virus has a genome of about 11000 bases that encodes a single large polyprotein that is subsequently cleaved into several structural and non-structural mature peptides. The polyprotein is divided into 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 (see figure 2). The structural proteins are the capsid (C) protein, the envelope (E) glycoprotein and the membrane (M) protein, itself derived by furine-mediated cleavage from a prM precursor. The E glycoprotein is responsible for virion attachment to receptor and fusion of the virus envelope with the target cell membrane and bears the virus neutralization epitopes. In addition to the E glycoprotein, only one other viral protein, NS1, has been associated with a role in protective immunity. NS3 is a protease and a helicase, whereas NS5 is the RNA polymerase in charge of viral RNA replication.

Figure 2. Dengue virus genome structure with the structural and nonstructural genes

The life cycle of dengue involves endocytosis via a cell surface receptor (see video 1 and figure 3). The virus uncoats intracellularly via a specific process. In the infectious form of the virus, the envelope protein lays flat on the surface of the virus, forming a smooth coat with icosahedral symmetry. However, when the virus is carried into the cell and into lysozomes, the acidic environment causes the protein to snap into a different shape, assembling into trimeric spike. Several hydrophobic amino acids at the tip of this spike insert into the lysozomal membrane and cause the virus membrane to fuse with lysozome. This releases the RNA into the cell and infection starts.

Video 1. Dengue virus entry in cell

Figure 3. Dengue virus life cycle.

The DENV RNA genome is in the infected cell translated by the host ribosomes. The resulting polyprotein is subsequently cleaved by cellular and viral proteases at specific recognition sites. The viral nonstructural proteins use a negative-sense intermediate to replicate the positive-sense RNA genome, which then associates with capsid protein and is packaged into individual virions. Replication of all positive-stranded RNA viruses occurs in close association with virus-induced intracellular membrane structures. DENV also induces such extensive rearrangements of intracellular membranes, called replication complex (RC). These RCs seem to contain viral proteins, viral RNA and host cell factors. The subsequently formed immature virions are assembled by budding of newly formed nucleocapsids into the lumen of the endoplasmic reticulum (ER), thereby acquiring a lipid bilayer envelope with the structural proteins prM and E. The virions mature during transport through the acidic trans-Golgi network, where the prM proteins stabilize the E proteins to prevent conformational changes. Before release of the virions from the host cell, the maturation process is completed when prM is cleaved into a soluble pr peptide and virion-associated M by the cellular protease furin. Outside the cell, the virus particles encounter a neutral pH, which promotes dissociation of the pr peptides from the virus particles and generates mature, infectious virions. At this point the cycle repeats itself.

 

After a person is infected with dengue, they develop an immune response to that dengue subtype. The immune response produced specific antibodies to that subtype specific surface proteins that prevents the virus from binding to macrophage cells (the target cell that dengue viruses infect) and gaining entry. However, if another subtype of dengue virus infects the individual, the virus will activate the immune system to attack it as if it was the first subtype. The immune system is tricked because the four subtypes have very similar surface antigens. The antibodies bind to the surface proteins but do not inactivate the virus. The immune response attracts numerous macrophages, which the virus proceeds to infect because it has not been inactivated. This situation is referred to as Antibody-Dependent Enhancement (ADE) of a viral infection. This makes the viral infection much more acute. The body releases cytokines that cause the endothelial tissue to become permeable which results in Dengue Haemorrhagic Fever (DHF) and fluid loss from the blood vessels.

There are several possibilities to explain the phenomenon:

1. A viral surface protein laced with antibodies against a virus of one serotype binds to a similar virus with a different serotype. The binding is meant to neutralize the virus surface protein from attaching to the cell, but the antibody bound to virus also binds to the receptor of the cell, the Fc-region antibody receptor FcγR. This brings the virus into close proximity to the virus-specific receptor, and the cell endocytoses the virus through the normal infection route.

2. A virus surface protein may be attached to antibodies of a different serotype, activating the classical pathway of the complement system. The complement cascade system instead binds C1q attached to the virus surface protein via the antibodies, which in turn bind C1q receptor found on cells, bringing the virus and the cell close enough for a specific virus receptor to bind the virus, beginning infection. This mechanism as not been shown specifically for the dengue virus infection, but is supposed to occur with Ebola virus infection in vitro.

3. When an antibody to a virus is present for a different serotype, it is unable to neutralize the virus, which is then ingested into the cell as a sub-neutralized virus particle. These viruses are phagocytosed as antigen-antibody complexes, and degraded by macrophages. Upon ingestion the antibodies no longer even sub-neutralize the body due to the denaturing condition at the step for acidification of phagosome before fusion with lysosome. The virus becomes active and begins its proliferation within the cell.

In 1997, 205 cases of DHF/DSS occurred in Cuba, all in people older than 15 years, after an infection with DENV-2 serotype. All but three cases were shown to have been previously infected by DENV-1 virus, during the epidemic of 1977–1979. Two outbreaks of the disease occurred after the first epidemic in 1977-1979, one in 1981 and one in 1997. People who had been infected with DENV-1 during the 1977-79 outbreak and secondarily infected with DENV-2 in 1997 had 3 to 4 more chances to develop a severe disease than those secondarily infected with DENV-2 in 1981. While heterotypic antibody titers decrease, homotypic antibody titers increase during long time periods (4 to 20 years). This could be due to the preferential survival of long-lived B memory cells producing homotypic antibodies, thanks to their bigger affinity. This cross-reactive protection does not persist more than 3 months. The decrease of cross-reactive neutralizing antibodies titers in the serum could be the reason for more severe secondarily infections.