Mechanisms Of Disease Caused By Dengue
Dengue is a globally established arbovirus found mainly in tropical and subtropical regions of the world and has both endemic and epidemic cycles of transmission. It causes an acute systemic viral infection that is often asymptomatic, but can also result in mild to serious clinical manifestations, including hemorrhagic fever or dengue shock syndrome. Dengue is now a major cause of morbidity in Asian and Latin American countries. As of today, there is no vaccine or specific therapeutic medicine on the market to treat the disease. This is in part due to mechanisms at work during the process of viral entry and replication inside of host cells. Viral interaction with immune cells can cause the host to become susceptible to other serotypes of the virus through antibody-dependent enhancement. The focus of this review will be to explain the process of dengue viral replication, the factors that influence that process, and the disease symptoms associated with dengue viral infection.
The most rapidly spreading mosquito-borne viral disease in the world is dengue. According to guidelines published by the World Health Organization (WHO), there are approximately 2.5 billion people living in areas where dengue is endemic, with 50 million new infections every year (Special Programme for Research and Training in Tropical Diseases & World Health Organization, 2009). Another more recent study has indicated even higher numbers of infection, with 390 million new infections annually, of which 96 million develop symptoms (Bhatt et al., 2013). A study performed on returned travelers diagnosed with dengue coming from Southeast Asia, South Central Asia, South America, and the Caribbean found that among the 522 participants 68% had returned from Asia. The remaining were ranked in order of South America, the Caribbean, and Africa (Schwartz et al., 2008). Hospitalizations for people with dengue have also increased in the United States (Streit, Yang, Cavanaugh, & Polgreen, 2011). A diagnosis of dengue should be considered if a patient develops fever within 14 days of a trip to tropical or subtropical regions (Simmons, Farrar, van Vinh Chau, & Wills, 2012).
What is common to all areas where dengue is present is the Aedes mosquito vector. Three types of mosquito are known to be carriers of dengue: Aedes aegypti, Aedes albopictus, and Aedes polynesiensis. The Aedes aegypti mosquito has adapted to humans living in urban environments and transmits the disease from human to human. The latter two have both human and non-human hosts. Dengue is the only known arbovirus that can directly affect humans without using an animal reservoir. It has also been observed circulating among non-human primates (“WHO | Vector-Borne Viral Infections,” 2011). In urban or endemic settings, humans are the only known hosts for dengue. Forested regions report mosquito-borne transmission between non-human primates (Simmons et al., 2012). Sylvatic transmission has also been observed but is a highly rare occurrence. In 2009, a 20-year-old man was reported to have contracted a sylvatic lineage of dengue virus that closely matched a strain isolated from a sentinel monkey in Malaysia in 1970. This was the first case of sylvatic dengue since 1975 (Cardosa et al., 2009).
Dengue viral infection is most often asymptomatic, however there are a number of disease manifestations that can occur. These range from mild illness, such as dengue fever, to more severe and potentially fatal conditions such as dengue hemorrhagic fever or dengue shock syndrome (Rodenhuis-Zybert, Wilschut, & Smit, 2010; Simmons et al., 2012). Symptoms of viral infection typically occur after about 3 to 7 days of incubation in the host. An initial febrile phase of illness in the host is observed after incubation, with the most notable symptom being dengue fever. This phase of illness typically resolves itself in 3 to 7 days without issue. The critical phase occurs in a small percentage of patients. It starts as a systemic vascular leak and can lead to complications such as dengue shock syndrome and dengue hemorrhagic fever. Critical phase symptoms usually resolve themselves between 48 to 72 hours, after which the patient enters the recovery phase. The patient may feel symptoms of fatigue and a mild to severe rash during this phase (Simmons et al., 2012).
Dengue belongs to the family of viruses called Flaviviridae and the genus flavivirus (Simmons et al., 2012) . This genus is shared by several other pathogenic arboviruses such as yellow fever, West Nile, St Louis encephalitis, Japanese encephalitis, tick-borne encephalitis, Kyasanur Forest disease, and Omsk haemorrhagic fever (Gould & Solomon, 2008). There are four known serotypes of dengue found worldwide and their designations are dengue virus type 1, type 2, type 3, and type 4 (Simmons et al., 2012). Rumors of a fifth serotype began circulating in 2007, when blood and serum samples from a severe case in Borneo contained a virus strain that did not respond to diagnostic tests for the four serotypes. A sustained transmission cycle of serotype 5 in humans has not been observed so far, but it is suspected of circulating between non-human primates in Borneo (Normile, 2013). Dengue is an enveloped, positive strand RNA virus that codes for three structural proteins (C, E, and M) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). These proteins are targets for the host immune response, particularly the antibody response. The antibody response to the E structural protein is associated with antibody dependent enhancement (Rodenhuis-Zybert et al., 2010).
Vectors of Dengue Virus
Aedes aegypti is the primary arthropod vector for dengue. It has adapted to the urban environment by being able to lay eggs in small containers near the homes of people. Urban Aedes aegypti exclusively feeds on humans. Two other carriers of dengue virus, Aedes albopictus and Aedes polynesiensis, are not exclusive to humans (“WHO | Vector-Borne Viral Infections,” 2011). Aedes albopictus has received attention in recent decades for its spread around the world through the sale and shipment of used tires. Aedes albopictus is able to lays eggs in these tires, which enabled them to travel using trade (Reiter, 1998). Despite its spread around the world, the impact of Aedes albopictus on dengue virus transmission has been deemed minor. This is according to a study published in 2010 that demonstrated reduced vector competence and host preference to be key factors that reduce its effectiveness as a dengue vector (Lambrechts, Scott, & Gubler, 2010).
Infection of the Aedes aegypti mosquito vector starts after feeding upon a human that is experiencing a period of viremia during dengue infection. The virus incubates within the mosquito and passes from the intestines to the salivary glands in approximately ten days (Guzman et al., 2010). This incubation time is reduced in environments with higher temperature (Watts, Burke, Harrison, Whitmire, & Nisalak, 1987). Immature dendritic cells in the skin are infected by dengue through the DC-SIGN receptor and are transported to the nearest lymph node within the body (Guzman et al., 2010).
Virology of Dengue
Targets for viral entry of dengue in humans include monocytes, macrophages, and dendritic cells. The C-type lectin receptor of myeloid cells interact with the viral particles, and it has been observed that the viral particles diffuse toward a clathrin coated pit for clathrin-mediated endocytosis (Rodenhuis-Zybert et al., 2010). Alternate entry methods besides the clathrin-coated pit have been observed which depend mainly on serotype and target cell (Acosta, Castilla, & Damonte, 2009). The membrane fusion process of the virus is not well understood, and it has been suggested that acidic pH endosomes cause the dissociation of the E protein. This exposes hydrophobic fusion peptides which facilitate the fusion process (Stiasny, Allison, Schalich, & Heinz, 2002).
Once viral entry and uncoating has been completed, the viral RNA is translated into one polyprotein. This polyprotein is modified co and post translationally to form structural proteins C, E, and prM. Nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 are also formed, and begin the process of replicating the viral genome. The viral genome is then packaged by C proteins into a nucleocapsid (Clyde, Kyle, & Harris, 2006; Rodenhuis-Zybert et al., 2010). In the ER of the host cell, prM and E proteins form heterodimers that orient themselves into the lumen. These proteins guide the process of virion budding by associating into trimers and inducing a curved surface lattice (Kuhn et al., 2002; Zhang et al., 2004). Interactions between capsid proteins and the prM/E proteins are not clear. Encapsulation of the nucleocapsid does not seem to play an important in the dissemination of the virus, as capsidless subviral particles are documented often (Rodenhuis-Zybert et al., 2010). Immature particles formed in the ER travel through the secretory pathway to the trans-Golgi network. The slightly acidic pH of the trans-Golgi network causes the dissociation of the prM/E heterodimers and enables cleavage of the prM protein by cellular endoprotease furin. The pr peptide remains associated with the virion and acts as a chaperone that stabilizes the E protein until the virion buds out of the cell (Rodenhuis-Zybert et al., 2010).
Host Immune Response to Dengue
Dendritic cells and the majority of infected cells produce interferons upon infection with dengue, which numerous studies have claimed to be vital in combating the infection (Diamond et al., 2000; Shresta et al., 2004). Both type I (α, β) and type II (γ) interferons are produced. The early activation of natural killer cells also has been shown to play a crucial role. Viral interaction with pattern recognition receptors such as C-type lectins DC-SIGN, MR, CLEC5, and toll-like receptors TLR3 and TLR 7 induce the production of interferons. Binding of interferons to receptors initiates the JAK/STAT pathway and lead to the production of proteins which produce an antiviral state. Dendritic cell maturation and the activation of B and T cells is also observed (Rodenhuis-Zybert et al., 2010).
Studies have shown that dengue virus is able to inhibit interferon α antiviral responses by reducing STAT activation. The nonstructural proteins NS2A, NS4A, NS4B, and NS5 are thought to be the proteins responsible for the inhibition (Ho et al., 2005; Jones et al., 2005). Inhibition of the interferon α response has not been correlated with an increased risk of developing dengue hemorrhagic fever (Takhampunya et al., 2009).
The antibody response to dengue virus typically begins about six days into the infection and is mainly generated against E and prM glycoproteins. Antibodies to the NS1 protein are also produced due to the fact that this protein is expressed on the surface of infected cells and also secreted (Rodenhuis-Zybert et al., 2010). High levels of early viremia and NS1 in the blood stream has been associated with more severe clinical manifestations. NS1 is also the basis for commercial diagnostic assays for dengue (Simmons et al., 2012). Antibodies to the NS1 particle are able to activate complement mediated lysis of infected cells, while antibodies to E and prM proteins affect the infectivity of the circulating virus. Virus inactivation of dengue occurs only when the number of antibodies docked on the virus exceeds a certain threshold. Strongly neutralizing antibodies require less epitopes to bind to than weakly neutralizing antibodies. The most potent neutralizing antibody for dengue and other flaviviruses has been observed to bind to domain III of the E protein. The human antibody response is typically dominated by antibodies to domain I and II (Rodenhuis-Zybert et al., 2010).
Antibody-mediated enhancement of dengue has been observed but is not yet fully understood. Virion opsonization that has not reached the threshold of neutralization can be taken up by cells carting the Fc receptor. These cells include monocytes, dendritic cells, and macrophages, which are the primary targets of dengue infection. Uptake of these viral complexes increase the number of infected cells and the amount of virus in each cell (Rodenhuis-Zybert et al., 2010). Studies have shown that TH-1 can be infected with dengue which result in decreased production of IL-12, IFN-γ, TNF-α, and NO, and enhanced expression of IL-6 and IL-10 (Chareonsirisuthigul, Kalayanarooj, & Ubol, 2007). This results in the suppression of the antiviral state. Antibody-meditated enhancement is the reason infection with a different serotype after the first infection can result in a more severe illness. Antibodies from the previous infection are not able to opsonize the circulating virus to the neutralizing threshold. However, it should be noted that third infections are rarely reported, and a fourth infection has never been observed (“WHO | Vector-Borne Viral Infections,” 2011).
Pathogenesis into dengue hemorrhagic fever or dengue shock syndrome is marked by a loss of endothelial integrity due to a cytokine storm. “Clinical studies have shown that the levels of cytokines and immune mediators such as TNF- α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-18, MCP-1, and IFN-γ and IFN-α are significantly increased in patients suffering from DHF/DSS” (Rodenhuis-Zybert et al., 2010). It has been claimed that cross-reactive low-avidity T cells that exhibit suboptimal degranulation, altered cytokine production and cytolytic activity are the culprits for creating the storm. It should also be noted that this activity does not explain hemorrhagic fever and shock syndrome in infants (Rodenhuis-Zybert et al., 2010).
Signs and Symptoms
Clinical manifestations of dengue are more severe in children than adults, with the most severe cases being in infants (Whitehorn & Simmons, 2011). Studies have revealed several other risk factors associated with severe dengue which include young age, female sex, high body-mass index, virus strain, and genetic variants of the human major-histocompatibility-complex class I–related sequence B and phospholipase C epsilon 1 genes (Simmons et al., 2012). As mentioned previously, most cases of illness from dengue resolve themselves during the febrile phase within 3 to 7 days of symptoms. Symptoms of the febrile phase include dengue fever (≥38.5°C), headache, vomiting, myalgia, joint pain, and occasionally a transient macular rash (Simmons et al., 2012).
The critical phase of illness is typically observed about 4 to 7 days after the development of symptoms. This phase is marked by defervescence and increasing hemoconcentration, hypoproteinemia, pleural effusions, and ascites within the body. Physiological compensatory mechanisms attempt to maintain adequate circulation to critical organs, which result in the narrowing of pulse pressure. When the pulse pressure drops below 20 mmHg with signs of peripheral vascular collapse, dengue shock syndrome is diagnosed. Hypotension may eventually develop which results in a massive drop in systolic pressure that can result in shock and death. Physicians monitoring patients with dengue should be look for signs of progression into the critical phase during the late stage of the febrile phase. These signs may include persistent vomiting, increasingly severe abdominal pain, tender hepatomegaly, a high or increasing hematocrit level that is concurrent with a rapid decrease in the platelet count, serosal effusions, mucosal bleeding, and lethargy or restlessness (Simmons et al., 2012).
Hemorrhagic manifestations are rare in children and are often associated with prolonged shock. Adults may experience major skin bleeding, mucosal bleeding (gastrointestinal or vaginal), or both with only minor plasma leakage and without any signs of precipitating factors. Thrombocytopenia and decreased fibrinogen are commonly reported, while other severe manifestations such as liver failure, myocarditis, and encephalopathy are less common (Simmons et al., 2012). The altered vascular permeability is resolved within 48 to 72 hours, after which the patient is in the recovery phase. As mentioned previously, the patient may experience a mild to severe rash and/or fatigue for a period of time after the illness. There is currently no available treatment for dengue. No effective antiviral medication is available. Vaccines are currently in development for dengue, but none have been approved so far. Treatment remains supportive, with an emphasis on fluid resuscitation. (Simmons et al., 2012).
The rapid spread of this disease, particularly that of its viral vectors, has put dengue at the forefront of deadly arboviruses affecting humanity. A lack of available treatment and the phenomenon of antibody-mediated enhancement associated with dengue has confounded expert’s ability to treat the disease, which includes the development of vaccines. Based on the mechanisms discussed, a vaccine developed for one of the serotypes would leave the patient vulnerable to infection with the other serotypes. Our understanding of this disease and the methods of treatment has a long way to go. Until then, perhaps the best protection may the preventative use of bug spray in tropical areas.