Dengue Virus

Introduction

Dengue fever (DF) and the more serious forms of DENV infection, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), are currently found throughout much of the world. DF causes extreme bone pain. DHF and DSS are life-threatening, primarily in individuals who have been infected with one, and then another, of the four DENV serotypes. Dengue-related diseases are the most common vector-borne infections of humans, with 50–100 million cases of DF reported per year, primarily in tropical regions. Several hundred thousand cases of DHF occur as well, with a fatality rate of 5% in treated individuals. This severe condition is most common in children and young adults. A very severe epidemic of DF and DHF occurred in 1958, involving 1.2 million people in 56 countries. Over time, the epidemics have become larger and more frequent and, during the last 50 years, dengue-related diseases have risen almost 30-fold. This increase is due, at least in part, to increased human populations in urban centers and rapid transit of infected people via air travel, allowing the virus to become endemic in previously uninfected, local mosquito vectors’ populations.

The Pan American Health Organization reported 3.1 million cases of dengue-related diseases in the Americas in 2019, 28,000 of which were severe and led to over 1500 deaths. These diseases are increasing rapidly in the Americas: the number of cumulative cases in the 1980s was 1.5 million, while 16.2 million cases were reported from 2010 to 2019. In addition to South and Central America, DENV diseases are found in North America. The Centers for Disease Control and Prevention (CDC) reported 20 locally transmitted cases in the mainland of the United States in 2019. As to be suspected, the majority of these cases occurred in Florida, but Texas, North Carolina, and Washington, DC, also reported locally transmitted cases. DENV is also endemic in Puerto Rica and Guam. The World Health Organization reported many endemic cases throughout Latin America in 2019, including over 2 million in Brazil, over 100,000 in Mexico, and over 140,000 in Nicaragua. DENV-1, DENV-2, DENV-3, and DENV-4 are found in Brazil and Mexico, putting the population in those countries at high risk for DHF and DSS. Only DENV-2 was found in Nicaragua.

DENV is usually transmitted to humans by two species of Aedes mosquitoes ( Figs. 2.1 and 2.2 ).

Aedes aegypti is the primary vector for DENV and two other flaviviruses, yellow fever virus (YFV) and Zika virus (ZIKV), as well as for chikungunya virus. Ae. aegypti prefers to use humans as its primary host and, accordingly, lives in urban areas, especially near people’s residences. This anthrophonic behavior may be due to vector domestication. When humans move into an area and alter its fauna and flora, local life forms, including insects, must adapt or face possible removal from the area. Some blood-sucking insects evolve and adapt to the new ecosystem by changing from their preferred, former host animal species to preferring humans, who serve as a readily available, constant source of blood. During this process, the insect vector introduces microbes formerly associated with animals into human populations.

Different strains of Ae. aegypti have a great degree of genetic variation that sometimes leads to large differences in their ability to successfully transmit DENV and YFV to humans. Genetic variations among mosquito strains may give some mosquitoes an adaptive advantage over other strains. Once a mosquito species has gained the ability to utilize humans as its host, it is likely to be introduced over large distances into new geographical regions due to the great mobility of their human hosts. Vector domestication, resulting from human entry into and alteration of ecosystems, thus may contribute to the emergence of new microbial diseases. As humans continue to enter or increase their presence in new regions, this type of indirect zoonotic infection will, most likely, also continue. Additionally, just as Ae. aegypti evolved to use humans as their primary hosts, DENVs may be evolving in a manner that allows them to better utilize either Ae. aegypti or Aedes albopictus as their vector. Some DENV serotypes or strains within a serotype may thus become better adapted to a particular mosquito host and gain a survival advantage over other DENV strains.

Ae. albopictus has less of a biting affinity for humans, but is becoming more important as a DENV vector since it is adapting to life in cooler environments, thus expanding the range of DENV and the number of potential human hosts. This cold-adaptation may allow Ae. albopictus to exploit a new niche where it does not need to compete with Ae. aegypti . It is likely that the mosquito species that are native to this more temperate environment will now also ingest DENV during their blood meals. Over time, these local mosquito species may also become able to serve as vectors that transmit DENV to the local human population. As was discussed earlier, some of the DENV serotypes or strains may become better adapted to Ae. albopictus and may become the predominant type of DENV in more temperate regions.

History

DF has been occurred in humans for at least several millennia. During the Chin Dynasty of China (265–420 AD), a disease with DENV-like symptoms was recorded. This disease was called “water poison” and was linked to exposure to flying insects. The description of a dengue-like illness is also found in a Chinese medical encyclopedia from 992 AD. Similar reports were recorded between 1635 and 1699 in the West Indies and Central America. By the end of the 1700s, periodic epidemics of a disease whose symptoms are very similar to DF were reported in Asia and the Americas. Early reports of epidemics of DF in Asia and North Africa appeared in 1770. Philadelphia, a large population center in the northeastern United States, experienced a large outbreak of DF in 1780. These epidemics were periodic and infrequent. Often intervals of 10–40 years occurred between epidemics due, in large part, to its slow spread by sailing vessels. Ae. aegypti appear to have originated in Africa and then entered and become endemic in tropical or subtropical regions through trading ships carrying freshwater storage containers that could harbor and maintain mosquito breeding colonies.

DF was first reported in Queensland, Australia, in 1873, and the first known dengue-associated death in this continent occurred in 1885. At least 13 major dengue outbreaks have occurred in Queensland since then. DENV subsequently spread into other regions of the continent. However, the last known cases in Western Australia were in the 1940s, and the last epidemic in the Northern Territory, in 1955.

The seeds of the DF pandemic were planted in SE Asia during the Second World War as a result of the movement of military equipment that trapped rainwater containing Ae. aegypti eggs and larvae. This led to the spread of the mosquito vector to previously uninfected regions of the world. During the war, due to the destruction of many water systems, the numbers of water storage facilities increased, providing new niches for mosquito larvae to exploit, especially if the water storage facilities were not adequately covered. The dengue outbreak in Southeast Asia was additionally spurred by the transit of hundreds of thousands of uninfected Japanese and Allied soldiers into the DENV-infected areas. The mosquito vector hosts moved into most of the Central and South Pacific Islands that, due to their isolation and small human populations, could not support dengue epidemics for long periods of time. People living in larger Pacific islands were also infected and, between 1953 and 1954, an epidemic of DHF occurred in Manila in the Philippines, following its appearance in Bangkok in 1950.

Several decades later, DHF stuck Asia in Sri Lanka, India, Pakistan, Taiwan, China, and Singapore as the ranges of the mosquito vector and virus increased. Urbanization was becoming increasingly common. This process moved the human hosts into a more compact mosquito feeding ground. The entry of infected people into these urban centers helped to spread the virus throughout these large populations of immunologically naïve human hosts. Multiple DENV serotypes, predominately DENV-3, were present during an epidemic in the region. The DENV-3 strain associated with the epidemic differed from previous DENV-3 isolates. By 1975, DHF was the leading cause of hospitalization and death in children in the affected regions. Some cases of DENV infection in other Pacific islands also manifested symptoms of DHF. Additionally, about 900,000 cases of either DHF or DSS occurred in Thailand between the years of 1958 and 1990, with a fatality rate of 1.6%. The largest single epidemic in the region occurred in Vietnam in 1987 and resulted in 370,000 cases of DENV-related diseases. In the years since 1990, epidemics in SE Asia have continued to increase in intensity as the presence of multiple viral serotypes in a given region continues to become more frequent. Four times as many cases of DHF were reported in the last 15 years than had been detected during the previous 30 years. Recent modeling suggests as many as 390 million people are infected by DENV worldwide each year.

In several areas of Africa and the Middle East, dengue-related diseases are also increasing. While the surveillance systems in the region are imperfect, the incidence of DF epidemics has been rising since 1980, most notably in East Africa (Kenya, Mozambique, Sudan, Djibouti, and Somalia) and in Saudi Arabia. All four DENV serotypes are active in these regions. While sporadic cases of DHF have been reported in several countries, no epidemic of severe disease has yet been reported in this part of Africa. In addition to humans in western Africa and parts of SE Asia, DENV also circulates in local monkey populations. Also, in addition to Ae. aegypti and Ae. albopictus , Aedes africanus and Aedes luteocephalus are common DENV vectors in some parts of Africa.

In the early 1980s, dengue-related diseases reached pandemic levels and have remained so ever since. The rapid expansion of dengue-related diseases was fueled by the corresponding expansion of the geographical range of the mosquito vectors along with all four virus serotypes. It should be noted that in the late 1980s, more cost-effective and less laborious serotype-specific diagnostic tests were available that allowed the number of people infected with each serotype to be more accurately determined. Even excluding more accurate testing protocols, hyperendemicity is continuing to grow as multiple DENV serotypes are occupying the same region, resulting in the emergence of DHF and DSS. This may not be the first time that DHF and DSS have emerged, since previous clinical reports of illness similar to DHF and DSS are present in medical records from northeastern Australia in 1897 and Greece in 1928. Since these reports are based upon symptoms rather than modern diagnostic techniques, it is possible that these earlier disease reports were produced by other, similar flaviviruses that are no longer circulating in the human population.

Dengue was unknown in the Western Hemisphere until the advent of European colonization and the introduction of the African slave trade into the Americas. DENV’s primary vector, Ae. aegypti , whose ancestor is believed to have originated in Africa, entered the Americas soon after the arrival of African slaves. Programs directed by the Pan American Health Organization greatly reduced the numbers of Ae. aegypti . This mosquito species was eradicated in 18 regional countries and the number of DENV cases and the number of related YFV flavivirus plummeted. At that time, only sporadic cases of DF were found in some of the Atlantic islands that had failed to eradicate the mosquito vectors. Ae. aegypti was reestablished in the Americas in the 1970s due to the failure or abandonment of mosquito control measures, including banning the use of DDT. As the numbers of mosquitoes increased, mosquito-borne diseases in humans also increased. By 1995, the incidence of DF was greater than that reported before the vector control program began. Between 2011 and 2017, another series of DENV outbreaks occurred due to a large influx of tourists to see the 2011 Pan American Games in Mexico, the 2013 Confederations Cup, the 2014 World Cup, and the 2016 Olympics in Brazil.

In 1970, DENV-2 and DENV-3 serotypes were detected in the Americas. DENV-3 caused several epidemics in Puerto Rico and Columbia in the mid-1970s. DENV-1 was introduced into the area in 1977, resulting in epidemics of DF in Jamaica, Cuba, Puerto Rico, and Venezuela in 1977–78 before spreading throughout the Caribbean, northward into Mexico and Texas, and southward into Central America and northern South America. DENV-4 was imported into the eastern Caribbean in 1981 and caused major epidemics throughout the surrounding areas. The same year, a new and more virulent strain of DENV-2 that had originated in SE Asia (most likely from Vietnam) was imported into Cuba, leading to the first major epidemic of DHF in the Americas. Over 10,000 cases of DHF occurred during this outbreak; however, large-scale hospitalization and effective fluid replacement therapy limited the number of deaths to 158 people.

The next major epidemic of DHF in the Americas began soon afterward in Venezuela, in 1989–90, resulting in over 6000 cases of disease and 73 deaths. Between 1990 and 1995, the Pan American Health Organization reported that DENV-2 led to a series of smaller epidemics in Columbia, Brazil, Puerto Rico, and Mexico. By 1997, 18 countries in the region had reported DHF cases. The CDC reported that a novel strain of DENV-3 from Sri Lanka had entered the Western Hemisphere in 1994 and led to an outbreak of DF in Costa Rica and Panama and an outbreak of DHF in Nicaragua. Yellow fever incidence increased in tropical areas of the Americas as well.

The United States reported a small number of imported cases of DF in Texas in 1980, 1986, 1995, and 1997. While the virus has not yet become endemic in most of the continental United States, it is a major public health concern in Puerto Rica and other American-controlled islands in both the Atlantic and Pacific Oceans, including the US Virgin Islands, Samoa, and Guam. The southern part of the United States is home to two species of appropriate mosquito vectors, most notably, Ae. aegypti , which is found along the Gulf Coast states from Texas to Florida. The virus’s secondary vector, Ae. albopictus , entered the United States in the early 1980s and spread to over 25 states since then.

Several factors have contributed to the spread of DENV. RNA viruses, including DENV, have a high mutation rate that allows them to evolve in response to environmental changes and further expand into and exploit new niches. Additionally, the numbers of regional mosquito vectors rise as urbanization increases and adequate access to sewage treatment facilities decreases. Pools of stagnating standing water also fuel the growth of mosquito populations. As stated previously, aggressive vector control programs greatly reduced the numbers of DENV vectors from most of Latin America during the 1940s and 1950s; however, increasing urbanization led to the reinfestation of many areas during the 1970s. Globalization, air travel, and commercial trade rapidly spread microbes, including DENV, into new locations with naïve, susceptible human hosts. Between 1983 and 1995, the number of international travelers leaving the United States increased from 20 to 40 million people, half of which were bound for tropical regions.

The Diseases

The name “dengue” is derived from the Swahili “ki denga pepo” or seizure caused by an evil spirit. Most DENV infections are asymptomatic and the virus is usually cleared from a person in approximately a week. In those people who are symptomatic, the illness may manifest itself as different forms: DF, DHF, and DSS, as well as causing neurological disease in some patients. All forms of the disease are associated with generalized vascular damage. Diffuse hemorrhage, edema, and congestion of organs occur in DHF and DSS, but not in DF. In these two serious disease manifestations, antibodies produced during a prior infection with one DENV serotype, followed by infection with a different serotype, may lead to the formation of large immune complexes in the blood. These complexes contain large numbers of antibodies bound to microbial components. Due to their size, immune complexes may partially occlude blood vessels, resulting in the production of disseminated intravascular coagulation. The widespread coagulation depletes the blood of platelets and clotting factors. This decreases the ability of blood to clot and allows for the formation of hemorrhagic lesions. The mortality rate is 1%–10%, depending on the quality of the available health care. Additionally, DENV infection is associated with neurological disease in both children and adults with an incidence rate of 0.5%–20%. DENV RNA is present in the brain and cerebrospinal fluid of patients with acute neurological disease.

The Viruses

DENVs belong to the mosquito-borne group of the Flavivirus genus and are structurally related to other mosquito-borne flaviviruses, such as ZIKV, YFV, and Japanese encephalitis virus. Both mammalian and mosquito protein kinase G, a cGMP-dependent protein kinase, phosphorylate the DENV-2 NS5 protein, a nonstructural viral protein that replicates flavivirus RNA. Correct protein phosphorylation greatly enhances DENV production in both mammals and mosquito hosts. Additionally, phosphorylation increases flight activity in Ae. aegypti mosquitoes.

DENV exists in four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), which are believed to have emerged approximately 2000 years ago. The existence of a fifth DENV serotype has also been suggested. The nucleoside sequences of the different serotypes’ genomes vary by as much as 40% in the envelope genes whose protein products are the major targets for both antibody- and T cell-mediated immune responses. DENV-1 and DENV-3 are the most closely related members of the group, while DENV-4 has the greatest extent of genetic diversity. Some of DENV-2 and DENV-3 strains found in the Americas appear to be less virulent than the corresponding Asian strains. No immunologic cross-reactivity exists among the different dengue serotypes, thus allowing hyperendemicity (simultaneous infection with more than one DENV serotype) to arise. The four serotypes have undergone an explosion in genetic diversity over the past 200 years, in some ways similar to the more recent explosion of HIV strains. This is likely due to the increased availability of new groups of susceptible human hosts who had not previously been exposed to the virus, perhaps reflecting increases in and interactions among human populations. Rapid movement, such as air travel, and intermingling of human groups who had formerly been separated geographically and culturally may have facilitated this process.

Several mammalian cell types are infected by DENV, including dendritic cells (DCs) and Langerhans cells (immature DCs of the skin’s epidermis), monocytes/macrophages, B lymphocytes, endothelial cells, mast cells, and hepatocytes. Interestingly, many of these target cells are part of the immune system. Their infection induces the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in primary DCs, monocytes/macrophages, and B lymphocytes. TRAIL is protective, regulating DENV replication in infected monocytes in a nonapoptotic manner. Interferons (IFNs), cytokines that serve as one of our primary antiviral defense systems, enhance TRAIL expression, and, in a positive feedback manner, TRAIL then enhances the expression of IFN-β and IFN-inducible antiviral genes.

Early during the course of infection, mosquito-derived DENVs attach to Langerhans cells in a process that relies heavily upon the binding of the viral envelope glycoprotein (E protein) to a DC surface molecule, the mannose-specific lectin, DC-specific ICAM3-grabbing nonintegrin (DC-SIGN) in the presence or absence of a co-receptor. Interestingly, hepatitis C virus, a nonflavivirus member of the Flaviviridae family, also uses DC-SIGN early during the cellular infection process. Langerhans cells are believed to serve as the primary mammalian host cells soon after DENV transmission from its mosquito vectors. Although Langerhans cells typically have antiviral activity, in the case of DENV, when infected, these cells serve as hosts for viral replication and as the primary means of travel through the lymphatic system to lymph nodes at distant sites, as is also the case in other flaviviruses. Langerhans cells are approximately 10 times more susceptible to infection than blood or tissue monocytes or macrophages, respectively.

Other mammalian cell surface molecules serve as receptors for the binding of viral E protein in types of target cells that lack DC-SIGN. DENV binds to L-SIGN in liver endothelial cells and the mannose receptor in macrophages. A highly sulfated type of the heparin sulfate glycosaminoglycan acts as a receptor in epithelial cells. In cells that bear both heparin sulfate and DC-SIGN, heparin sulfate does not alter interactions between DENV and DC-SIGN. The removal of the heparin sulfate from DC-SIGN-bearing cells does not decrease DENV infection. Infectious DENV-2, however, is not able to bind to cultured kidney cells after the removal of heparin sulfate or its desulfuration. Dermatan sulfate, but not chondroitin sulfate A, also plays a role in the infection of DCs. Still, other cells bind to members of the cell surface receptor families TIM and TAM. These receptors normally function in the phosphatidylserine-dependent removal of cells undergoing apoptosis.

DENV gains entry into its various target cells via clathrin-mediated endocytosis, during which the viruses are engulfed by clathrin-coated pits in the plasma membrane of the host cell. The pits then invaginate, carrying the bound DENV into the cell in clathrin-coated vesicles. Experimental inhibition of clathrin-mediated uptake blocks viral entry. The viruses are then taken to Rab5-positive early endosomes, which then mature into late endosomes that lose Rab5 expression while gaining that of Rab7. The acidic environment of the endosomes stimulates an irreversible trimerization of viral E proteins. Next, the DENV and endosomal membranes fuse, allowing the release of genomic RNA from the endosomes into the cytosol. This process requires acidic conditions as well as bis(monoacylglycero)phosphate. Raising the endosomal pH blocks viral escape and inhibits the continuation of the DENV life cycle.

As is the case with other flaviviruses, DENV capsid proteins associate with both nuclear and cytoplasmic endoplasmic reticulum (ER)-derived subcellular compartments and with lipid droplets on the ER surface in both the virus’s mammalian and mosquito hosts. These droplets provide a scaffold for viral genome encapsulation during the production of infectious viral particles. During DENV infection, the number of lipid droplets increases. Inhibition of lipid droplet formation reduces DENV replication. In the ER membranes, translation and glycosylation also occur. The E protein of mosquito-derived DENV contains one to two N-linked high-mannose glycosylation sites. All DENV genotypes contain a possible glycosylation site at Asn 67, a trait that is unique among flaviviruses, as well as a glycosylation site at Asn 153 in DENV-1 and DENV-3.

The nucleocapsid is assembled and remodeled in the ER, producing vesicle-like structures where double-stranded RNA and the viral nonstructural proteins NS2B, NS3, NS4A, and NS4B are produced. Prior to budding from the ER, the DENV genomic RNA associates with viral capsid proteins and then surrounds itself with part of ER lipid bilayer that contains viral E and premembrane proteins (prM). It is estimated that the DENV envelope contains approximately 8000 lipid molecules, making lipids the most abundant component of the flavivirus particle. The virus particles then are passed along to the Golgi apparatus where the prM is cleaved to generate mature and immature virions that are then secreted from the host cell.

Transmission

DENV is transmitted to humans primarily by the bite of female Ae. aegypti or Ae. albopictus mosquitoes. The outbreaks of dengue correlate to the size of the vectors’ population in a given area. They usually occur during the wet season and following heavy rain since these events increase the number of potential mosquito breeding sites. Ae. aegypti are black-and-white mosquitoes that are generally aggressive, domestic, day-biting insects that prefer to use humans for their blood meals. Their biting activity begins in the 2 h after sunrise and ends prior to sunset. They adapt well to urban settings and lay their eggs in small stagnant pools of water or in water that has collected in vessels used to trap and store rainwater, flower vases, pet water bowls, and urban castoffs, such as nonbiodegradable plastic containers, cellophane, and discarded tires. Ae. aegypti are found south of 35°N latitude.

Ae. albopictus , by contrast, is a rural mosquito originating in Asia that has been adapting to urban settings. It is believed to have been introduced into the United States in the 1980s in Asian truck tires brought into the country to be retreaded. Ae. albopictus are much more cold-tolerant than Ae. aegypti , allowing them to extend their range by 7°N latitude. The increased tolerance to cold results from the process of diapause, a suspension of egg hatching during winter months, and is similar to hibernation. Ae. albopictus is currently found throughout much of the eastern United States. Humans are less targeted by these mosquitoes than by Ae. aegypti . Several other mosquito species may also serve as DENV vectors: Ae. scutellaris in Polynesia, Ae. niveus in Malaysia, and Ae. furcifer–taylori in West Africa help to maintain the mosquito–monkey cycle.

Once a mosquito has ingested blood containing DENV from an infected person, it remains infective to humans for the remainder of its life without itself suffering any ill effects from its viral passenger. In addition to acquiring DENV during blood meals from infected human hosts, female mosquitoes may pass DENV to their offspring through their eggs or to their mates through sexual transmission.

Other than mosquito-to-human transmission, several other relatively rare instances of human-to-human transmission have been reported. Several cases of vertical transmission from infected mothers to newborns have been reported and are most likely due to the presence of DENV in breast milk. Nosocomial transmission is also possible via needlesticks contaminated by the virus from an infected patient’s blood. Rare instances of infection have been reported following renal or bone marrow transplantation. A small number of instances of transmission via blood transfusion are known to have occurred as well. Many countries require a deferral period from blood donation following DENV infection.

The Immune Response