Dengue
Marı́a G Guzman, Omar Fuentes, Eric Martinez, and Ana B Perez, PAHO/WHO Collaborating Center for the Study of Dengue and
Its Vector, ‘Pedro Kouri’ Tropical Medicine Institute, Havana, Cuba
Ó 2017 Elsevier Inc. All rights reserved.
This article is an updated version of the previous edition article by María G. Guzmán, Ana B. Perez, Omar Clemente Fuentes González, Gustavo Kouri,
volume 2, pp. 98–119, Ó 2008, Elsevier Inc.
Introduction
Vector-borne diseases are some of the most common and devas-
tating maladies. Currently, vectors that spread disease are an
important public health concern; however, they have been
neglected, with major consequences for health and socioeco-
nomic development. During the 1960s, most vector-borne
disease control programs deteriorated. By the 1980s, many
important diseases such as dengue emerged in new areas or ree-
merged in locations they had once called home. Dengue hemor-
rhagic fever and dengue shock syndrome (DHF/DSS) extended
from Southeast Asia and the Western Pacific to the American
region. The 1998, 2002, and more recently the 2013 dengue
epidemics were unprecedented in terms of number of reported
cases and the large geographic affected areas.
Today dengue is the most rapidly spreading mosquito-borne
disease in the world. In the past 50 years, dengue incidence has
increased 30-fold with increasing expansion to new countries
including extension from urban to rural areas (WHO, 2010).
The Concept
Dengue is an acute mosquito-transmitted disease of humans
caused by any of four serotypes of the dengue virus, with Aedes
aegypti (Ae. aegypti) the most important vector.
Historical Summary
According to some, the origin of the word ‘dengue’ has its roots
in the Swahili term ‘ki denga pepo,’ or a disease characterized by
the sudden cramp-like seizure caused by an evil spirit, applied to
an outbreak in Zanzibar, 1870. The term ‘denga’ or ‘dyenga’ was
used to name the disease on the east coast of Africa in 1823. The
term ‘dengue’ spread with the slave trade from East Africa to the
Caribbean islands and particularly from Cuba during the
outbreak of 1828. Dengue means ‘affectation.’
Epidemics of classical dengue have been recognized for more
than 200 years and doubtless occurred long before that. The first
accurate clinical description of dengue during the Philadelphia
epidemic (1780) was published by Benjamin Rush in 1789.
Outbreaks of possible dengue illness were reported during
the eighteenth and nineteenth centuries along the Atlantic
coast, Caribbean islands, and in the Mississippi basin. Also in
tropical Asia and Australia, dengue pandemics occurred at
periods of 15–20 years during the nineteenth century, evolving
to shorter intervals among pandemics during the first half of
the twentieth century. Table 1 summarizes the major dengue
pandemics and epidemics up until World War II.
Bancroft in 1906 first identified Ae. aegypti mosquitoes as
dengue vectors. This observation as well as the viral etiology
of the disease was confirmed later by studies in human volun-
teers (Halstead, 2012).
International Encyclopedia of Public Health, 2nd edition, Volume 2
During epidemics in Australia, 1897, Greece, 1928, and
Taiwan, 1931, a severe syndrome characterized by shock,
hemorrhagic manifestations, and death was reported.
However, the association of hemorrhagic illness with dengue
Table 1 Major dengue epidemics and pandemics up to World War II
Years
Pre-nineteenth China (992)a; French West Indies (1635); Panama
century (1699); Egypt, Indonesia, Philadelphia, India, Arabia,
and Persia (1779–80)
1818 Lima, Peru
1824–28 Suez, Egypt; north of Calcutta, India; Curacao; Virgin
Islands; Jamaica; Lesser Antilles; Cuba; northern
Colombia; Vera Cruz, Mexico; Bermuda; southern
United States
1845–68 St. Louis, United States; Senegal; Cairo, Egypt; Rio
Janeiro, Brazil; Calcutta and Kanpur, India; Hawaii;
southern United States; Tahiti; Lima, Peru; Cadiz,
Spain; Canary Islands; Port Said, Egypt; Tanzania;
Kenya
1870–77 East African coast; Arabian peninsula; Bombay, Pune,
and Calcutta, India; Singapore; Java, Indonesia;
Taiwan; Mauritius Islands; southern United States;
Lima, Peru
1883–90 Gibraltar; Greek Islands; southern Turkey; Syria;
Palestine; the Nile delta of Egypt; Istanbul on the
Bosporus; Varna and Trabzon on the Nlack Sea;
Israel; Zanzibar; Tanzania; Fiji
1894–99 Townsville and Charters Towers, Brisbane, Australia;
Indochina and Hong Kong; India; Texas and Florida,
United States; Havana, Cuba; San Juan, Puerto Rico;
Eritrea; Somalia
1901–07 Hong Kong; Bangkok, Thailand; Rangoon, Burma;
India; Singapore and Penang, Malaysia; Hawaii; Java,
Indonesia; the Philippines; Taiwan; Brisbane,
Australia; Galveston and Houston, United States;
Havana, Cuba; Natal, South Africa; Egypt
1912–18 Panama; Meerut, northern India; Iquique, Chile;
northern Argentina; Queensland, Australia; San Juan,
Puerto Rico; Taiwan; Rio Grande do Sul, Brazil;
Sudan
1920–28 Australia; India; Manila, Philippines; Taiwan; Ghana;
Niteroi, Brazil; from Texas to Florida and Georgia,
United States; Venezuela; Yemen; Senegal; South
Africa; Athens, Greece; Egypt; Vietnam; Durban,
South Africa
1930–36 Pacific Islands; Taiwan; Japan; Java and Sumatra,
Indonesia; Malaysia; Burma; India; Florida, Georgia
and Alabama, United States; India; Ghana; Egypt
1940–45 Australia; Hawaii; Caribbean and South-East Asian
countries; Japan; India; Philippines
a
Chinese Encyclopedia of Disease and Remedies (AD 265–420) was edited in AD 610
and again in 992.
Adapted from Gubler, D.J., Kuno, G. (Eds.), 1997. Dengue and Dengue Hemorrhagic
Fever. CAB International, Wallington, UK, with permission.
http://dx.doi.org/10.1016/B978-0-12-803678-5.00103-X 233
234 Dengue

Figure 1 Annual number of dengue cases and countries with dengue report to PAHO/WHO, 1980–2013.

viruses was recognized during outbreaks of viral hemorrhagic Today severe dengue is observed in all countries with dengue
fever in Manila and Bangkok during the 1950s. Indeed, during transmission.
the 1950s, DHF/DSS was recognized as a syndrome associated Figure 1 shows the reported dengue cases and countries
with dengue viruses throughout Southeast Asia. with transmission by year in the Americas.
According to Gubler and Kuno (1997) and Gubler (1997), Table 2 presents the most important features reported in the
the ecologic disruption and demographic changes occurring Americas during the past 50 years of the twentieth century.
during and after World War II were conducive to increased Dengue surveillance in Africa has been very poor. At the end
transmission of dengue. The need for stored water contributed of the twentieth century, some epidemics were reported;
to the increased number of Ae. aegypti. Moreover, war facilitated however, currently the four serotypes have been isolated from
the transportation of the mosquitoes and their eggs to new Africa, and available data suggest that DENV is endemic in at
geographic areas through the transport of supplies and other least 34 countries (Were, 2012; Figure 2).
war materials. Conditions were thus created for the emergence
of DHF/DSS in Southeast Asia.
Current Epidemiological Situation
The second half of the twentieth century was characterized
by the geographic expansion of the disease, having been The beginning of the new millennium was characterized by an
preceded by the extension of the mosquito vector and the expanding distribution of Ae. aegypti and Ae. albopictus to most
dengue viruses. DHF/DSS extended first throughout Southeast tropical and subtropical areas of the world, co-circulation of
Asia and later to the Western Pacific to finally reach the Amer-
ican region. Countries evolved from a no endemic or Table 2 Main features of dengue epidemiology in the past 60 years
hypo-endemic situation to that of high endemicity. During in the American region
the 1980s and the 1990s, DHF/DSS became a major pediatric
Year Features
problem in Southeast Asia and the Western Pacific. By 2000,
dengue epidemics reached African and Eastern Mediterranean 1953 First dengue virus isolation (DENV-2 isolation in Trinidad
countries (Guzman and Kouri, 2003). During the 1950s, an and Tobago) in no epidemic situation
Ae. aegypti control program targeting urban yellow fever preven- 1963–64 DENV-2 and DENV-3 epidemic
tion in the American region had eliminated the vector from 1968–69 DENV-2 and DENV-3 epidemic
many countries, and consequently there was a decrease or elim- 1977 DENV-1 introduction in Jamaica
ination of dengue epidemics. However, due to the deteriora- 1981 First DHF/DSS epidemic, Cuba
tion of the mosquito control programs, the reinvasion by Ae. 1981 DENV-4 introduction in Caribbean countries
1985 Introduction of Ae. albopictus mosquito
aegypti started in some countries by the 1970s, continuing
1989 Second DHF/DSS epidemic, Venezuela
during the 1980s and the 1990s (Guzman and Kouri, 2003; 1994 Reintroduction of DENV-3 in Nicaragua, Panama, and
San Martin et al., 2010; Brathwaite et al., 2012). Costa Rica
The first DHF/DSS epidemic in the Americas was reported in 2013 More than 2 million cases reported
1981 in Cuba. More than 344 000 patients with more than Current Expansion of Ae. aegypti mosquito, dengue
10 000 severe and very severe cases and 158 fatalities (101 chil- situation hyperendemicity with co-circulation of multiple
dren) were reported (Kouri et al., 1989). After the second DHF serotypes, endemicity of DHF
epidemic occurred 8 years later in Venezuela, an increase in the
Adapted from Guzman, M.G., Kouri, G., 2003. Dengue and dengue hemorrhagic
number of DHF cases and epidemics and in the number of fever in the Americas: lessons and challenges. J. Clin. Virol. 27, 1–13 with
countries reporting the severe syndrome has been observed. permission.

Known endemic
Suspected endemic
Figure 2 Known or suspected dengue endemic in Africa. Reproduced from Were, F., 2012. The dengue situation in Africa. Paediatr. Int. Child
Health 32, 18–21.
multiple serotypes of dengue virus, development of endemicity
of severe dengue in many countries of the tropical world, and
increased frequency of dengue activity (Simmons et al.,2012;
Figure 3).
According to WHO, dengue has been recognized in over
100 countries and causes an estimated 50–100 million infec-
tions annually with more than 2.5 billion people at risk. There
are about 500 000 DHF cases and an average case fatality rate of
5% (around 20 000 deaths). Recent estimates report
390 million dengue infections with 96 million cases annually
worldwide, more than three times the dengue burden esti-
mated by WHO (Bhatt et al., 2013). Although the distribution
and burden of dengue is still not well characterized, the disease
is endemic in Africa, the Americas, the Eastern Mediterranean,
Southeast Asia, and the Western Pacific. Circulation of the
four serotypes has been reported in these regions.
Particularly, an increase in dengue transmission in Africa,
India, The Americas, and Eastern Mediterranean countries
such as Pakistan, Saudi Arabia, Sudan, and Yemen character-
ized the last decade. An example of the dengue situation is
the recent report in 2013 of more than 2 million cases in
the American region, the highest figure in the history of
dengue in this geographic area. Dengue has resurged in
Hawaii, Galapagos Islands, Easter Island, and Buenos Aires.
Autochthonous dengue transmission has been recently
confirmed in Hawaii (2015), Japan (2014), Florida
Dengue 235
(2009–10; 2013), and southeastern France (2010), with an
epidemic of more than 2000 cases in Islas Madeiras, Portu-
gal. Coinfections of dengue with malaria, HIV, leptospirosis,
influenza, chikungunya, and Zika virus have been increas-
ingly reported.
In this last decade, travelers have played an important role
in global dengue epidemiology, carrying viruses from one
region to another (Wilder-Smith et al., 2014). Figure 4 exem-
plifies travellers from dengue endemic countries that Islas
Madeiras received in 2012.
Finally, a very interesting observation is that while malaria
is declining, the number of dengue cases is rising. This fact
supports the serious epidemiological global dengue situation
(Table 3; Brady et al., 2012)
Dengue burden determination is still a research priority. As
not all infections are symptomatic, asymptomatic infections
should be considered as well as those infected individuals
with a mild illness not detected by the surveillance systems.
Figure 5 shows the estimated number of symptomatic and
inapparent dengue infections by geographic region (Bhatt
et al., 2013). The real impact of asymptomatic infections in
dengue transmission is not well known, although a very recent
study has supported the contribution of individuals with an
asymptomatic DENV infection to the dengue transmission
dynamic by efficiently infecting mosquito vector (Duong
et al., 2015). This issue constitutes a research priority.
236 Dengue

Figure 3 Dengue risk areas. Simmons, Cameron P., Farrar, Jeremy J., Van Vinh Chau, Nguyen, Wills, Bridget, 2012. Dengue. N. Engl. J. Med. 366,
1423–1432.

Figure 4 Map of air travel volume from endemic countries and cities to islas Madeiras, 2012. Reproduced from Wilder-Smith, A., Quam, M.,
Sessions, O., Rocklov, J., Liu-Helmersson, J., Franco, L., Khan, K., 2014. The 2012 dengue outbreak in Madeira: exploring the origins. Euro Surveill.
9 (8), 19. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId¼20718.
 Dengue 237

Table 3 Comparison of dengue and malaria burden and possible recombination. The major drivers of geographic
dengue spread and increased incidence are (Gubler, 2011):
Dengue
l lack of effective mosquito control,
Brady et al. WHO malaria l changing life styles,
WHO (2012) report, 2013
l unplanned urbanization,
Population at risk 2.5 billion 3.97 billion 3.4 billion l globalization.
Number of endemic >100 128 97
countries
Number of infections 50 million 70–500 million 219 million The Agent
per year
Hotta and Kimura in 1943 were the first to isolate dengue
viruses through inoculating the brains of suckling mice with
the serum of clinically ill dengue patients. Almost simulta-
neously (1944), Sabin isolated viruses from US soldiers in
Global
India, New Guinea, and Hawaii. These first isolations were later
classified as serotypes 1 and 2 (DENV-1 and DENV-2). Sero-
Oceania types 3 and 4 (DENV-3 and DENV-4) were isolated by Ham-
Symptomac
Americas mond during the epidemic of Manila, Philippines in 1956.
Inapparent
These closely related virus serotypes belong to the genus fla-
Asia
vivirus, family Flaviviridae. This family of enveloped RNA
Africa
viruses causes significant disease both in humans and animals.
0 100 200 300 400 Flavivirus, the largest of the three genera of the family,
comprises over 70 viruses, mostly arthropod-borne viruses,
Figure 5 Estimates of symptomatic and inapparent dengue infections with yellow fever virus the prototype.
by geographic regions. Adapted from Bhatt, S., Gething, P.W., Brady, Mature particles of dengue viruses are spherical with a diam-
O.J., Messina, J.P., Farlow, A.W., Moyes, C.L., Drake, J.M.,
eter of 50 nm, containing multiple copies of the three structural
Brownstein, J.S., Hoen, A.G., Sankoh, O., Myers, M.F., George, D.B.,
virus proteins, a host-derived membrane bilayer, and a single
Jaenisch, T., Wint, G.R., Simmons, C.P., Scott, T.W., Farrar, J.J.,
Hay, S.I., 2013. The global distribution and burden of dengue. Nature, copy of a positive-sense, single-stranded RNA genome. The
496, 504–507. genome of the four viruses contains a single open reading frame
of 10 233 nucleotides encoding for a polyprotein ordered
as 50 -C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5–30 . The
The current pandemic and the increase in dengue epidemics full-length polypeptide is processed by viral and host proteases
are the direct result of changes in demographic and economic into the capside (C), membrane (M), and envelope
trends, such as (E) structural proteins that make up the virus and seven
l the global population explosion, with an increased pop- nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B,
ulation density; and NS5). The C protein binds strongly with RNA to form the
l unplanned urbanization (resulting in substandard housing nucleocapsid. The pre-membrane (prM) glycoprotein
and inadequate water, sewer, and waste management represents the precursor of the mature M protein. It has
systems); a crucial role, as it prevents the E glycoprotein from
l rapid movement of individuals potentially infected with undergoing an irreversible acid-catalyzed conformational
dengue, both within and between countries, favoring the change during its transportation through host cell acidic
introduction of dengue viruses; compartments. The major structural protein (E) represents the
l deterioration and breakdown of the public health infra- target for neutralizing antibodies, protective immunity, and the
structure that through surveillance monitors for diseases antibody-dependent enhancement (ADE) phenomenon. It also
such as dengue; mediates receptor binding and pH-dependent membrane
l deterioration of vector control programs that had controlled fusion activity. E glycoprotein is divided into three functional
populations of Aedes and other mosquitoes: domains, termed domain I, II, and III. Domain I, the hinge
region, is linked to the two other regional functional domains
B climate change and particularly temperature and
(this region is responsible for the changes in E structure due to
humidity changes are factors that favor increased
variation of external pH). Domain II has a hydrophobic-rich
dengue transmission;
peptide sequence featuring the membrane fusion activity.
B social and economic factors (e.g., poverty, illiteracy,
Domain III is probably involved in the binding to receptor
and low-risk perception).
molecules on the host cell membrane. NS1 glycoprotein can
Finally, new dengue strains can enter into a specific popula- be found on the cell surface, inside the cell, and as a secreted
tion via migration (gene flow), as dengue viruses are often form. It may have a role in RNA replication and in the
transported large distances by hosts and vectors. This fact, in pathogenesis of clinical disease.
conjunction with the increase in the size and density of the The NS3 protein has three enzymatic activities, a trypsin-like
human host population, provides more opportunities for viral serine protease, an RNA helicase, and an RNA triphosphatase
transmission with an extension of serotypes and genotypes activity. Together with NS2B, it forms the protease domain of
with a wider geographic distribution, greater mixing of strains, the virus.
238 Dengue

Protein MW Function
E (kDa)

E 55-60 Cell receptor binding, envelope fusion.

M
prM 18-19 Proper E folding and assembly. Avoid E
premature fusion during secretion.

C C 9-12 Binds the RNA to form the nucleocapsid

NS1 42-50 Viral replication??

NS2a 22 RNA synthesis and virus assembly??

RNA
NS2b 14 Cofactor of serine protease activity of NS3.

NS3 67-70 Serine protease (polyprotein processing),

NTPase and helicase (synthesis of viral RNA),
5´ Positive single-stranded RNA (11 kb) 3´ and triphosphatase (capping pathway).

Cap ORF NS4a 16 RNA synthesis and virus assembly??

Structural proteins Non structural proteins NS4b 27 RNA synthesis and virus assembly??

NS5 104- RNA dependent- RNA polymerase (synthesis

106 of viral RNA), and methyltransferase
C prM E NS1 NS2 NS3 NS4 NS5 (capping pathway).

Cytoplasm
Host signalase
Endoplasmic Viral serine protease (NS2b-NS3)
reticulum Host furin
Unknown

Intralumen space

Figure 6 Dengue virus genome, viral proteins and their functions and polyprotein processing in cell endoplasmic reticulum.

Functions of NS2A, NS4A, and NS4B are not well known
but are believed to play a role in RNA replication.
NS5 is the largest and most conserved viral protein, and it is
considered the putative RNA-dependent RNA polymerase
responsible for viral RNA replication. The methyltransferase
activity of this protein is probably involved in the
methylation of the 50 cap structure (Figure 6).
Dendritic cells (skin Langerhans cells), monocytes, and
macrophages seem to be the primary target cells in vivo for virus
replication. In vitro studies suggest that fibroblasts, keratinocytes,
endothelial and hepatic cells, and neurons can also be infected.
Mastocytes are also targets for DENV infection (Figure 7).
Several putative receptors have been identified on human
and mosquito cell types. Studies suggest that heparin sulfate
and DC-SIGN are indispensable for dengue virus infection in
humans. Heparan sulfate is thought to be a co-receptor,
which associates with other molecules to form functional
complexes and enhances the efficiency of virus infection into
host cells. DC-SIGN serves as an important attachment factor
for dengue virus on dendritic cells. Apparently carbohydrate
molecules in the extracellular matrix are strongly related to
DENV receptors (Hidari and Suzuki, 2011).
After attachment of E protein to surface host receptors,
virions enter cells via receptor-mediated endocytosis. Later,
virions are found in uncoated prelysosomal vesicles, where
an acid-catalyzed membrane fusion releases the nucleocapsid
into the cytoplasm. The viral genome is thus released into the
cytoplasm and is used as a template for a large precursor poly-
protein. In the process of virus attachment to cell surface and
membrane fusion, domains I, II, and III of the E glycoprotein

DC-SIGN FcγR Y IgG antibody Virus

Figure 7 Major target cells of dengue virus infection.


Figure 8 Replication cycle of dengue virus.
play an important role. After synthesis of RNA strands of nega-
tive polarity, the plus-stranded RNA molecules are generated.
These genomic plus-stranded RNAs (RNSs) serve as templates
for further negative strand production, or they are packaged
into the new virus particles.
Cleavage at the amino-terminal portion of the polyprotein by
host cell enzymes produces the structural proteins that form
superstructure for the enveloped virus. The remaining viral poly-
protein is cleaved by the virus protease, producing the NS
proteins that are required for producing more virus copies.
Translocation of prM, E, and NS1 into the lumen of the
endoplasmic reticulum occurs co-translationally and is initi-
ated by a signal sequence at the C terminus of the C protein.
Host signalized cleavages at the prM–E and E–NS1 junctions.
Soon after translation, the prM and E proteins associate as a het-
erodimeric complex. The prM acts as a ‘chaperone’ to aid the
folding and maturation of E protein. These heterodimers drive
the assembly and budding of immature particles at the rough
endoplasmic reticulum membrane. Prior to or during release
of the infectious virus from cells, the host enzyme furin cleaves
the prM protein, probably dissociating the prM/E heterodimer,
resulting in rearrangement of the E proteins into dimers on the
surface of the mature particle. The pr protein is secreted and the
M protein remains anchored in the virus membrane. The
matured virions are transported through an exocytic way into
the plasma membrane and released to extracellular space via
secretion (Figure 8). Both mature and immature virions are
produced during in vitro DENV propagation; however, the
maturation state of dengue virions in humans is unknown.
Differences in the conformation of E and prM/M proteins on
the surface of mature and immature virions may have impor-
tant implications in dengue pathogenesis and vaccine develop-
ment. Recent studies have supported that infectivity of
immature particles is restored by prM-specific antibodies,
highly cross-reactive among the dengue serotypes, permitting
virus entry via the Fc receptor expressed on the surface of
immune cells and presumably targeting the virus sites where
furin-mediated cleavage of prM occurs. As these antibodies
are not present during a primary DENV infection, enhanced
secondary infection might be due to these antibodies (Fischil
Dengue 239
1. Binding
2. Receptor-mediated
endocytosis
3. Low ph-mediated
membrane fusion
4. Uncoating
5. Translation
6. Polyprotein processing
and RNA replication
7. Virion assembly
8. Virion transportation and
maturation
9. Virion release
and Bartenschlager, 2011). Therefore, partially immature parti-
cles expand the pool of infectious virus and likely contribute to
the pathogenesis of the illness as they probably are involved in
ADE and the neutralization phenomena (Voorham et al., 2012;
Rodenhuis-Zybert et al., 2011a,b).
Dengue viruses show substantial genetic diversity (Holmes
and Twiddy, 2003; Holmes and Burch, 2000). In addition to
the four serotypes, clusters of variants (known as genotypes)
have been identified within each serotype. Molecular epidemi-
ologic approaches have demonstrated the extensive genetic
variability of these viruses (Table 4). DENV-1, DENV-2, and
DENV-3 classify into five genotypes, while DENV-4 into 4
genotypes. In addition to genotypes, also intra-genotypic
groups within each genotype have been suggested (Amarilla
et al., 2009). There is some evidence that recombination in
natural populations of DENV serotypes could contribute to
the genetic variation of DENV. The circulation of different sero-
types and genotypes of DENV in a particular geographic area
has been documented as well as the coexistence of two different
serotypes in a given mosquito or patient, making feasible the
recombination phenomena.
At the population level, the replacement of one viral geno-
type for another of the frequent lineage replacement within one
genotype and mutations of nonstructural proteins have been
associated with a higher virus transmissibility and epidemic
severity, respectively. Studies in Mexico support that, for each
Table 4 Genotypes within dengue serotypesa
Serotype Genotypes
DENV-1 Sylvatic/Malaysia, Americas/Africa, South Pacific, Asia,
and Thailand
DENV-2 American, American/Asian, Asian 1, Asian 2, Cosmopolitan,
and Sylvatic
DENV-3 Southeast Asia/South Pacific, Thailand, Indian subcontinent,
and Americas
DENV-4 Sylvatic/Malaysia, South-East Asia, and Indonesia
a
Classification in genotypes uses nucleotides from the entire E gene region.
Rico-Hesse, R., 2003. Microevolution and virulence of dengue viruses. Adv. Virus
Res. 59, 315–341.
240 Dengue
serotype, multiple introductions of viral lineages have occurred
with little co-circulation (Carrillo-Valenzo et al., 2010). Virus
evolution in Mexico is typified by frequent lineage replace-
ment, with only a single lineage dominating a specific serotype
at a specific time point. A different situation was reported in
a highly endemic area such as Vietnam where multiple lineages
of DENV-1 were observed during the period of 2003–08 sug-
gesting equivalent viral fitness (Raghwani et al., 2011). In the
same area, previous studies suggested that a DENV-2 genotype
replacement occurred (Asian-American genotype by Asian I
DENV-2 genotype). The higher viremia produced during the
Asian I DENV-2 infections would likely increase the probability
of human to mosquito transmission and in consequence facil-
itate greater population diffusion and possibly an increased
incidence of more severe disease (Hang et al., 2010). Recently,
Rodriguez-Roche et al. (2012) studying isolates from Vene-
zuela found that changes at nonstructural proteins such as
NS1, NS2A, and NS4B may play an important role in virus
evolution. In the same study they have demonstrated the
co-circulation of several lineages of DENVs and an association
of DENV-2 with severe cases (Rodriguez-Roche et al., 2012).
Dengue Vectors
Dengue viruses are transmitted to humans by mosquitoes from
the Aedes genus and mainly by Ae. aegypti. They are widely
distributed generally within the limits of 40 North or South
latitude, being most frequently found in the tropics (Figure 9).
Females feed on any vertebrate host, but prefer humans
(Christophers, 1960). They fly upwind, following chemoattrac-
tant odors. The first step can be to enter a house. Blood feeding
and oviposition occur mostly in the morning and late in the
afternoon. Only the female bites for blood, which she needs
to mature her eggs.
She takes a complete blood meal of 2–3 ml of blood, and
will produce a batch of about 100 eggs in about 3 days.
Stomach distention triggers ovarian development. Thus,
smaller blood meals produce fewer eggs, and refeeding with
repeated biting by the same female occurs when the volume
of ingested blood is too small for efficient egg production.
Figure 9 Ae. aegypti mosquito.
Older populations, having taken many blood meals, have
a greater potential for virus transmission.
Females usually fly no more than 50 m, but they can easily
fly 100–200 m, and can travel 3 km in search for a site to
oviposit in. They can be transported by cars, trucks, aircraft,
and even hurricanes for still longer distances. Ae. aegypti
mosquitoes are peridomestic; that is, they prefer to rest inside
the house, rather than in the garden. Most resting is on walls.
These mosquitoes can live for months; yet most usually
survive only a few weeks. Half of them die in the first week
and 95% in the first month of life.
The mosquito’s preferred breeding sites are in areas of stag-
nant water, such as flower vases, uncovered barrels, buckets,
and discarded tires. The most dangerous areas are wet shower
floors and toilet bowls, as they allow the mosquitoes to breed
right in the residence. Universally, automobile and truck tires
are the main breeding sites for these mosquitoes.
Other mosquitoes such as Ae. albopictus, Ae. polynesiensis,
and some forms of Ae. scutellaris can also transmit the disease.
Generally Ae. aegypti is the main vector in urban transmission,
but Ae. albopictus is shown to be more competent and more
susceptible to experimental infection. Ae. albopictus may be
a less selective feeder so it is of less epidemiological signifi-
cance, as it may bite nonhuman hosts as well as humans,
diluting its capacity to acquire and transmit dengue.
The DENVs originated and are maintained in a forest trans-
mission cycle involving forest-dwelling Aedes mosquitoes and
lower primates from Asia and Africa. Sylvatic cycles in Asia
are associated with Macaca and Presbytus sp. monkeys and
mosquitoes of the genus Ochlerotatus, and in Africa with Eryth-
rocebus patas monkeys and several sylvatic mosquitoes such as
Ae. taylori, Ae. furcifer, Ae. luteocephalus, and Ae. opok. In Asia,
circulation of sylvatic DENV-1, DENV-2, and DENV-4 has
been detected in sylvatic mosquitoes and/or sentinel monkeys.
In Africa, sylvatic DENV-2 has been associated to sylvatic
mosquitoes (Franco et al., 2011).
The urban cycle, mosquito to human to mosquito, is the most
important in terms of human transmission and disease outcome;
however, some sylvatic DENV isolates from patients have been re-
ported including one from a DHF case (Franco et al., 2011).
The Disease
Dengue infection may be clinically unapparent or cause an
illness with varied intensity, including patients with fever
and body pains to the severe picture of shock and large
hemorrhages with or without organ impairment. Plasma
leakage rather than bleeding is a major determinant of most
cases of dengue shock, associated with significant hematocrit
elevations and accumulation of fluid in serous cavities, such
as pleural effusions, ascites, and pericardial leakage. Dengue
severity can also be associated with encephalopathy, cardio-
myopathy, or hepatopathy, as well as kidney dysfunction
(Simmons et al., 2012).
Dengue is a very dynamic illness, in spite of its short dura-
tion (no more than 1 week in nearly 90% of the cases). Its clin-
ical expression can change as the days go by and can also
worsen suddenly. Dengue illness can evolve into three phases:
the febrile phase – observed in most of the patients – and the
critical and the recovery phases (WHO, 2010).

Figure 10 Petechiae in a DHF patient. Kindly supplied by S. Zagne,
Niteroi, RJ, Brasil.
The febrile phase is variable in duration and is associated
with the level of viremia (virus in blood). Fever is generally
the first clinical manifestation of illness with a variable inten-
sity. It is associated with headache and vomiting, as well as
body pains. In children, fever is frequently the only clinical
manifestation or is associated with rash and/or unspecific
digestive symptoms. The pharynx may become reddened,
but other symptoms and signs of the respiratory system are
not frequent or important (Capeding et al., 2010). A slight
abdominal pain and diarrhea may occur. Diarrhea is more
frequently reported in patients younger than 2 years of age
and in adults. The leukocyte count is usually decreased. Pete-
chiae or ecchymosis can be present, with or without thrombo-
cytopenia (Figure 10).
Most cases recover after defervescence, however in some, the
patient’s state worsens when the fever drops. Therefore, the
period during which the fever subsides indicates the beginning
of the critical phase.
Dengue is a dynamic disease (Balasubramanian et al., 2012).
The critical phase coincides with the leakage of plasma that
can lead to shock, with coldness in the teguments, weak pulse,
delayed capillary filling, tachycardia, and hypotension. At the
beginning, not all clinical signs of shock are observed being
enough to detect a narrowing of the differential arterial tension
(AT) or pulse pressure (difference of 20 mmHg or less between
the maximum or systolic AT and the minimum or diastolic AT).
At this stage, the hematocrit level is increased and the platelets
– which were decreasing already – reach their lowest figure.
With an adequate and appropriate fluid administration,
dengue shock is rapidly reversible, although it may recur during
the next 12–24 h, and sometimes more. The prognosis depends
primarily on the early recognition and treatment of shock,
which depends on careful monitoring and prompt action, for
preventing other complications such as metabolic acidosis,
multiorgan failure, major hemorrhages, and disseminated
intravascular coagulation (DIC) (Khan et al., 2008). At the
recovery phase, the patient’s improvement is usually evident,
but on occasions a state of fluid overload occurs, as well as
secondary bacterial infections.
The differential diagnosis of dengue includes influenza,
exanthematous infections (measles or rubella), severe bacterial
infections (meningococcemia, bacterial sepsis, typhoid fever,
or leptospirosis), and other viral hemorrhagic fevers. Details
of the treatment of dengue and severe dengue are out of the
Dengue 241
scope of this review; however, a very good description appears
at the WHO guidelines for dengue prevention and control
(WHO, 2010).
New Dengue Clinical Classification
For three decades the WHO recommended the classification of
dengue as dengue fever (DF) and DHF with or without DSS. Clin-
ical manifestations of DF should include fever and two of the
following symptoms: headache, retroocular pain, osteomyoartic-
ular pains, rash, leukopenia, and bleeding (WHO, 1997).
Four criteria characterize DHF: (1) fever or history of fever in
the previous week; (2) spontaneous bleeding – usually pete-
chiae, or other – or, at least, a positive tourniquet test; (3)
thrombocytopenia less than 100 000 per mm3; and (4) plasma
leakage, evidenced by a 20% elevation of the hematocrit level,
or by a 20% decrease of the hematocrit level after the critical
phase of illness, or by the presence of pleural effusion, ascites,
or pericardial leakage determined by imaging studies, usually
sonography (WHO, 1997).
In recent years several authors questioned the usefulness of
the former WHO dengue clinical classification in DF and DHF
(Bandyopadhyay et al., 2006; Deen et al., 2006; Rigau-Perez,
2006). According to them, this classification fails as it is too
dependent on laboratory results, and also it does not include
severe dengue patients with organ impairment such as patients
with encephalitis, myocarditis, and severe hepatitis. Addition-
ally, the classification is not useful for patient clinical manage-
ment, either. Taking into account these arguments the Special
Programme for Research and Training in Tropical Diseases
(TDR/WHO) conducted an international study as part of the
DENCO (Dengue Control) projects, one of whose components
was clinical, with the main purpose to obtain information from
a high number of patients with a laboratory confirmed dengue
infection and to find a better way to clinically classify them, as
well as to identify warning signs of severity useful to improve
patient management.
Clinical information was obtained from nearly 2000 patients
(from seven countries in two continents) with confirmed
dengue. The study concluded that 18–40% of the cases could
not be classified by the WHO Classification as DF or DHF,
and over 15% of cases with shock could not be classified as
severe cases of dengue either, since they did not fill the criteria
of DHF/DSS. Another consistent result was the binary classifica-
tion of the disease as dengue and severe dengue (Alexander et al.,
2011; Figure 11), considering that a spectrum of disease exists
rather than two distinct entities (Farrar et al., 2013).
Criteria for severe dengue were (1) severe plasma leakage,
expressed by hypovolemic shock, and/or difficulty breathing
due to the fluid accumulated within the lungs; (2) severe
hemorrhage, according to the criterion of the assisting physi-
cian; and (3) organ damage: severe hepatitis (transaminases
higher than 1000 units), encephalitis, or a serious damage of
other organs, such as myocarditis. These severity criteria had
95% of sensitivity and 97% specificity (Alexander et al.,
2011). The new WHO Dengue Classification was validated in
18 countries (Barniol et al., 2011). Further studies have sup-
ported the usefulness of the new classification (Basuki et al.,
2010; Narvaez et al., 2011; Lima et al., 2013).
242 Dengue
Dengue ± Warning signs
With
Without
warning
signs
Presumptive diagnosis
•Fever, and two or Warning signs*
more> •Abdominal pain or tenderness
•Vomiting, nausea •Persistent vomiting
•Rash
•Clinical fluid accumulation
•Body pains •Mucosal bleed
•Leucopenia
•Lethargy; restlessness
•Petechiae or Tourniquet
•Liver enlargement >2cm
test
•Laboratory: Increase in HCT
Neighbourhood concurrent with rapid decrease
dengue/history of travel in platelet count
to dengue endemic area
Figure 11 WHO dengue classification, 2010. Reproduced from WHO, 2010. Working to Overcome the Global Impact of Neglected Tropical
Diseases. First WHO report on neglected tropical diseases, Geneva.
‘Warning signs’ such as intense and continuous abdominal
pain, frequent vomiting, sleepiness, and/or irritability, as well
as a sudden decrease in temperature leading to hypothermia
sometimes associated with fainting due to orthostatic hypoten-
sion are indicators of the presence of shock. These signs appear
more frequently at defervescence, and allow identification of
the loss of fluid to the extracellular space, which the patient
can hardly compensate or cannot compensate at all (Gibson
et al., 2013; de Andrade et al., 2014). The revised WHO/TDR
(2009) classification shows a very high sensitivity for identi-
fying severe dengue and is easy to apply (Prasad et al., 2009).
The better performance of the revised classification in the detec-
tion of severe clinical manifestations allows for improved
detection of patients with severe dengue and may reduce deaths
(Pamplona et al., 2014).
Risk Factors for Dengue Transmission and DHF/DSS
Transmission dynamics of dengue virus are determined by the
interaction of the environment, the agent, the host population,
and the vector. These components can be divided into macro-
determinants and microdeterminants. The new WHO dengue
clinical classification was introduced in 2010, hence most path-
ogenesis studies, including risk factor studies for severe disease,
have been done using the old WHO clinical classification,
namely, DF and DHF/DSS.
In particular, DHF occurs as a consequence of a very
complex mechanism where the virus, host, and environment
interact in such a way that this severe form of the disease
occurs in 2–4% of individuals with a secondary infection.
An integral hypothesis for the development of DHF
epidemics was early proposed and later documented
(Kouri et al., 1989). The intersection of the three groups of
factors determines the occurrence of the severest dengue
Severe dengue
1.Severe plasma leakage
2.Severe hemorrhage
3.Severe organ impairment
1. Severe plasma leakage leading to
•Shock (DSS)
•Fluid accumulation with
respiratory distress
2. Severe bleeding
as evaluated by clinician
3. Severe organ involvement
Liver: AST or ALT>=1000
CNS: Impaired consciousness
heart and other organs
* Requiring strict observation and medical intervention
outbreaks. The intensity of dengue transmission and the
simultaneous circulation of several serotypes are also of
importance. An eco-biosocial conceptual framework useful
for research investigation has been proposed. Figure 12 inte-
grates both the integral hypothesis proposed for the develop-
ment of DHF/DSS and the eco-biosocial conceptual
framework (Arunachalam et al., 2014).
Environmental Factors
In general, the epidemiological and ecological factors are
important determinants for the development of a dengue
outbreak. Figure 12 shows the macrodeterminants of dengue
transmission. Vector factors such as abundance and types of
mosquito production sites, density of adult females, age of
females, frequency of feeding, host preference, host availability,
and innate susceptibility to infection are among the microde-
terminants of dengue transmission.
Host Factors
Individual risk factors determine the expression of the disease
in a particular person in a given population. The presence or
absence of these individual risk factors in the matrix of epide-
miological and viral factors not only defines whether or not
the persons with a secondary dengue infection develop a clin-
ical picture of DHF, but also determines the infection outcome.
Individual Risk Factors
Infection with one serotype gives long-lasting immunity to this
serotype and short-lived protection against the others. Homo-
typic immunity is probably based on neutralizing antibodies.
The transient nature of heterotypic immunity is probably due
to cross-reactive antibodies to E protein. Over time, these anti-
bodies decline and an individual is susceptible to infection
with a different DENV serotype.
 Dengue 243

Social context
Vector exposition
Risk perception
Health system: case
Age. Sex management, early detection,
chronic diseases quality of care, availability of
Nutritional status medical supplies, equipment
Ethnicity
Viral factors
Circulating serotypes/
Immunity genotypes
Previous infection Viral load: viraemia titres
ADE Viral tropism
Original antigenic sin Virulence
Host Virus evolution
Transient autoimmunity Virus
Unbalanced T-cell Sequence of serotypes
responses

Genetic factors
Vector control
Genes involved in cell
Policies: Legislation related to
entry, replication, Vector control activities
immune response
Control actions: surveillance,
type/coverage of actions,
costs, human resources,
Vector ecology eficiency/sustainability.
Climate variability Social context Stakeholders
Availability/quality of Population density,
breeding sites urbanization, migration
Feeding opportunities Housing, habits, traditions
Vector capacity Water supply
Vector species Poverty

Figure 12 Eco-biosocial conceptual framework for the development of dengue. Adapted from Kouri, G.P., Guzman, M.G., Bravo, J.R., 1987.
Why dengue haemorrhagic fever in Cuba? 2. An integral analysis. Trans. R. Soc. Trop. Med. Hyg. 81, 821–823 and Arunachalam, N., Tana, S.,
Espino, F., Kittayapong, P., Abeyewickreme, W., Thet Wai, K., 2010. Eco-bio-social determinants of dengue vector breeding: a multicountry study in
urban and periurban Asia. Bull. World Health Organ. 88, 173–184. http://dx.doi.org/10.2471/BLT.09.067892..

A secondary heterotypic infection (e.g., a second infection
caused by a different serotype of dengue virus) is associated
with the development of DHF/DSS, and is considered the main
individual risk factor for severe disease. Maternally transferred
or naturally acquired antibodies against one serotype do not
prevent against infection with another serotype. In fact, previous
exposure to one serotype may exacerbate disease caused by expo-
sure to a second serotype. The ADE phenomenon is mainly medi-
ated by IgG cross-reacting but not cross-neutralizing antibodies
specific to E and prM dengue proteins. Uptake of the virus by
FcgR expressing cells (monocytes/macrophage) increases
through ADE, and severe disease may result from higher levels
of virus load in blood and the fast dissemination of the infection
(Figure 13). The outcome of a secondary DENV infection is
modulated by the individual’s age, infection with a different
(second) serotype of DENV, sequence of infection, and interval
between infections.
Longer intervals between sequential infections, when
heterologous sequential infections occur, generally do not
protect against clinical disease. Guzman et al. (2002b) have
reported a higher disease severity after more than 20 years
of the primary infection in individuals secondarily infected
by DENV-2 and DENV-3. Recently, this observation has
been extended to symptomatic dengue infection as a long

Binding of virus/Ab Enhancement of Higher viral load

Aiding of virus
complexes to FcR virus replication and number of
entry
infected cells
Y
Y

Monocyte
Fc γ R Y Non-neutralizing IgG antibody Virus

Figure 13 Antibody-dependent enhancement.

244 Dengue

DSS Fatal cases

14 14

12 12

10 10

8 8

6 6

4 4

2 2

0 0
<1 2 4 6 8 10 12 14 <1 2 4 6 8 10 12 14
Age Age

Figure 14 Age distribution in DSS and fatal children cases, 1981 DHF/DSS epidemic. Adapted from Bravo et al., 1987. Trans. R. Soc. Trop. Med.
Hyg. 81, 816–820 with permission.

interval between sequential DENV infections has been asso-
ciated with symptomatic as opposed to inapparent infection
(Montoya et al., 2013). In children, primary infection with
any DENVs varies from inapparent to mild disease while in
adults the disease is often overt mostly as dengue. Both age
and DENV serotypes are important determinants of infection
outcome. Primary DENV-2 and DENV-4 infections in
children are usually silent. In adult, primary DENV-2 infec-
tions are also silent (Guzman et al., 2000, 2002a; Halstead,
2012).
DHF presents primarily as a childhood disease in South-
east Asian countries, while in the tropical Americas all age
groups are involved. Epidemic adult DHF/DSS was observed
for the first time during the Cuban DHF epidemic of 1981.
Subsequently, reports of DHF in adults have progressively
increased, initially in the Americas and now in some South-
east Asian countries. In children, DHF can present in infants
born to dengue-immune mothers and in children with
a secondary infection. Figure 14 shows the age distribution
of DSS and fatal children cases during the Cuban 1981
epidemic. No severe or fatal cases were observed in children
aged 1–2 years who developed a primary dengue infection.
This observation supports the role of secondary infections as
major risk factors for DHF/DSS.
Age was demonstrated to be an important variable in the
outcome of secondary DENV-2 infections. During the 1981
DHF Cuban epidemic, DHF/DSS case fatality and hospitalization
rates were highest in young infants and the elderly. Indeed, the
risk that a child would die during a secondary DENV-2 infection
was nearly 15-fold higher than adults aged 15–39 years
(Figure 15). Similar observations have been reported in Vietnam
and Singapore (Whitehorn and Simmons, 2011). Differences in
baseline microvascular permeability between children and adults
could contribute to this phenomenon (Gamble et al., 2000).
A higher frequency of DHF/DSS in females has been re-
ported by some (Anders et al., 2011).

30

25

20 Deaths/havana
Deaths/10 000

Deaths/cuba

15

10

0
3−4 5−9 10−14 15−19 20−24 25−29 30−34 35−39 40−44 45−49 50−54 55−59 60−64 65+
Ages

Figure 15 Age-specific DHF/DSS death rates per 10 000 secondary DENV-2 infections in Havana, and Cuba, 1981. Adapted from Guzman, M.G.,
Kouri, G., Bravo, J., Valdes, L., Vazquez, S., Halstead, S.B., 2002a. Effect of age on outcome of secondary dengue 2 infections. Int. J. Infect. Dis. 6,
118–124 with permission.

Table 5 Chronic diseases as risk factors for DHF/DSS
DHF DHF
epidemic, epidemic
1981a 1997
Fatal cases DSS Fatal cases
Children Adults Children Adults
Bronchial 23% 13% 25% 8.3%
asthma
Sickle cell 6% 17% – 8.3%
anemia
a
Significantly different compared to general population.
Data from Bravo et al., 1987. Trans. R. Soc. Trop. Med. Hyg. 81, 816–820; Valdes
et al., 1999. Pan Am. J. Public Health 6, 16–24; Gonzalez, D., Casdtro, O.E., Kouri,
G., et al., 2005. Classical dengue hemorrhagic fever resulting from two dengue
infections spaced 20 years or more apart. Havana, Dengue 3 epidemic, 2001–2002.
Int. J. Infect. Dis. 9, 280–285.
Some studies suggest that moderate to severe malnutrition
reduces the risk of DHF/DSS while others suggest that under-
and overnutrition DHF patients have either a greater risk of
shock or unusual presentations and complications (Thisyakorn
and Nimmannitya, 1993; Nguyen et al., 2005; Kalayanarooj
and Nimmannitya, 2005).
Asian individuals are highly susceptible to DHF. During
the DHF epidemics of 1981, 1997 and 2001–02 in Cuba,
hospitalization rates for Blacks were consistently lower than
that for Whites; however, dengue infection rates were the
same. In agreement with these epidemiological observations,
Sierra et al. (2006) found a reduced risk of DHF in Blacks
compared to Whites. These differences in susceptibility to
DHF/DSS among racial groups coincide with epidemiolog-
ical observations in African and Black Caribbean
populations.
Bronchial asthma, sickle cell anemia, and diabetes melli-
tus have been associated with a higher risk of DHF/DSS
(Table 5). A chronic medical condition has been observed
in 70% of fatal adult cases in Puerto Rico (Rigau-Perez and
Laufer, 2006). A case–control study in adult patients showed
that diabetes mellitus was associated with increased risk of
DHF. In addition, individuals with diabetes mellitus and
hypertension had a higher risk of developing DHF compared
with individuals with no diabetes and no hypertension
(Pang et al., 2012).
Genetic factors have been associated with protection against
or susceptibility to DHF/DSS. These factors include allelic vari-
ants of genes that encode cellular receptors (DC-SIGN, FcgRIIA,
vitamin D receptor, mannose-binding lectin, G6PD deficiency,
TNFa, IL-10, TGF-b1, DC-SIGN (CD209) promoter),
molecules involved in antigen recognition (HLA I and II
molecules), and cytokines (TNFa) among others (Lan and
Hirayama, 2011; Perez et al., 2010; Garcia et al., 2010). Also
alleles of MHC class I polypeptide-related sequence A
(MICA) and MHC class I polypeptide-related sequence B
(MICB) associated with symptomatic versus asymptomatic
infection as well as MICB and PLCE1 alleles associated with
susceptibility to DSS have been identified (Garcia et al.,
2011; Khor et al., 2011). How many genes contribute to
dengue susceptibility, how they interact to cause severe
illness, and how the pathogenesis mechanisms might be
genetically predicted remain unknown.
Dengue 245
Viral Factors
The pathogenic potential of distinct dengue virus serotypes and
genotypes have been investigated. In secondary infections,
DHF epidemic
a gradient of severity has been described for the four serotypes
2001–02
DSS
(from the most to least severe): DENV-2, DENV-3, DENV-1,
Adults and DENV-4. The sequence of the infecting viruses is also asso-
ciated with severity. Severity during a secondary infection has
22.2% been related to antecedent primary DENV-1 infection.
Secondary DENV-2 infection has been associated with a greater
11.1% pleural effusion index and more severe hemorrhagic manifesta-
tions. A higher risk of DHF/DSS has been linked to sequential
DENV-1 then DENV-3, as opposed to DENV-2 then DENV-3
infections. In addition, Asian genotypes of both DENV-2 and
DENV-3 seem to be more virulent than those found in the
Americas, having been associated with DHF outbreaks.
Full-length sequencing of the DENV-2 Asian and American
genotypes expose several nucleotide differences, specifically of
the E gene and at the 50 and 30 UTRs. Amino acid differences in
the prM and E proteins have also been determined and corre-
lated with virulence and particularly with the higher replicative
ability of some genotypes in human dendritic cells (DC) and
macrophages, and in the efficiency to infect and disseminate
in Ae. aegypti. These data support the hypothesis that differ-
ences among genotypes exist, with possible implications for
pathogenesis and transmission.
Furthermore, mutant variants of dengue viruses can escape
antibody neutralization, and also seem able to contribute to
the complexity of the DHF phenomenon.
The association of genetic variation and disease severity in an
individual is not well documented. Recent studies have been
directed to determining the viral population structure in both
mosquitoes and patients. Although the sequences of the major
variants are the same, the extent of sequence variation in
mosquitoes is lower than in patients. In addition, intra-host vari-
ation in patients is apparently lower than expected (Lourenco
and Recker, 2010). Further studies on intra-host virus popula-
tion and its implication on disease severity are needed.
Kinetics of Dengue Infection
Dengue disease begins 5–7 days following the bite from an
infected mosquito. After subcutaneous injection of the virus
into the skin, released viral particles infect neighboring cells,
mainly resident skin immature dendritic cells (DCs) and Lang-
erhans cells, which act as sentinels, sensing the antigenic micro-
environment and capturing antigens. Infected DCs in the dermis
migrate to the paracortex of regional draining lymph nodes,
where the virus localizes and replicates primarily likewise in cells
such as monocytes and macrophages. Later, replication also
occurs in other target organs, like the liver, spleen, or brain.
The virus is then released from those tissues and spreads through
the blood to infect white blood cells and other lymphatic tissues.
DENV genome and protein antigens are found mainly in phago-
cytic cells of lymph node, spleen, lung, in perivascular cells of
brain, in hepatocytes, and in endothelial cells in spleen. Viremia
in peripheral blood can be detectable 24–48 h before the onset
of clinical symptoms, peaks during the early days (first 2–3 days)
of acute illness and then falls suddenly. Symptoms of illness can
last 2–7 days. Higher levels of viremia have been detected in
246 Dengue

Dengue virus skin Spreading of Immune response Disease outcome

inoculaon infecon to ssues
IgM / IgG Viral
antibodies clearance and
recovery

Complement Bening evoluon

Complicated evoluon

CD4+ T cells
CD8+ T cells Immune-
1st 2nd pathogenesis
viremia viremia Y

Y
Effects on vascular endothelium
Cytokines and
chemokines
releasing

Plasma leakage/hemorrage

Incubation period Acute phase Recovery

Figure 16 Kinetic of dengue infection.

patients with severe disease compared to uncomplicated Table 6 Pathological findings observed in severe dengue illness
patients. Immune response seems to be required for the resolu-
Tissue organ Pathology
tion of a DENV infection; however, as severity is associated with
secondary infections and the critical phase of illness appears Skin Swelling endothelial cells, perivascular edema,
when viremia has declined, immune mechanisms have been infiltration of mononuclear cells
involved in the pathogenesis of the severe clinical picture Lymphoid organs Proliferation of reticulo-endothelial cells in the
(Figure 16). spleen and lymph nodes
Table 6 shows some of the pathological findings observed Liver Degeneration with focal necrosis of hepatic
in distinct tissues and organs observed in fatal DHF cases. cells and hyaline necrosis in Kupffer cells
In addition to the particular serotype, genotype, and dengue Kidney A mild immunocomplex-type
strain, specific viral proteins play an important role in dengue glomerulonephritis
Myocardium Left ventricle thick and hard, and the chamber
pathogenesis (Ye et al., 2013; Table 7).
is not filled to the full volume
Lungs Thickening of interstitial septa
Immune Response against DENV Infection Brain and spinal cord Perivascular edema
Bone marrow Depression of hematopoietic cells is observed
Innate Immunity during febrile phase
Vessel walls Deposition of serum complement,
The mechanisms of innate immunity provide early defense
immunoglobulin, and fibrinogen
against DENV infection. The principal components of innate Mucosa of Hemorrhage occurs in the subcutaneous
immunity are cells such as DCs, phagocytic cells (monocytes gastrointestinal tract tissues
and macrophages) and natural killer (NK) cells, and soluble Pleural and abdominal Serous effusion with high protein content
mediators as blood proteins, comprising members of the cavities (mostly albumin)
complement system and other pro-inflammatory mediators Adrenal glands Depletion of lipid in the cortex
and cytokines that regulate and coordinate most of the func-
tions of the cells of innate immunity.
DCs are target cells in the skin following a bite by a dengue-
infected mosquito. They may also facilitate the spread of the IRF transcription factor. This factor stimulates type I interferon
virus within the host. These cells express receptors which ensure (IFN I) gene expression, and NF-kB transcription factor that
that the innate immune system can respond to the presence of stimulate the expression of genes encoding molecules required
genome or proteins of the dengue virus. In a dengue infection, for inflammatory responses including some mediators like
the TLR3 in the endosomal membrane and RIG-I and MDA5 inflammatory cytokines and chemokines. IFN I functions to
in the cytoplasm are sensors of double-stranded viral RNA that inhibit viral replication in both infected and uninfected cells
activate signal transduction events which, in turn, activate the by inducing an ‘antiviral state’ (Costa et al., 2013).

Table 7 Role of viral proteins in the pathogenesis of dengue
infection
Viral protein Role in the pathogenesis
E Target of ADE-mediating antibodies
Mimicry with plasminogen
prM Target of ADE-mediating antibodies
Apoptosis induction in infected cells
Generates cross-reacting antibodies with endothelial cells
NS1 Massive complement activation and vascular leakage;
apoptosis of endothelial cells
Share common antibodies epitopes with human blood
clotting, integrin/adhesin proteins expressed on
platelets and endothelial cells
Binds to C4b and modulates complement activation
Apoptosis induction in hepatocytes and endothelial cells
NS2A Blockade IFN-mediated signal transduction
Autophagia induction
NS2B Apoptosis induction in infected cells
NS3 Apoptosis induction in infected cells
NS4A Blockade IFN-mediated signal transduction
NS4B Potent inhibitor of IFN signaling by decreasing STAT1
phosphorylation
NS5 Induces transcription and translation of IL-8
Blockade IFN-mediated signal transduction
Local inflammatory response in the skin stimulates the
recruitment of leukocytes from the vasculature, including NK
cells. Due to its ability to rapidly limit viral dissemination,
NK cells kill infected cells and seem to be a key early mecha-
nism of immunity against DENV infection, before an adaptive
immune response has developed (Beltrán and López-Vergès,
2014). Activated DCs and monocytes produce IFNa, IL-12,
IL-15, and IL-18, the major cytokines that stimulate NK func-
tion enhancing their cytotoxic activity. DCs are also considered
the most important antigen-presenting cells (APCs) after they
migrate to the draining lymph nodes where they present viral
antigens to naive T cells.
Adaptive Immunity
DENV-specific B and T cells are generated about 6 days
postinfection, and they help to completely control the
infection. DCs in the draining lymph nodes present viral
antigens to naive T cells, which results in the proliferation
and in the differentiation of specific T cells into effector cells,
triggering the adaptive cellular immune response. T cells
recognize a complex of a particular viral epitope presented by
a specific MHC molecule. MHC molecules are extremely
polymorphic, with several 1000 variants known in humans,
implying a wide diversity of peptides that could be presented
to T cells. More than 350 antigenic regions on DENV proteins
identified in humans have been described in the literature,
most of them restricted by HLA MHC class I alleles. CD4þ
T cells recognize epitopes from C, E, and NS3 proteins,
whereas the NS3, NS4B, and NS5 proteins are the dominant
targets for CD8þ T cells. Cytotoxic T lymphocytes (CTLs) are
predominantly CD8þ T cells and recognize cytosolic, usually
endogenously synthesized, viral peptides presented by class I
Dengue 247
MHC molecules, whereas helper CD4þ T lymphocytes
recognize exogenously processed viral peptides presented by
class II MHC molecules (Weiskopf and Sette, 2014).
Once CD4þ T cells are activated, they proliferate and differen-
tiate to a Th1 pattern, releasing important cytokines for the
control of the infection such as IFNg, IL-2, and TNFa. Higher
frequencies of DENV-specific IFNg-producing T cells are
observed in children who subsequently develop a subclinical
infection, compared with those who develop a symptomatic
secondary DENV infection. Full differentiation of CD8þ CTLs
often requires cytokines produced by CD4þ helper cells. They
undergo massive proliferation during infection recognizing
a few viral peptides. Effectors’ CTLs kill infected cells by necrosis
or apoptosis. They can activate nucleases within infected cells
that degrade viral genomes and also produce cytokines, having
a pivotal role in the control of the viral infection.
Virus or virus-infected cells stimulate B lymphocytes to
produce antibodies. B cells from lymph nodes and spleen are
activated and differentiated to plasmablasts and plasma cells
to secrete antibodies. The plasmablast numbers peak consis-
tently at day 6 or 7 and drop to baseline level within 2–3 weeks
after the onset of disease. The first isotype of antibodies
detected during a dengue infection is IgM, 5 days after the onset
of clinical signs of illness. A role for IgM antibodies in the
control of a DENV infection through complement fixation
and/or favoring virus uptake by phagocytic cells has been sug-
gested. Thus, the viremia reduction most likely is due to virion
clearance by IgM antibodies.
The neutralization capacity of antibodies in the serum is
broadly cross-reactive against the four DENV serotypes, both
during the acute phase of illness and at 3 months post-onset
of symptoms.
Antibodies protect the host through the neutralization of
viruses, lysing infected cells, and opsonizating viral particles,
which facilitate phagocytosis by monocytes and macrophages
and antigen clearance. Most DENV-specific antibodies
generated in humans are serotype-cross-reactive, weakly
neutralizing, and directed against the immature prM, and E
and NS1 proteins.
Neutralizing antibodies seems to be the main correlate of
protection. Antibody-mediated neutralization of viral infec-
tivity may occur at two levels: at the level of cell binding,
through the inhibition of the interaction between the virus
with cell-surface receptors and at the level of viral fusion,
through binding of antibodies to the E protein fusion loop,
or blocking the conformational changes of the E protein that
are required for membrane fusion. However, after a primary
infection, an individual is vulnerable to new infections with
other serotypes. Cross-immune protection is observed after
a sequential infection, with a different serotype occurring
shortly after the primary infection (some weeks to few months)
(Friberg et al., 2012).
Sequential Infection: Secondary Immune Response
Upon a secondary, heterologous, DENV infection, preexisting B
memory cells are activated to rapidly produce antibodies, but
these are predominantly directed against the viral serotype of
the primary infection, a phenomenon known as ‘original anti-
genic sin’ (Figure 17). Massive expansion of memory B cells
248 Dengue
Figure 17 Original antigenic sin phenomenon.
IgG
Viremia
NS1
IgM
IgA IgE
Primary infection
Figure 18 Kinetic of the antibody response against dengue virus sequential infection.
occurs during days 4–6 of symptoms and the magnitude of the
plasmablast response is higher during a secondary infection as
well as in severe dengue cases (Smith et al., 2014).
The titer of DENV-specific antibodies in serum rapidly
increases within days of the onset of fever and will bind to the
heterologous serotype; they have less neutralizing capacity
against the new infecting serotype than to the serotype of the
primary infection. Although there is detectable IgM production,
IgG1 and IgG3 serum antibodies are the predominant
immunoglobulins throughout the course of illness.
Neutralization of virion infectivity by antibodies binding
to the extracellular virion and blocking its interaction with
the host target cells represents the major antibody function
of protection during a secondary infection. Serotype-specific
neutralizing antibodies target diverse regions of the E protein,
but mainly recognize a region in the E protein domain I/II
IgG
Viremia
NS1
IgM
IgA IgE
Secondary infection
containing the fusion loop, including epitopes present only
on the intact fully assembled viral particle.
Additionally, antibodies recognize viral antigens on the surface
of infected cells, killing them through complement-mediated
cytotoxicity (CMC) or by using effector cells as NK cells and
macrophages, via antibody-dependent cell cytotoxicity (ADCC).
Both mechanisms could be of importance in the elimination of
the infected cells in a secondary infection. The antibodies that
mediate CMC and ADCC recognize viral proteins expressed on
the plasma membrane of infected cells, presumably E, prM, and
NS1 proteins could be their main targets. Moreover, IgM and
IgG bind viral-soluble antigens forming immune complexes
and act as opsonins favoring antigen removal by the phagocytic
cells. The kinetic of the different classes of antiviral-specific anti-
bodies after a primary or a secondary dengue infection are shown
in Figure 18.
 Dengue 249

Homologous Heterologous Y Homologous Ab

secondary infection secondary infection Y Heterologous Ab
FcγR
Y Y Y Virus nucleocapsid

Y
Y Y Y

Y
Y Y Virus
Y

Y
Y Y Viral proteins
Y

Y
Cell proteolitic enzyme
Y

Y Y Y

Y Y

Y
Y

Monocyte/macrophage

Neutralization
Y Antibody-dependent
Y Y Y Y enhancement

Y
Y Y
Y

Y
Y

Protection Increase of viral load and spread of infection

Figure 19 Role of antibody during homologous and heterologous secondary infection.

It has been proposed that cross-reactive T cells raised against Table 8 Immune effector mechanisms against dengue infection
the original infecting serotype (primary infection) also domi-
Classes or
nate during a secondary heterologous infection, being impli-
Effector subpopulation Mechanism Target protein
cated in the ‘original antigenic sin’ phenomenon (Figure 19).
The expansion of preexisting lower avidity cross-reactive Antibodies IgM, IgG Virus neutralization E, prM
memory T cells dominates the responses over the naive IgG Antibody-dependent ?? E, prM, NS1
T cells that show a higher avidity for the new DENV serotype. cell cytotoxicity
It is further hypothesized that peptide variants derived from IgM, IgG Complement-mediated ?? E, prM, NS1
the secondary infecting serotype can induce a response that is cytotoxicity
qualitatively different from the response induced by the orig- T cells CD4þ Cytotoxicity NS3, E, prM, C
CD8þ Cytotoxicity NS3, C, NS1,
inal antigen (primary infecting serotype), such as inducing
NS4b and NS5
a different pattern of cytokine production. However, high titers
of cross-reacting neutralizing antibodies could protect those
individuals who are exposed to a heterotypic secondary infec-
tion. Very early blockade of the inoculated virus and control interaction of the virus–antibody complex with Fc receptors.
of the viral infection may serve to prevent development of This phenomenon, called ADE, was originally proposed by Hal-
the disease or modulate or progression to severe outcomes. stead as an important pathogenic mechanism in DHF/
The most important effector mechanisms and their viral targets DSS (Figure 19).
are shown in Table 8. ADE increases the efficiency of the virus infection and favors
the early spread of the infection, thus increasing the number of
DENV-infected cells. Cell infection via ADE appears to remodel
Immunopathogenesis of Dengue
and to suppress the innate immune response with increasing
Severe disease has been associated with a heterologous secondary virus particle production in infected cells. This phenomenon
infection as the most important human risk factor. Preexisting called intrinsic ADE can occur along both TLR-dependent and
antibodies from a previous DENV infection play an important -independent pathways. It seems to suppress IFN I-mediated
role in this predisposition. When non-neutralizing properties antiviral responses. The suppression of the innate immune
against the new serotype predominate in these antibodies, system during ADE likely facilitates longer survival of infected
dengue virus infection of macrophages may be enhanced. The cells with the possible increase of virus output. In addition,
enhancement of the infection is facilitated through an efficient other molecules produced by infected cells may induce
250 Dengue
a pathological host response. For example, C-type lectin
domain family 5 member A (CLEC5A), which directly
interacts with the dengue virion, results in the stimulation of
the release of pro-inflammatory cytokines (Costa et al., 2013).
Higher viremia titers in severe illness compared to that in
mild disease have been observed. In general, immune
responses are essential for the resolution of a DENV infection;
however, as severe dengue is associated with secondary infec-
tion and the complications emerge at the time when viremia is
declining, severity has been explained as a consequence of
immunopathology. The activation of different immune func-
tions and the duration and magnitude of the immune
response depend on how the virus interacts with the host
target cells and on how much the virus spreads. The more viral
antigens are present in different parts of the body, depending
on the virus spreading in the early phases of the infection, the
more mechanisms of the host immune response will be
involved in stopping and clearing the infection. These mech-
anisms depend on the kind of infection (primary, secondary,
tertiary, or quaternary), the infecting serotype and the
sequence of infection.
During a secondary infection, the expansion of preexisting
lower avidity cross-reactive memory T cells dominates the
responses over the naive T cells, and peptide variants derived
from the second serotype seems to induce a response qualita-
tively different from the response induced by the original
antigen, such as inducing a different pattern of cytokine
production. These altered T cell responses may contribute to
the ‘cytokine storm’ observed during a heterologous secondary
infection, and thus to the immunopathogenesis of DHF/DSS.
Different HLA alleles are associated with differential magnitude
of anti-DENV responses; some of them are associated with an
increased risk of severe illness and a weaker CD8þ response
(St. John et al., 2013).
The absence of structural damage in the vasculature found
in autopsy studies suggests that circulating factors are primarily
responsible for the existence of microvascular permeability
during a DENV infection. Therefore, soluble mediators
produced by leukocytes in response to the infection represent
crucial mediators to enhance plasma extravasation seen in
severe cases. Consequently, cytokines and other inflammatory
mediators act on the endothelium, potentially altering normal
functions of endothelial cells. Although elevated levels of
pro-inflammatory cytokines and chemokines occur in patients
with DF, most of them are found at higher levels in patients
experiencing severe disease. Key inflammatory cytokines
involved in the massive increase of vascular permeability
include TNFa, IL-6, IFNg, IL-8, and ligand (CCL)-2, CCL-3,
CXCL-8, CXCL-10, which activate macrophages or nearby
vascular endothelium to increase the expression of adhesion
molecules that promote leukocyte and plasma extravasations.
TNFa and IL-10 could also be involved in the hepatic dysfunc-
tion, hypotension, thrombocytopenia, and hemorrhagic mani-
festations. Besides the effects of cytokines and chemokines,
leading to increased vascular permeability, it was also demon-
strated that overproduction of soluble matrix metalloprotei-
nase (MMP) 9 and 2 and HMGB1 produced by infected cells
increases endothelial permeability (Sun and Kochel, 2013).
Alternatively, elevated LPS levels detected during a DENV infec-
tion in correlation with disease severity may suggest
a disruption of the intestinal barrier inducing microbial trans-
location. This has been associated with extensive immune acti-
vation that could play a role in dengue pathogenesis. On the
other hand, apoptosis is observed with impairment of CD8þ
T-cell cytokine production, decreased circulating of CD4þ
and CD8þ T cell counts, impairment of T cell proliferation,
and increased T cell apoptosis.
NS1 protein–antibody complexes and several cytokines also
activate the release of complement activation products such as
C3a and C5a that have a direct effect on the vascular perme-
ability. Other soluble mediators such as PAF, prostaglandins,
and stress oxidative metabolites may also be involved in severe
disease. Molecular mimicry has also been associated with
platelet and endothelial injury. Antibodies elicited by NS1,
prM, or E proteins recognized in a cross-reactive fashion host
‘self’ epitopes contained in blood-clotting elements, plasmin-
ogen, or proteins expressed on platelets and endothelial cells.
These antibodies could mediate the decrease of the circulating
levels of these coagulation factors, as well as cytotoxicity or
apoptosis of cells (Wan et al., 2013). Table 9 shows the main
mechanisms associated with vascular leakage, liver damage,
leukopenia, and hemorrhagic manifestations during a severe
dengue infection.
In summary, the presence of enhancing antibodies
combined with a misbalanced T cell response and deficient
control of the infection contribute to the immunopathogenesis
of dengue and development of severe disease in an individual
with a susceptible genetic background.
Dengue Diagnosis
Dengue diagnosis is important for clinical care, surveillance
support, pathogenesis studies, vaccine and drug development,
and clinical trials. Dengue laboratory diagnosis can be per-
formed through virus isolation, genome and antigen detection,
and serology, with the last the most widely applied in routine
diagnosis (Figure 20). Assay choice depends on the timing of
sample collection and the purpose of testing (Guzman et al.,
2014; Guzman et al., 2010a).
Virus Isolation
Viremia is present 2–3 days before, and then for 3–4 days after
the onset of fever. The decrease in viremia coincides with the
appearance of specific IgM antibodies and the remission of
fever. Dengue viruses can be isolated in serum collected in
the acute phase of illness and preferably before fever disap-
pears. Viruses can also be isolated from tissues (liver, spleen,
lymph nodes, lung, and thymus) obtained at necropsy.
Mosquito cell lines (Ae. albopictus C6/36 and Ae. pseudoscu-
tellaris AP61) are the cell culture systems of choice for dengue
virus isolation. Mammalian cell cultures such as Vero cells,
LLCMK2 cells, and others have been employed with less
efficiency.
Direct mosquito inoculation improves the sensitivity of
virus detection; however, insectaria facilities and technical skill
are required. The intracerebral inoculation of suckling mice is
the oldest and least sensitive method for isolating virus, and
is only used when no other method is available. Inoculated
mice develop encephalitis. Figure 21 shows the biological
systems for dengue virus isolation.
 Dengue 251

Table 9 Associated mechanisms to the main signs of severe disease

Phenomenon Associated mechanisms

Plasma leakage Endothelial cell apoptosis via production of nitric oxide or inflammatory activation
induced by antibodies anti-NS1
Immune recognition of infected endothelial cells
Dysfunction of endothelial cells caused by high levels of TNFa and IL-8
Complement activation and systemic generation of anaphylatoxins and SC5b-9 that
increase vascular permeability
Direct infection of endothelial cells modulates differently the cell surface expression of
molecules critically involved in the interactions between endothelial and inflammatory
cells
Changes in surface and soluble VEGFR2 expression induced by DENV, which regulates
vascular permeability via binding VEGF
Liver damage Hepatocyte infection
Apoptosis of hepatocytes induced by DENV infection
Immune cytotoxicity
Leukopenia Bone marrow suppression
Apoptosis of infected cells
Apoptosis of leukocytes by immune mechanisms
Hemorrhagic manifestations Thrombocytopenia Infection of platelets
Consumption of platelets
Cross-reactive anti-NS1 and anti-prM antibodies
Platelet dysfunction
High levels of IL-10 and soluble TNF receptor-II
Deficiency in coagulation Cross-reactive anti-E antibodies that recognize
plasminogen
Cross-reactive anti-NS1 antibodies recognize fibrinogen
Fibrinolysis activation induced by DENV infection, which
degrades fibrinogen directly and causes secondary
activation of pro-coagulant homeostatic mechanisms
High levels of IL-6 associated with elevated levels of tPA
and a deficiency in coagulation
Marked hepatic dysfunction

Opportunity

Direct methods Indirect methods

Virus isolaon Genome Angen Serology Serology

detecon detecon IgM IgG

Confidence

Figure 20 Laboratory diagnostic methods employed for diagnosis of a dengue infection. Guzman et al., 2011. Nat. Rev.
252 Dengue
Cytopathic effect in mosquito cells
inoculation
Figure 21 Main systems for viral isolation.
The immunofluorescence assay with serotype-specific
monoclonal anti-dengue antibodies on squashed mosquito
heads, infected cells, or brain tissues from inoculated mice is
the choice method for virus identification; however, PCR,
ELISA, and immunohistochemical techniques can also be
employed.
Antigen Detection
Secreted NS1 glycoprotein is a diagnostic marker during the
early phase of infection. Its detection in sera may be a valuable
surrogate marker of viremia and may also serve as a prognostic
marker of disease progression. NS1 detection is influenced by
the day of illness, kind of infection (primary or secondary
infection), and infecting virus. At present, several commercial
kits for NS1 detection, both as ELISA and rapid test formats,
are available which have different sensitivity and specificity
profiles. NS1 glycoprotein can also be detected in urine
collected from febrile patients, tissues collected from fatal
cases and in infected mosquitoes (Guzman et al., 2010b;
Duong et al., 2011; Hunsperger et al., 2014). Sensitivity of
diagnosis increases when both markers, NS1 and IgM, are
studied during the first 4 days of acute illness (Guzman
et al., 2010b; Figure 22). Immunohistochemical techniques
have been shown to be useful for dengue antigen detection
in formalin-fixed paraffin-embedded tissue samples from
fatal cases.
Figure 22 NS1 and IgM dengue detection by day of illness.
Increasing in diagnostic sensitivity. Data collected from Guzman et al.,
2010. PLoS NTD.
Mosquito inoculation Sick newborn mice
Genome Detection
Dengue virus RNA can be detected by PCR directly from clinical
samples such as serum, plasma, and autopsy-fresh and
paraffin-embedded tissues, and from the supernatant of
dengue virus–infected mosquito cell cultures and infected
adult mosquito and mosquito larvae. DNA amplification is
preceded by a reverse transcription (RT) reaction, producing
cDNA from the target RNA.
Because of its high sensitivity, PCR allows the detection of
concurrent infections by multiple serotypes, detection of
dengue in stored samples over long periods, and detection in
mosquito pools (groups of captured mosquitoes tested
together), favoring its application in entomological surveil-
lance (Figure 23). PCR in conjunction with nucleotide
sequencing or restriction enzyme analysis has allowed the
study of genetic strain variability in order to identify the origin
of epidemics and reveal markers of virulence.
The introduction of real-time PCR is supplanting conven-
tional PCR, allowing the rapid detection (in less than 2 h)
and the quantification of minimum copies of the RNA.
Currently, many RT-PCR and real-time RT-PCR protocols are
available. A recent CDC RT-PCR assay has been produced
that enables dengue diagnosis in the first 7 days of illness
(Santiago et al., 2013). Protocols for RT-PCR and real-time
RT-PCR for multiplex detection of several arboviruses and
hemorrhagic fever viruses are also available.
D2 D3 D4 D1 MW
Figure 23 Standard RT-PCR and nested PCR for dengue RNA
detection as described. Lanciotti et al., 1992. J. Clin. Microbiol. 30,
545–551; Photo kindly supplied by D. Rosario, IPK, Habana.

Serological Diagnosis
During primary dengue infection, anti-dengue IgM is detected
5–6 days after onset of fever, and then persists for 30–
60 days. In some individuals, IgM may persist for more than
170 days. IgG levels slowly rise, are detectable by the 9th to
10th day after symptom onset, and then persist for life.
Unlike a primary infection, a secondary infection with
a heterologous serotype is characterized by a rapid (1 or
2 days after fever onset), high, and cross-reactive IgG response.
In some cases, an IgM response is not detectable.
Specific IgA and IgE antibodies also develop during both
primary and secondary infections. The IgA is broadly
cross-reactive to the four serotypes, and the level of IgE has
been related to disease severity. Anti-dengue IgA and IgE may
complement dengue diagnosis as possible markers of recent
infection.
The detection of anti-dengue IgM is considered the best indi-
cator of an active or recent infection. MAC-ELISA (IgM
antibody–capture ELISA) diagnosis represents one of the most
important advances in dengue diagnosis, being an invaluable
tool for routine diagnosis and surveillance. Serum, blood on
filter paper, and saliva are useful for IgM detection if samples
are taken during the appropriate time period, around the 5th
day after the onset of fever. Different commercial kits for
anti-dengue IgM and IgG detection are commercially available,
with variable sensitivity and specificity (Hunsperger et al., 2009).
The presence of anti-dengue IgG in serum is a criterion for the
diagnosis of a past infection. However the presence of high titers
of IgG in an acute serum sample, or seroconversion or fourfold
increase in paired sera from a suspected dengue case are criteria
for a recent or confirmed dengue infection, respectively. ELISA
has gained acceptance as a faster and more convenient alternative
to determine IgG antibodies, although hemagglutination inhibi-
tion (HI) assay is still considered the gold standard technique.
Due to the presence of cross-reactive antigens shared by flavi-
viruses, specific diagnosis is not possible in most occasions.
When a serological specific diagnosis is required, neutralization
assay and particularly plaque reduction neutralization test
(PRNT) is employed. Neutralizing antibodies inhibit dengue
virus infection and provide greater specificity; however, neutral-
ization assay is not recommended for routine diagnosis, as it is
labor intensive and time consuming. In addition, reproducibility
among laboratories is low, as several factors such as cell line,
virus strain, and expression of receptors and attachment factors
that complement virus propagation cell line can contribute to
this heterogeneity. Nonetheless, this assay is recommended
when a different flavivirus is suspected (Guzman et al., 2014).
Table 10 shows the laboratory criteria of dengue infection.
The identification of prognosis markers for DENV disease
severity is currently a research priority. Besides clinical
warning signs recommended by the WHO (WHO/TDR,
2009), other studies are conducted to identify some
biomarkers for disease severity. Higher levels of viremia,
greater consumption of von Willebrand factor and its cleaving
enzyme ADAMTS-13, elevated levels of cell-free circulating
DNA, and the increased expression of mitochondrial ribo-
somal proteins in DSS patients have been associated with
severe disease (Ha et al., 2011; Djamiatun et al., 2012). A
dengue pathogenesis assay may become a reality in the near
future (Loke et al., 2010).
Dengue 253
Table 10 Laboratory criteria of dengue infection
Criteria
Confirmed dengue infection Isolation of dengue virus from serum or
autopsy samples
IgG or IgM seroconversion or
demonstration of a fourfold or greater
change in reciprocal IgG titers in
paired serum samples
Demonstration of dengue antigen in
serum or autopsy tissue
Demonstration of positive PCR/real-time
PCR in serum or autopsy tissue
Highly suggestive or probable Positive IgM antibody test on a single
dengue infection late acute- or convalescent-phase
serum to dengue antigen
Reciprocal HI antibody titer 1280 or an
equivalent IgG ELISA titer
Control and Prevention
Vector Control
Vector surveillance provides important evidence for the presence
and density of vector populations including vector resistance to
insecticides. It also targets the evaluation and analysis of the
impact of control programs (Kay, 1999). Sampling surveys
include larval, adult, standard and sticky ovitrap surveys.
Indoor fogging (intradomiciliary) or out fogging in the
streets by airplane, trucks, or other types of equipment
(thermo-nebulization or ultra-low volume) using different
insecticides especially pyrethroids, are the most important
way up to now for the control of Ae. aegypti (Esu et al.,
2010). However, the use of airplanes or other methods of
out-fogging in the urban ecosystem is not efficient for species
that live inside houses and its use must be analyzed according
to the conditions and the possible environmental damage. The
targets are the infected mosquito females that maintain the
virus in circulation. Biological control methods, such as Bacillus
thuringiensis var. israelensis, are used to reduce vector larvae
(Boyce et al., 2013). In recent years, the use of biological agents
such as Wolbachia (family Rickettsiaceae), a bacterium that
affects the metabolism and capacity of transmission of DENV
in Ae. aegypti mosquitoes has caught the attention of many
researchers. This bacterium was found in naturally infected
Ae. albopictus mosquitoes but the possibility of obtaining
strains of Wolbachia-infected Ae. aegypti and its propagation in
nature would be an alternative method (McMeniman et al.,
2009). On the other hand, the use of transgenic mosquitoes
to compete with normal mosquitoes in nature is a new method
and could be a successful strategy for the control of Ae. aegypti
populations in some settings. Some experiments have showed
good results in a short time (Alphey, 2008).
The use of larvicides such as temephos, an organofosforade,
is changed by insect growth regulators as piryproxifen with very
low toxicity (WHO, 2003).
However, the main problem of Ae. aegypti control is its
ecology, linked to humans. If people refuse the use of any
control method, they will fail and if the environmental condi-
tions and water distribution are inadequate, the mosquito can
254 Dengue
find breeding sites in houses and other dwellings because of the
existence of containers of water.
In summary, Ae. aegypti control has different components,
where the use of insecticides must be accomplished with effi-
cient public service of garbage, residual water, distribution of
water, and citizen responsibility.
Education and Community Participation
Sanitary public health education leading to low mosquito pop-
ulations through cleanup campaigns with significant public
involvement and action is a necessary step. The public deserves
to know the risks and how to reduce them.
The community must be involved in the prevention, surveil-
lance, and control of the vector to ensure program sustain-
ability. Communities with effective vector control integration
are cleaner, and do not have the large mosquito populations
that communities with reproduction of the mosquito in
containers do.
In each country, the maximum authority must organize
a control program with fully integrated activities into the estab-
lished administrative structures, mass social organizations,
churches, schools, business, and factories in order to make
a significant contribution to vector control (Kroeger et al.,
2006). The WHO and the Pan-American Health Organization
(PAHO) have developed the Integrated Management Strategy
for Prevention and Control of Dengue to face the challenge
of the control of dengue (WHO/TDR, 2009).
Vaccine Development
Dengue vaccines have been under study since the 1940s;
however, in recent years their development has accelerated
dramatically. Constructing a dengue vaccine has been a chal-
lenge. A potential vaccine must provide a balance between the
immunogenicity it evokes and attenuation of pathogenicity of
the virus. The vaccine should be safe and also provide
ChimeriVaxTM
Chimeric Dengue -Yellow fever 17D genome cDNA
5’ C prM E C
DENV
Chimeric recombinant DENV attenuated
NIH: Den4/Den
5’ CC prM E NS
DENV 1,2 or 3
CDC: Den2/Den
5’ C prM E NS
DENV 1,2 or 3
Figure 24 Chimeric dengue virus vaccine candidates.
complete long-lasting protection against the four serotypes
in order to avoid any enhancement of dengue illness after
subsequent infection. These requirements, in addition to the
lack of an animal model and our elementary knowledge of
disease pathogenesis, have negatively influenced dengue
vaccine development.
Conventional live-attenuated and inactivated vaccines,
subunit vaccines based on recombinant strategy, chimeric,
and DNA vaccines are among the main dengue vaccine strate-
gies (Thomas, 2014). Figure 24 shows the chimeric dengue
vaccine candidates.
DENV themselves have been employed as backbones for
chimeric vaccines. In addition to chimeric vaccine candidates,
dengue genes of structural and nonstructural proteins, particu-
larly the E gene including its domain III, have been expressed in
systems such as E. coli bacteria, baculovirus, yeast, and
Drosophila flies. Candidates have been evaluated both in mice
and monkeys, showing variable grades of immunogenicity
and protection.
Several candidates are or have been in stages of human
testing. The Sanofi Pasteur tetravalent live–attenuated
chimeric dengue/yellow fever vaccine, which is based on
a yellow fever vaccine 17D backbone expressing the prM
and E genes of each of the four DENV serotypes, has been eval-
uated in Phase III efficacy trials in humans. The CYD 23 trial
showed that vaccinated Thai children developed neutralizing
antibodies to the four viruses after 2 or 3 doses. However the
protective efficacy (30%), which differed by serotype was
lowest for DENV-2 (Sabchareon et al., 2012). In 2015, results
of two Phase III studies developed in children from Latin
American and Southeast Asian countries were published
with a vaccine efficacy of 56.5% (Asian study) and 60.8%
(American study). Higher protective efficacy figures were
observed against dengue hospitalization, disease severity,
and in dengue seropositive individuals. DENV-2 showed the
lowest serotype-specific efficacy (35–42.3%), while DENV-3
and DENV-4 showed figures around 74–78%. These studies
NS 3’
YFV genome
3’
DENV 4 genome
3’
DENV 2 genome

Table 11 Current dengue vaccine candidates in clinical stages
Strategy Candidates
Live-attenuated chimeric DENV-1 to DENV-4, prM and E genes inserted into the
vaccine prM and E regions of the yellow fever 17D vaccine
DENV-1 to DENV-4, prM and E genes inserted into the
prM and E regions of attenuated DENV-2
(16681, PDK 53)
DENV-1 to DENV-3 prM and E genes inserted into the
prM and E regions of DENV-4 backbone (attenuated by
deleting 30 nucleotides in the 30 UTR)
Recombinant subunit vaccines E/NS1 protein subunit expressed in Drosophila
Inactivated vaccine Whole purified inactivated virus
DNA vaccine prM and E protein genes
represent the first Phase III dengue vaccine studies (Villar
et al., 2015; Capeding et al., 2014) contributing new informa-
tion on this important topic.
WHO, through the Initiative for Vaccine Research, and the
Dengue Vaccine Initiative in conjunction with the TDR/WHO
made joint efforts to accelerate the development and introduction
of a dengue vaccine (Hombach et al., 2005). Table 11 shows
some of the vaccine candidates and their current clinical stage.
Conclusion
Today, dengue ranks as the most important mosquito-borne
viral disease in the world. To address this situation, WHO
recently published a global strategy for dengue prevention
and control in the period 2012 to 2020 with the main goal
to reduce the disease burden and with two principal objectives,
to reduce mortality and morbidity from dengue by 2020 by at
least 50% and 25%, respectively (WHO, 2012). To achieve
these objectives, it is important to move on from a reactive
response to an emergency situation to proactive risk assess-
ment, to develop early warning systems, and preventive
measures, guided by entomological as well as epidemiological
surveillance. Research continues playing a crucial role. In this
scenario, commitments and obligations from partners, organi-
zations, and countries, as well as leadership by WHO are of
utmost importance (WHO, 2012).
See also: Arboviruses.
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