Kumpulan Jurnal Dengue

KUMPULAN JURNAL
DENGUE
Dengue, M aj or John G Aaskov, bsc, phd, FASM , f r cpat h, RAAM C
Vi r us dengue,di agnosi s & mol ecul ar epi demi ol ogycal
Dengue vi r al i nf ect i ons; pat hogenesi s and epi demi ol ogy
Neur ol ogi cal mani f est at i ons of dengue i nf ect i on
kangmasmant r i @gmai l .com
Review
Dengue viral infections; pathogenesis
and epidemiology
William J.H. McBride
a*
, Helle Bielefeldt-Ohmann
b
a
Department of Pathology, Cairns Base Hospital, The Esplanade, Cairns, Queensland 4870, Australia
b
Department of Microbiology and Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia
ABSTRACT Dengue viral infections affect up to 100 million individuals per year. Dengue
haemorrhagic fever is a clinical form of disease characterised by intravascular uid loss. There has been
a marked increase in the incidence of this form of the disease over the last few decades, associated with
signicant mortality, particularly in the paediatric population. A number of theories relating to the
pathogenesis of dengue haemorrhagic fever exist that have evolved from the analysis of the
epidemiology of this disease. Virological and immunopathological factors are both important but the
exact mechanisms for the disease are unknown. 2000 ditions scientiques et mdicales Elsevier
SAS
dengue / pathogenesis / epidemiology
1. Introduction
Dengue fever is caused by one of the four serotypes of
dengue virus (serotypes 14). It is transmitted from human
to human by the mosquito Aedes aegypti. Infection with
one of these viruses characteristically results in fever,
headache and rash. The clinical spectrum can vary, how-
ever, from asymptomatic to more severe infections with
bleeding and shock. In areas where more than one sero-
type co-circulate, or when an area is subject to sequential
epidemics caused by different serotypes, a more severe
form of infection called dengue haemorrhagic fever (DHF)
may occur. The manifestations of DHF include haemor-
rhage and shock, which is the result of a sudden loss of
intravascular volume consequent to vascular leakage.
Although classical dengue fever has been recorded for
many centuries, DHF appears to be a more recent phe-
nomenom. Epidemics of DHF have become more frequent
since the 1950s in Southeast Asia and since the 1980s in
Central America. This coincides with a change in the
pattern of dengue viral infections. Dengue viral infections
now cause more illness and death than any other arboviral
illness. It has become a leading cause of paediatric mor-
bidity and mortality in some Southeast Asian countries [1].
2. Pathogenesis of classical dengue viral
infections
2.1. Host range and transmission
All four serotypes of dengue virus have a similar natural
history, including humans as the primary vertebrate host
and Aedes mosquitoes of the subgenus Stegomyia (espe-
cially Ae. aegypti, Ae. albopictus and Ae. polynesiensis) as
the primary mosquito vectors [2]. In Africa, and perhaps
the Indian subcontinent, dengue viruses also exist in
enzootic and epizootic forest cycles with nonhuman pri-
mates as the vertebrate host [3, 4]. Other vertebrate spe-
cies are generally not susceptible to dengue viruses, with
the exception of neonatal mice, challenged intracere-
brally.
Dengue infection does not have a direct pathogenic
effect on vectors. After ingestion of a blood meal contain-
ing virus, there is infection of the epithelial cells lining the
midgut. The virus then escapes from the midgut epithe-
lium into the haemocele and infects the salivary gland.
Finally, virus is secreted in the saliva, causing infection
during probing. The genital tract is also infected and virus
may enter the fully developed egg at the time of oviposi-
tion [5].
For transmission to occur, the female Ae. aegypti must
bite an infected human during the viraemic phase of the
illness which generally lasts 4 to 5 days but may last up to
12 days [1]. Ae. aegypti may be infected with 2 different
viruses without affecting the yield of either virus [6]. The
extrinsic incubation period refers to the time required from
when a viraemic human is bitten to when the mosquito * Correspondance and reprints mcbrij@health.qqld.gov.au
Microbes and Infection, 2, 2000, 10411050
2000 ditions scientiques et mdicales Elsevier SAS. All rights reserved
S1286457900012582/ REV
Microbes and Infection
2000, 1041-1050
1041
http://tugas-pbw.comuf.com/penyakittropis/upload/D1.pdf
itself becomes infective. This period is about 8 to 12 days
[7]. Figure1 illustrates the time periods in the cycle of
dengue virus transmission. The feeding behaviour of the
mosquito is characterized by easily interrupted feeding
and repeated probing of one or several hosts [8].
Whilst the Ae. aegypti has a generally lowsusceptibility
to oral infection with dengue virus, it remains the most
important vector because of its highly domesticated hab-
its. The persistence of dengue virus therefore depends on
the development of high viral titres in hosts to ensure
transmission in mosquitoes. This vector/virus relationship
may be a major factor in selecting and propagating patho-
genic strains of dengue in the urban setting [9].
2.2. Cellular targets of the virus
Dengue virus antigen has been detected in cells of the
monocyte-macrophage lineage in the lymphoid organs,
lung and liver of patients with dengue infection [10, 11],
and there are reports of isolation of dengue virus from
peripheral blood mononuclear cells during the viraemic
period [10, 12]. The possibility that dengue virus may
infect and replicate in epidermal-dermal cells at the site of
the mosquito bite remains to be shown.
Liver involvement in the clinical presentation of DHF
[10, 13] has been corroborated by demonstration of den-
gue virus RNA by reverse transcription (RT)-PCR in archi-
val liver and lymphoid organ samples obtained from indi-
viduals who had succumbed to dengue virus infection
[14]. However, since virus could only be re-isolated from
the liver, it was speculated that the liver might be the major
site of virus replication, whereas the presence of virus
RNA and antigen in lymphoid tissues reected local virus
inactivation [14]. Another recent study found that dengue
virus can infect but not replicate in human Kupffer cells
[15]. Rather these cells undergo apoptosis and are phago-
cytosed. Taken together these results suggest that the hepa-
tocytes may be the primary target cells in the liver, at least
in severe, fatal cases of dengue virus infection [14, 16]. It
remains to be shown that hepatocytes are also produc-
tively infected in nonfatal dengue fever.
In vitro, dengue virus can infect and replicate in a wide
range of cells of endothelial and epithelial derivation.
Notably however, the dengue viruses infect and replicate
comparatively poorly in primary leukocytes and estab-
lished leukocyte cell lines, unless the viruses have been
previously adapted or subneutralizing levels of virus-
specic antibodies are present ([1719] and unpublished
data). The ability of subneutralising concentrations of anti-
bodies to enhance infection has been described for other
virus/cell culture systems [20]. The role of antibody-
dependent enhancement (ADE) in the pathogenesis of
DHF is discussed later in this review.
2.3. Putative receptor/s for dengue virus
The identication of dengue virus receptors on target
cells is still not denitive, although the involvement of the
virus envelope protein in the process is undisputed [19,
21]. What remains disputed is the location of the receptor-
engaging epitopes on the virus envelope protein [19,
2224] as well as the identity of the host cell surface
moieties involved in the virus-binding and infection pro-
cesses. Early studies described a cell surface protein on
human monocytes responsible for binding of dengue virus
in the absence of virus-specic antibodies, i.e., a non-FcR
molecule [25], while Chen et al. [26] using a recombinant
dengue virus envelope-Fc fusion-protein were unable to
detect binding to human monocytes other than via the
FcR. Lately, a series of reports describing dengue virus-
binding molecules on human hepatocytes [27], simian
Vero and COS cells, hamster CHO and BHK cells [23, 27,
28], and on the C6/36 insect cell line [29] have implicated
both glycoproteins [27, 29] and glycosaminoglycans
(GAGs) [23, 28] in the process (table I). In studies using
human peripheral blood [30] and human leukocyte cell
lines ([19, 31] and Bielefeldt-Ohmann and Meyer, unpub-
lished data), respectively, a CD14-associated cell surface
molecule as well as non-FcR proteins were found to be
involved in the binding and/or internalization process-
es(table I). What emerges from these and other studies
(Bielefeldt-Ohmann and Meyer, unpublished data) is that
the dengue virus binding moieties on the target cell surface
membrane may vary between cell types as well as for the
different dengue virus serotypes.
Based on the data so far available it seems evident that
binding and internalization of the dengue viruses are a
multistep process involving the ordered and sequential
engagement of several target cell surface molecules by
multiple epitopes on the envelope protein [19, 32]. This
has also been found for several other viruses, notably
herpes simplex-, adeno- and human immunodeciency
viruses [33] The rst, and less specic step may, in some
virus-cell combinations, be binding to cell surface GAGs,
i.e., a bridging molecule, followed by engagement of one
or more specic protein moieties and internalization of the
virus [32]. Notably, the GAG-binding may be neither
necessary nor sufficient for virus infection to occur ([19]
and unpublished data).
Figure 1. Stages in the transmission of dengue fever from indi-
vidual to individual. A period of between 13 and 31 days is
estimated to elapse between successive cases in an epidemic.
Review McBride et al.
1042 Microbes and Infection
2000, 1041-1050
2.4. Replication of dengue virus
Once the dengue virus is bound to cell surface recep-
tors, uptake by endocytosis follows. Exceptions to this may
occur in insect cells and with some virus mutants in
vertebrate cells, where direct fusion of the viral cellular
membranes may take place [34, 35]. Once inthe endocytic
vesicle and following lowering of the pHin the endosomal
milieu the virus envelope protein undergoes an irrevers-
ible conformational change, from a dimer to a trimer [22,
36]. This change facilitates the subsequent fusion of the
virus envelope and host cell endosomal membrane, and
the nucleocapsid is released into the cytoplasm. This is
followed by the immediate translation of uncoated viral
genome, an 11-kb single-stranded positive-sense RNA
molecule coding for three structural proteins (core, pre/
membrane and envelope) and seven nonstructural (NS1,
NS2a, NS2b, NS3, NS4a, NS4b and NS5) proteins in one
open reading frame. The resulting polyprotein is posttrans-
lationally cleaved and modied.
Early translation occurs in association with the rough
endoplasmic reticulum (RER), thereby facilitating local-
ization of viral proteins in their characteristic luminal,
membrane or cytoplasmic context [36]. Following pro-
cessing, several NS proteins associate to form a viral
replicase complex. The complex binds specically to the
3' untranslated region of the viral genome and subse-
quently copies the positive-strand RNA into a negative-
sense intermediate RNA strand. Positive-strand synthesis
occurs from the thus formed RNA duplex. Early in the
infection negative- and positive-strand synthesis occur at
similar rates, but the ratio becomes asymmetric, favouring
positive-strand synthesis as infection progresses [37].
Extensive proliferationof membranous organelles, prob-
ably RER- and Golgi-derived, appears to be a unique
feature of avivirus-infected cells [38, 39], with these
structures apparently compartmentalizing various aspects
of avivirus replication [39]. Nucleocapsids may eventu-
ally become enveloped by budding through RER mem-
branes, followed by accumulation of virions in intracyto-
plasmic vesicles [38, 39].
3. Pathogenesis of DHF
3.1. Denitions
DHF and dengue shock syndrome (DSS) are dened by
a range of clinicopathological manifestations. The World
Health Organisation has dened DHF as comprising con-
tinuous fever lasting 2 to 7 days, haemorrhagic tendencies,
thrombocytopenia (100 000 cells per mm
3
or less) with
haemoconcentration (haematocrit increased by 20% or
more) [40].
The severity of DHF is further graded according to
clinical criteria. Grade I: fever accompanied by nonspe-
cic constitutional symptoms. The only haemorrhagic
manifestation is a positive tourniquet test; grade II: spon-
taneous bleeding, usually skin, nose or gum, in addition to
manifestations of grade I; grade III: circulatory failure
manifested by rapid, weak pulse with narrowing of pulse
pressure (< 20mmHg) or hypotension; grade IV: moribund
patients with undetectable blood pressure and pulse.
Grades III and IV are called DSS. DHF encompasses all
four grades.
DHF usually begins with abrupt onset of fever accom-
panied by dengue-like symptoms of fever, headache and
myalgias. Four to ve days later, during or shortly after the
fall in temperature, the condition of the patient suddenly
deteriorates, the skin becomes cold, the pulse rapid, and
the patient lethargic and restless. In some individuals the
pulse pressure progressively narrows, the patient becomes
hypotensive and, if not treated, may expire in as little as
four to six hours.
Minor haemorrhagic phenomena may be seen during
the febrile stage such as a positive tourniquet test, pete-
chiae, epistaxis or bruising. A maculopapular rash or
conuent petechial eruption may be seen after the tem-
perature falls. Many patients have hepatomegaly. Pleural
effusions, particularly on the right, may develop.
Pathophysiologically it is the sudden increase in vascu-
lar permeability that results in the loss of intravascular
uid volume, with consequent raised haematocrit,
hypotension and serous effusions. The pathogenesis of the
Table I. Putative dengue virus receptor molecules on human and nonhuman target cells.
Cell type (species) Virus serotype Receptor characteristics Reference
C6.36 (insect) D4 Proteins (40 and 45 kDa) 29
CHO (hamster) D2 Glycosaminoglycans 26
BHK (hamster) D2 Glycosaminoglycans 28
Vero (simian) D1 Protein 27
Vero (simian) D2 Glycosaminoglycans 26
COS (simian) D2 Glycosaminoglycans 26
Monocytes (human) D2 FcR; protein 25
Monocytes (human) D2 CD14-associated molecule 30
K562 (human) D2 Protein (~ 100kDa) 31
HepG2 (human) D1 Protein 27
HL60, BM (human) Proteins (~ 40, ~ 70 kDa),
D2, D3 GAGs 19
Raji (human B cell) D1-4 Protein *
MOLT4 (human T cell) D1-4 Protein, glycosaminoglycans *
* H. Bielefeldt-Ohmann & M. Meyer, unpublished data 1999.
Dengue pathogenesis and epidemiology Review
2000, 1041-1050
1043
increased vascular permeability has yet to be dened but
is the subject of intense investigation. Several major theo-
ries have emerged.
3.2. The immune enhancement theory
Based on epidemiological and in vitro studies the
'anamnestic sensitisation' hypothesis, nowadays better
known as 'antibody-dependent enhancement (ADE) of
infection' theory, was invoked to explain the pathogenesis
of DHF/DSS. Epidemiological studies, conducted mainly
in Thailand in the 196070s, suggested that DHF occurred
predominantly in children experiencing a second infec-
tion with a dengue virus serotype different from the one
encountered in the rst infection (reviewed in [20, 41]).
The in vitro correlate of these observations was that in the
presence of cross-reactive but non-neutralizing antibod-
ies, cells of the macrophage lineage were more readily
infected with dengue virus [20].
The ADE hypothesis has since undergone several modi-
cations andrenements totake intoaccount other aspects
of the immune response, including various T lymphocyte
subsets and the cytokine cascade [42]. Briey, antibodies
to dengue virus bind to the virus, forming non-neutralized
antibody-virus complexes, which bind to the Fc receptors
of monocytes-macrophages, followed by a productive
infection. Viral antigens are presented by the infected cells
in the context of MHC antigens, leading to priming and
stimulation of CD4
+
and CD8
+
T lymphocytes. One of the
consequences of this T-cell activation is the production of
cytokines, notably interferon- (IFN-), which activates
other cells including the macrophages, resulting inupregu-
lation of Fc receptor and MHC expression. Thus, a chain
reaction is set in motion that results in immunopathology.
Other factors such as complement activation, platelet
activation, and the production of potentially cytotoxic
cytokines, including tumour necrosis factor-a, interleukin
(IL)-1 and -6, by macrophages, lymphocytes and
endothelial/epithelial cells will contribute to and exacer-
bate this cascade of inammatory events [4145].
It is, however, notable that this inammation/immune
response scenario does not differ signicantly from what
has been shown to occur in many other viral infections
[46, 47], most of which do not progress to a haemorrhagic/
shock syndrome unless secondary bacterial infections
occur. This suggests that other factors play a role, either
primarily or as contributing, in the progression from DF to
DHF/DSS.
3.3. Alternative theories for the DHF/DSS pathogenesis
3.3.1. Molecular mimicry
One such alternative or contributing factor is molecular
mimicry, resulting in an autoimmune reaction [48].
Molecular mimicry and autoimmunity have been invoked
to explain neurological lesions during infections with the
related rubella virus, and in the absence of viral replica-
tion in the brain [49]. Through computer-aided sequence-
homology searches it was found that a 20-amino acid
sequence in the dengue envelope protein shared sequence
similarity with a family of clotting factors, including plas-
minogen [50]. Furthermore, cross-reactive antibodies to
plasminogen appeared during the immune response to
dengue virus infection [50, 51]. However, while cross-
reactive antibodies were more frequently detected in chil-
dren with secondary than with primary infection, there
was no correlation between their presence and DHF/DSS
manifestations [51]. The presence of these antibodies,
which is short-lived, may therefore be nothing more than
an epiphenomenon of a vigorous immune response. Alter-
natively they may contribute to the haemorrhagic mani-
festations in, at least, some cases of DHF.
Similarly, the dengue virus NS1 has been found to elicit
antibodies in mice which cross-react with epitopes on
human blood clotting factors and integrins, and bind to
human endothelial cells [52]. It remains to be demon-
strated that similar antibody reactivities are induced in
humans following dengue virus infection, and if so, what
role such antibodies might play in DF, DHF and DSS. The
lack of a suitable animal model for dengue is a major
impediment for addressing such questions.
3.3.2. Viral factors
While many, perhaps even most, cases of DHF/DSS
occur in patients experiencing a second dengue virus
infection, or in very young children with remaining mater-
nal antibodies to dengue virus, DHF is also seen in primary
dengue infections [53, 54]. Conversely, DHF/DSS occurs
in only a relatively small fraction of individuals with
secondary infections. Despite the co-circulation of several
dengue serotypes in the Americas, it was not until the
1981 epidemic in Cuba that the rst DHF cases occurred
in the region. This event coincided with the introduction
of a new genotype of dengue virus serotype 2 from South-
east Asia [55]. Subsequent epidemics with DHF in South
America also coincided with the occurrence of Southeast
Asian dengue virus strains [55]. In contrast, in Peru, no
evidence of DHF was found during an epidemic caused by
dengue 2 virus, ve years after an epidemic of dengue 1.
Evidence for secondary dengue virus infections was found
in 60.5% of subjects tested [56]. The absence of DHF in
this population has been attributed to the American origin
of the dengue 2 strain causing the epidemic [57]. These
and other ndings suggest that viral virulence factors may
play an essential role in the pathogenesis of DHF/DSS [10,
24, 47].
Viral virulence factors may, amongst others, encompass
the ability to (i) infect more cells, (ii) generate more prog-
eny virus, (iii) cause a more severe inammation, and (iv)
evade immune response effector mechanisms. Different
strains of dengue 2 virus behave differently in assays for
ADE. Noting that dengue 2 isolates from the Caribbean
were not associated with DHF until the mid 1980s, Kliks
[58] demonstrated that virulent strains of dengue 2 virus
bound to monocytes and were internalised in the same
way as avirulent strains but that virulent strains were able
to fuse with the phagosomal membrane at a more basic
pH. It was proposed that antigenic variation on the enve-
lope glycoprotein fusion domain might be responsible for
this phenomenon. Avirulent strains had previously been
shown to replicate less well in ADE assays [59]. Other
recent studies suggest that there are strain differences in
the dengue viruses' ability to bind to and infect target cells,
and ability to generate more virus progeny in vitro, with
2000, 1041-1050
different viral gene products determining different aspects
of these phenomena ([19, 24], Bielefeldt-Ohmann and
Meyer, unpublished data 1999). The lack of a suitable
animal model for dengue has so far precluded testing these
and the immunological aspects of viral virulence in vivo.
Likewise, the effect of the immune response on the evolu-
tion of the viruses and the selection of more virulent strains
remain to be elucidated. Like other RNA viruses, dengue
viruses exist as quasispecies [60], albeit under greater
constraints than many other viruses due to the two-host
transmission cycle [47, 61]. It is therefore imperative for
the understanding of the pathogenesis of DF, DHF and
DSS that the factors inuencing the selection and mainte-
nance of (relatively) virulent and avirulent virus strains in
human, nonhuman primates and mosquito-host popula-
tions, respectively, be elucidated [47, 6264].
3.4. Pathological consequences of infection
Much of the descriptive pathology is based on studies of
tissues obtained from fatal cases of DHF. There is wide-
spread petechial haemorrhage and serous effusions in the
pericardial, pleural and peritoneal cavities. The liver may
be enlarged and discoloured. Histological changes in the
liver are characteristic, with midzonal necrosis of hepatic
cells, swelling of the Kupffer cells and formation of Coun-
cilman bodies (apoptotic cells). There is usually no gross
or microscopic evidence of severe organ pathology that
can explain the cause of death. Specically relating to
capillaries and venules, there is often perivascular oedema
and haemorrhage and an inltrate of lymphocytes and
mononuclear cells [10, 16, 65].
4. Epidemiology of dengue viral
infections
4.1. Distribution of infection
Dengue occurs principally in the tropical areas of Asia,
Oceania, Africa, and the Americas (see gure 2). The
distribution is constrained only by the range of the princi-
pal mosquito vector Ae. aegypti. In areas with year-round
vector activity and high population densities, one or more
dengue virus types may be maintained endemically. Else-
where, especially in small insular populations such as
exist in North Queensland of Australia, epidemics result
from the introduction of a new viral strain [5, 9]. Over 2.5
billion people are at risk for dengue infection and there are
an estimated 100 million infections annually [1]. Current
information concerning the status of dengue infections in
particular countries is available on the internet at http://
www.tropicalmedicine.org.au/dengue/status.htm.
4.2. Patterns of infection in the individual and community
An estimate of the impact of dengue epidemics is
obtained by assessing the proportion of a population that
is affected by any given epidemic. The actual morbidity,
however, is determined by the clinical/subclinical ratio.
Infection rates for some dengue epidemics have varied
from 5.6% in Taiwan [66] to an estimated 90% in Niue
[67]. Population infection rates of 20 to 50%are typical in
well-dened large epidemics [68]. Serological assessment
of infection rate is feasible where an epidemic is caused by
a single serotype in an immunologically naive population.
Figure 2. International distribution of dengue fever and DHF cases.
2000, 1041-1050
1045
Where continuous transmission of multiple serotypes
occur, a better measure of dengue infection in a popula-
tion is the age-dependent seroprevalence. For example,
during 1980 in Bangkok, 50% of seven-year-olds had
antibodies to dengue and 6.3% seroconverted during the
six-month dengue transmission season (June 1980 to Janu-
ary 1981) [69].
In the prospective study of dengue infections in school
children in Bangkok, 87% were found to be either asymp-
tomatic or minimally symptomatic (absent from school
only one day) [69]. The clinical attack rate in adults is
probably much higher, almost all nonindigenous people
in Thailand who had antibodies to dengue had a history of
a compatible illness [70]. However in a 1982 Puerto Rican
dengue epidemic only about 35% were considered to
have been symptomatic [71] and more recently another
Puerto Rican study reported that only 13% of dengue
infections were symptomatic [72]. In Cuba, 28% of white
people and 11%of black people with dengue 2 antibodies
could recall a dengue-like illness. Over 80%of this sample
were aged over 14 years [73]. A study of US soldiers
returning from Somalia found that of those with IgM
antibodies to dengue in their serum at the time of depar-
ture, 84%could remember symptoms consistent with their
having had dengue fever [74]. A similar gure of 86%was
found in an Australian population infected with dengue 2
[75]. The Cuban study suggests that genetic factors might
play a role. The variability in estimated subclinical infec-
tion rates in different populations has important implica-
tions for the way that interepidemic surveillence is con-
ducted. In populations with high estimated subclinical
infection rates, serological surveillance plays an important
role, whereas clinical surveillance (with serological con-
rmation) is appropriate where subclinical infection rates
are low.
4.3. Factors affecting transmission of the virus
In tropical areas dengue transmission occurs through-
out the year. Increased transmission, however occurs dur-
ing the rainy season. It is speculated that temperature and
humidity favour the survival of adult mosquitoes beyond
their extrinsic incubation period thereby increasing the
probability that viral transmission occurs. It has also been
shown that increased temperature shortens the extrinsic
incubation period [76]. Rainfall itself may not be particu-
larly important, as the principal breeding sites for Ae.
aegypti are present year round. The importance of Ae.
aegypti breeding sites in the Caribbean and Pacic, where
rainfall is less reliable, is highlighted as the most important
factor in these locations [9].
A number of factors have been proposed in the initia-
tion and maintenance of an epidemic: (i) the strain of the
virus, which may inuence the magnitude and duration of
viraemia in humans; (ii) the density, behaviour and com-
petence of the mosquito vector population; (iii) the sus-
ceptibility of the human population (both genetic factors
and pre-existing immunity); and (iv) the introduction of
virus into a receptive community [9].
Differences in the viral virulence were observed in two
epidemics in Tonga caused by dengue 2 virus in 1974 and
dengue 1 virus in 1975. The rst epidemic was manifest
clinically by mild disease of short duration and the second
by severe disease with haemorrhagic manifestations. Most
of these infections were primary [77]. Preexisting immu-
nity is a population factor that determines whether an
epidemic can proceed but is likely to be less important in
heavily populated areas where a pool of younger persons
susceptible to infection occurs. Introduction of new viral
strains into susceptible areas is the most important factor
for areas now free from dengue. It is clear that viraemic
humans can enter a receptive area, and even initiate
dengue transmission without causing a subsequent epi-
demic [78]. An aggressive approach to the prevention of
the initiation of an epidemic is clearly the best tactic for
receptive areas currently free of endemic dengue transmis-
sion.
Once an epidemic has started there are a number of
factors which have been identied as inuencing an
individual's risk of being infected. House-screening
reduces the risk of infection signicantly [66, 79]. One
study showed that communities with an average tempera-
ture of 30 C had a 32-fold increased rate of infection
compared with cooler locations. Infection rate decreased
withincreasing altitude independently of temperature [80].
Insecticide use has a variable effect. The presence of
larvae or water containers on the property is associated
with higher infection rates as is, paradoxically, the use of
mosquito nets at night [79, 80]. Incidence and prevalence
of infection in Puerto Rico was signicantly associated
with slum housing, poverty, lack of house-screening and
wooden house construction [71].
4.4. Epidemiology of DHF
The bimodal age distribution of DHF was one of the
earliest clues that alerted researchers to the immune
enhancement theory. The age peaks occur at seven months
and three to ve years. Furthermore, infants were present-
ing with DHF after primary dengue infections [20]. An
investigation of DHF in infants caused by dengue 2 virus
found that maternal dengue 2 neutralisation titre and age
of the infant when DHF occurred was strongly correlated.
The authors hypothesised that maternal antibody against
the homologous serotype was at rst protective and later,
enhancing [81]. Adults are also subject to DHF and may
be the predominant age group affected during an epi-
demic [82, 83]. In Cuba, where a dengue 1 epidemic
occurred from 1977 to 1980 and a dengue 2 epidemic
occurred in 1981, about two thirds of the fatal DHF cases
occurred in children. Another dengue 2 epidemic in 1997
was associated almost exclusively with adult DHF cases.
Most had evidence of secondary infection and the interval
between the dengue 1 and dengue 2 epidemics was
greater than 16 years [83].
The sequence of dengue infections has been studied. In
the 1980 Thailand epidemic, a dengue 1/dengue 2
sequence of infection was associated with a 500-fold risk
of DHF compared with a primary infection. For a dengue
3/dengue 2 sequence the risk was 150-fold and a dengue
4/dengue 2 sequence had a 50-fold risk of DHF [20, 84].
Other major DHF epidemics have involved dengue 2 as
the second infecting serotype including the Cuban epi-
demic in 1981, China in 1985 and India in 1988 [20].
2000, 1041-1050
Epidemics associated with a signicant DHF occurrence
have been recorded for dengue 3 [85] and dengue 4 [86].
During DHF epidemics in countries where dengue fever is
endemic, the isolation of all four viral serotypes is often
recorded [87].
Ethnicity, age and sex have been highlighted as possible
factors. In Cuba in 1981, only 14%of DHF cases occurred
in blacks, although 34%of the population are black. Cases
of DSS were uncommon in people more than 14 years of
age. In Thailand, females were hospitalised at twice the
rate for DHF [88], although in Singapore the male to
female ratio was 1.46:1 [89]. More recent studies demon-
strate a more even distribution between the sexes [90].
Genetic markers for DHF have been analysed. Blood
group and glucose-6-phosphatase status are not related
but HLA typing showed that HLA A1, HLA-B blank and
HLA-Cw1 were signicantly more common in DHF cases
than in controls [91]. Nutritional status has been identied
as a risk factor, with well-nourished children having a
higher risk [84, 92].
Considerable genetic divergence exists both within and
between the four dengue serotypes. Based on the genomic
sequence encoding the envelope protein, which deter-
mines most antigenic characteristics of the aviviruses,
each of the four dengue virus serotypes has been subdi-
vided into 2 to 6 subtypes [93, 94]. A major contributing
factor to this diversity is undoubtedly the relative high
mutation rate during RNA replication, a characteristic of
many RNA viruses which lack the proofreading enzymes
employedby DNAviruses [95]. Another important mecha-
nism may be recombination during simultaneous infec-
tion of humans or mosquitoes with two or more dengue
viruses, an event which has been documented [96], and
appears a highly likely occurrence in hyperendemic areas
where two or more serotypes circulate concurrently [24,
94, 97]. It is likely that both mechanisms of genetic evo-
lution play a role in the pathogenesis of dengue disease
5. Future trends
Understanding of the pathogenesis of severe DHF
remains incomplete despite many decades of research.
The antibody-dependent enhancement theory has strongly
inuenced the approach taken to the development of a
vaccine for dengue fever, with most effort concentrated on
the production of a tetravalent vaccine. Incomplete vac-
cine efficacy will be viewed with concern if there is a
possibility that heterologous antibody might enhance
infection. Recent studies have added weight to previous
suggestions that viral pathogenic factors are important and
further research to investigate these factors must be under-
taken. The molecular mechanisms of virus attachment and
replication will be a vital part of these studies.
Clinically, we need to be able to better predict which
individuals are at risk for DHF. A range of cytokines and
receptors are correlated with severity but none are suffi-
ciently specic at the moment. Gene display methods
could provide valuable information about how infected
cells respond to infection at a molecular level. The devel-
opment of quantitative PCR in the diagnosis of dengue
viral infections will allow the evaluation of viral load as
factor in disease severity.
Continued observations of epidemics and characterisa-
tion of the viruses responsible will help to further dene
the factors responsible for severe epidemics.
Internationally the impact of dengue viral infections has
relentlessly increased and the diseases caused by these
infections are assuming a growing public health impor-
tance. In few other areas of medicine have the answers to
such basic questions concerning pathogenesis been so
urgently required.
Acknowledgments
We thank Dr. Vincent Deubel and colleagues at the
Institut Pasteur, Paris, for making pre-publication data
available. Dr Bielefeldt-Ohmann is supported by the Fac-
ulty of Biological Sciences, University of Queensland, and
the Ramaciotti Foundation for Medical Research.
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66 ADF Health Vol 4 September 2003
ADF Health ISSN: 0025-729X 1 September 2003 4 2 66-71
ADF Health 2003 http://www.defence.gov.au/dpe/dhs/
Infectious diseases
DENGUE is a disease caused by four serotypes of a virus of the
same name (dengue 1, 2, 3 and 4). The severity of dengue
infections is influenced by the age and genetic background of
the host, the strain and serotype of the infecting virus and the
prior history of dengue infections of the host.
1-6
History
Dengue fever was accepted as an occupational hazard of living
and working in the tropics. The 1905 edition of Mansons
Tropical Diseases
7
states: In Europeans, an attack of dengue
very often leads to a condition of debility necessitating
temporary change of climate, or even return to Europe.
Perhaps the first reports of dengue haemorrhagic fever and an
associated mortality were made by Hare in Charters Towers in
northern Queensland in 189697 (Box 1).
8
He wrote: as
epidemic succeeds epidemic, the disease appears to be more
severe and fatal cases more frequent. Second attacks are as
severe or more severe than the first.
8
However, it took 60 years
and outbreaks of a new haemorrhagic fever in Thailand and
the Philippines
9
before the significance of these observations
was appeciated. There is now compelling evidence that severe
dengue occurs most commonly following infection with a
second or subsequent dengue virus serotype.
5,10
Several other seminal observations relating to dengue and its
pathogenesis also were made in Australia. Following a dengue
outbreak in Brisbane in 1905, in which it was estimated that one
third of the workforce was incapacitated, Bancroft, who was a
general practitioner in the then rural Brisbane suburb of
Alderly, demonstrated that Aedes aegypti mosquitoes which
had fed on a dengue patient were able to transmit virus to
previously healthy members of the Alderly community.
11
These observations subsequently were confirmed by Cleland,
Bradley and MacDonald in Sydney
12
and were extended to
determine the interval from infection to onset of symptoms, the
duration of viraemia in patients and to show that the virus was
present in both serum and blood cells. In some notes on failed
experiments, these authors mentioned the unexpected diffi-
culty in obtaining volunteers even with a considerable monetary
inducement and in two experiments the finding of a positive
Wasserman test (syphilis?) in a volunteer prevented further
utilisation of the virus in the blood. Outbreaks of dengue
continued in northern Australia on an annual basis until the
mid-1920s. Cases of dengue have been reported in Australia
since the 1980s, due to the arrival or return of human hosts who
have been infected with dengue virus in another country. Since
World War II, there also have been numerous examples of local
transmission of dengue viruses introduced into Australia
(19545, dengue 3; 19812, 19901, dengue 1; 19923, 1995,
19967, dengue 2; 19979, dengue 3; 2001, dengue 2).
13,14
The primary vector of dengue is the mosquito Aedes aegypti,
a peridomestic mosquito (ie, found in and around homes) with
a short flight range. It breeds in a variety of containers, usually
associated with human refuse or water storage. In Australia, it is
found in northern Queensland. A secondary vector, Aedes
albopictus, has similar habits to Ae. aegypti and has recently
invaded the south-west Pacific region. Ae. albopictus is
common in Papua New Guinea and poses a constant risk to
Australia.
Origin of dengue viruses
The origin of dengue viruses is uncertain. Some have
speculated that they originated in Africa and moved out of that
continent along with its mosquito vector Ae. aegypti as a result
Major John Aaskov is an Arbovirologist with the Army Malaria Institute and Senior
Lecturer in Immunology and Virology in the School of Life Sciences at the
Queensland University of Technology. He has dengue research programs in
Vietnam and Myanmar and is undertaking a dengue genotyping project with
collaborators throughout Asia.
Australian Army Malaria Institute, Gallipoli Barracks, Enoggera,
QLD.
John G Aaskov, BSc, PhD, FASM, FRCPath, RAAMC, Arbovirologist.
Correspondence: Major John G Aaskov, Australian Army Malaria Institute,
Gallipoli Barracks, Enoggera, QLD 4052. j.aaskov@qut.edu.au
Dengue
Major John G Aaskov, BSc, PhD, FASM, FRCPath, RAAMC
Abstract
An estimated 50100 million cases of dengue occur
annually in more than 100 tropical and sub-tropical
countries.
Dengue has been a source of unquantifiable morbidity in
many of Australian Defence Force campaigns in the
Asia-Pacific region. There were more confirmed dengue
cases in ADF personnel in Timor in the first six months of
operations (215) than were reported in Vietnam in seven
years (4).
A new generation of assays allows point-of-care
diagnosis of dengue infection by semi-skilled operators.
There are opportunities for the ADF to become involved
in the evaluation of the first dengue vaccines likely to be
effective.
Training of ADF medical practitioners might include
attachments to hospitals which handle significant
numbers of tropical infections, including dengue.
Communicable disease surveillance, including that for
dengue, should include an interaction with the civilian
community, particularly during peacekeeping
ADF Health 2003; 4: 66-71
deployments.
Infectious Diseases
http://www.defence.gov.au/health/infocentre/journals/ADFHJ_sep03/ADFHealth_4_2_66-71.pdf
ADF Health Vol 4 September 2003 67
of the slave trade.
15
Others have proposed that the viruses may
have evolved from a jungle cycle involving lower primates and
canopy dwelling mosquitoes in the Malay peninsula.
16
Whatever their origin, there has been an exponential diversifica-
tion in dengue virus genotypes which has paralleled the
increase in the human population over the last 200300 years.
17
While there is clear evidence of the introduction of dengue
viruses into non-endemic countries like Australia and Cuba and
the introduction of an Asian strain of dengue 2 into South
America,
6
most changes in virus genotypes in countries where
the virus is endemic appear to be due to local evolution.
18,19
Variation in the competence of mosquito vectors Ae. aegypti
and Ae. albopictus to transmit different strains of dengue virus
also suggest local co-evolution of virus and vector.
20
Nonethe-
less, in areas where there is extensive movement of human hosts
it may be possible to identify the introduction of new strains of
virus even if they do not become established.
19
From phylogenetic studies carried out by the Australian
Army Malaria Institute and the Queensland University of
Technology, it appears that all four serotypes of dengue virus
are circulating in East Timor (Box 2). The Timorese dengue 1
strains recovered between 1999 and 2001 were related to older
Pacific strains, but appear to be evolving locally. In contrast,
there were four genotypes of dengue 2 circulating in Timor in
1999, only one of which (Timor 2001 D2-79) appeared to
have continued in circulation. These data suggested that some
or all of the dengue 2 virus genotypes were introduced,
possibly with UN personnel from dengue endemic areas such
as India and Singapore. Too few dengue 3 isolates were
recovered to draw any conclusions about this virus serotype.
There appeared to be two distinct genotypes of dengue 4 in
Timor in 1999, which were quite distinct from dengue 4
viruses from other countries. One lineage may have
disappeared, while the Timor 99/00 D4-252 lineage appeared
to have continued to evolve locally until 2002. In an
illustrative example of the peridomestic nature of the
mosquito vector of dengue, four of these dengue 4 virus
isolates were recovered sequentially, at about weekly
intervals, from four defence personnel who were sharing
accommodation.
Clinical features
Most dengue infections are inapparent, but symptoms, when
they occur, vary in severity from a mild flu-like illness to a
haemorrhagic fever and hypovolaemic shock which, if
untreated, may be fatal.
21
The mildest form of clinical dengue
infection is dengue fever, but because of the broad spectrum of
signs and symptoms, the World Health Organization (WHO)
has suggested there should not be a detailed clinical definition
for dengue fever. Clinical features of dengue fever include
abrupt onset high fever, headache, retro-orbital pain, muscle
and bone or joint pain, nausea, rash and, occasionally,
petechiae.
Dengue haemorrhagic fever is characterised by four major
clinical manifestations: high fever, haemorrhage, hepatomeg-
aly and circulatory failure. An abbreviated WHO case
definition for dengue haemorrhagic fever is:
Fever, lasting 27 days and perhaps biphasic
Haemorrhage (bleeding from the mucosa or gut, positive
tourniquet test, petechiae, ecchymoses or purpura,
haematemesis or melaena)
Thrombocytopenia (<100 000 cells/mL)
Plasma leakage (> 20% rise in age and sex adjusted
haemocrit, pleural effusion, ascites)
Dengue shock syndrome usually occurs in patients with
dengue haemorrhagic fever after 27 days of fever. Patients
1: Dengue in northern Queensland, 1897
Dr Hare, of Charters Towers, made perhaps the first report of
dengue haemorrhagic fever in the medical literature,
8
as part of his
report of an epidemic of dengue fever that swept northern
Queensland in 1897.
Above: The medical staff of the Charters Towers Hospital in 1896. Dr
Hare, who recognised dengue haemorrhagic fever cases, is seated on
the extreme left in the front row. Below: the hospital.
I have collected some account of 60 fatal cases occurring in North
Queensland during the epidemic of 1897. Half the number were adults.
In many, pre-existing conditions appeared to determine the fatal issue.
Among these were old age, diabetes, chronic bronchitis, opium smoking,
pregnancy and especially alcoholism. There is a widespread popular
idea that alcohol has prophylactic influence against the disease, and
this, I am sure, acted disastrously at times. F E Hare
8
may have a rapid weak pulse, complain of abdominal pain and
become restless, and the skin may become cool and blotchy.
Blood pressure and pulse become imperceptible. The WHO
case definition for dengue shock syndrome is:
Rapid and weak pulse
Narrow pulse pressure (<20 mmHg[2.7kPa])
Hypotension for age
Cold, clammy skin and restlessness
21
None of the clinical signs and symptoms listed above are
specific for dengue and so the disease is frequently misdiag-
nosed, even by paediatricians and physicians who have worked
with these patients all their careers. Laboratory tests are
essential if a definitive diagnosis is to be made. For these
reasons, there are no reliable figures for the number of cases of
dengue the ADF experienced during the Pacific campaign of
World War II. In the first two years of the ADF deployment in
2: Phylogenetic relationships between dengue viruses recovered in East Timor and representative
examples of clades (ie, commonly descended branches) of each dengue serotype
Viruses are
identified as
country; year of
isolation; serotype-
strain number.
Timor, more laboratory confirmed cases of dengue were
reported (234) than there were unconfirmed dengue cases (4)
reported for the duration of the commitment to Vietnam.
22
Diagnosis
There are three criteria for a definitive diagnosis of a dengue
infection.
1. Detection of dengue virus or dengue virus RNA in an acute
phase serum/tissue sample or
2. Detection of anti-dengue virus IgM antibody or detection of
anti-dengue IgG antibody at a titre equivalent to a haema-
gglutination-inhibiting antibody titre of 1280, in serum
collected within 1014 days of onset of symptoms compatible
with dengue or
3. Detection of a four-fold or greater rise in anti-dengue virus
antibody titre in paired sera collected 714 days apart and tested
in parallel.
There remain a number of problems associated with the
laboratory diagnosis of dengue. Serological tests are easier and
often faster to perform than virological ones, and there are a
range of dengue serological tests available commercially.
However, it may be 56 days after onset of fever before
diagnostic levels of anti-dengue virus antibodies are produced.
Conversely, dengue viruses may not be detected in all
seronegative, acute-phase, serum samples. In a small percent-
age of cases, seroconversion may not occur.
If doctors or paramedical personnel suspect a case of dengue
among military personnel they can perform a rapid
immunochromatographic dengue assay
23
on serum as soon as
the patient presents. This test can be performed in a Regimental
Aid Post or similar facility, provided attention is paid to the
manufacturers recommended procedures for performing and
interpreting the test. If the test is performed on serum collected
before the fifth day of fever, and is negative, a second test
should be performed 67 days later. Suspected patients should
not be returned to their unit before this time because they may
still be viraemic and so act as a source of further infection.
While in medical care, these patients should be confined under
a mosquito net at all times that they are not moving about. There
is strong evidence from the number and type of dengue viruses
recovered from patients in the Dili Hospital, early in the
deployment of the ADF, that dengue virus transmission
occurred inside the hospital because these precautions were not
taken/enforced.
If a large dengue outbreak is suspected, it is far more efficient
to use enzyme-linked immunosorbent assays (ELISA),
24,25
which require basic laboratory facilities. These assays should
be read spectrophotometrically in an ELISA Plate Reader, but
in an emergency they can be read by eye (ELISAs were read by
eye in the early stages of the dengue outbreak in Australian
troops in Timor with almost 100% sensitivity and specificity.
Major Scott Kitchener, personal communication).
Most assays for the rapid detection of dengue viruses are still
research tools or are in house assays performed in large
specialised laboratories.
26
The one commercial assay for the
detection of dengue viruses in serum lacks sensitivity. A
number of polymerase chain reaction (PCR) based assays are
approaching commercialisation and these have the potential to
be rapid, sensitive and specific and to provide laboratory
confirmation of dengue virus infection in acute phase serum.
The Australian Army Malaria Institute and the Combatant
Protection and Nutrition Branch of the Defence Science and
Technology Organisation have adapted real time PCR
protocols for the detection of dengue viruses using the
Ruggedised Advanced Pathogen Identification Device
(RAPID) and have deployed this to Timor. The system worked
well in the laboratory and the field, but this technology is not at
a stage where it could be employed by paramedical staff at a
Regimental Aid Post.
Clinical management
Managing patients with dengue haemorrhagic fever, and even
dengue shock syndrome, does not require sophisticated medical
facilities. Perhaps the greatest risk to such a patient is
overcompensation for plasma leakage when giving intravenous
fluids. Early oral rehydration may be adequate for mild cases of
dengue haemorrhagic fever and is a useful initial treatment in
patients who may go on to severe disease requiring more
comprehensive management.
Dr Suchitra Nimmannitya from the former Bangkok
Childrens Hospital has developed a simple, effective, step-by-
step, treatment protocol based on decades of experience, which
has the endorsement of WHO, and is used extensively in
hospitals in dengue endemic areas.
21
This protocol is based on
regular monitoring of platelet counts and haematocrit to guide
treatment. Antipyretics may be given during the febrile phase of
dengue haemorrhagic fever but these will not reduce the
duration of fever. Salicylates should not be used because they
affect platelet function and they may precipitate Reye syndrome
in children. A rise in haematocrit of 20% or more is the trigger
to begin fluid replacement. Colloids (dextran 70 or gelafundin
35000) have been found to restore cardiac index and blood
pressure and to normalise haematocrit more rapidly than
crystalloids (Ringers lactate).
27
In severe cases of dengue shock syndrome, hyponatraemia
and metabolic acidosis may occur. Prompt fluid replacement
and correction of the acidosis with sodium bicarbonate usually
overcomes these complications. In patients experiencing
significant bleeding, fresh whole blood may be given to restore
a normal red blood cell volume. There appears to be a window
of about six months to five years after a dengue infection in
which a person is at greater risk of severe disease if infected
with a second dengue virus serotype. This may be due to
enhancement of the subsequent infection by dengue virus
cross-reactive antibodies produced following the first infection.
The magnitude of this risk for re-deployment of ADF personnel
who have experienced prior dengue infection(s) depends on the
interval between the most recent infection and deployment,
whether there are multiple dengue virus serotypes circulating in
the area of operations, the rate of infection in the area of
operations and the number of people with prior dengue
infections deployed. It is unlikely that there would be
significant numbers of cases of severe dengue in ADF
personnel re-deployed to Timor. People who have been infected
with three or four dengue virus serotypes are probably totally
immune to re-infection.
Vaccine development
The watershed in the study of dengue and its causative agent
was the isolation and culture of dengue viruses; first by Hotta in
Japan
28
and then by Sabin (of subsequent polio vaccine fame)
in the United States of America.
29
Hotta succeeded in growing
the virus in mice but had to inject infected mouse tissue into his
mother to confirm he had isolated dengue virus; whereas Sabin
relied on human volunteers to grow his virus isolates until he
too managed to adapt his viruses to grow in mice.
30
These
experiments took place during World War II, so each group was
unaware of the work of the other. Once the viruses could be
cultured, diagnostic tests were developed and the first candidate
vaccines were produced. Sixty years later there are still only
candidate dengue vaccines. Dengue poses some particular
challenges for vaccine development. There are four serologi-
cally distinct viruses and long-term immunity is specific for the
infecting serotype. Sequential infections with different sero-
types may result in severe disease.
5,10
Since the vaccine is to be
used in endemic areas, there must be no risk that pre-existing
anti-dengue virus antibody in a vaccine will enhance
31
the
vaccine infection and cause severe disease, and the vaccine
must induce simultaneous, life-long immunity to all four virus
serotypes if it is not to sensitise vaccinees to severe disease
following a natural dengue virus infection. Added to these
difficulties is the absence of an animal model of dengue
haemorrhagic fever in which to test a vaccine and the lack of
definitive markers of virus attenuation.
All of the tetravalent dengue vaccines in trial or about to enter
clinical trials
32,33
are derived from a single genome of each
dengue virus serotype or a plaque-purified population (ie, a
very homogeneous population of virus). In some cases, the
viruses used in the vaccines are those which were circulating
2030 years ago. Dengue viruses have RNA genomes and
because of the error-prone nature of RNA polymerases,
populations of virus might be expected to be diverse. Recent
experiments have confirmed this.
34
If a virus population is
diverse, it has subpopulations that may be ideally suited to
occupy new ecological niches or to escape the immunological
pressures of a host immune response (ie, the immune response
to a single dengue genotype in a vaccine might not protect
against all the viruses in a diverse, natural, virus population of
the same serotype).
Two other influences may be acting to force change on
dengue virus populations. There is extensive evidence of intra-
serotypic recombination occurring in dengue viruses.
34,35
This
occurs when a host is infected with two different dengue virus
populations and part of the genome of one replaces a
corresponding region of the second to give rise to a new virus.
Although recombinant dengue viruses have been identified for
some time, it was only in 2002 that we identified a single Ae.
aegypti mosquito which contained two different dengue virus
populations, along with a third which was a recombinant of
these two.
34
Ae. aegypti is easily disturbed when feeding and we
postulate that this insect fed on two patients in order to complete
a blood meal and acquired a virus population from each.
Viruses from each population then recombined.
Rapid and dramatic changes in dengue virus genotype have
been detected in Thailand
19,36
and Myanmar (unpublished
observations) due to what is believed to be genetic bottlenecks.
These occur at times of low virus transmission when there is a
possibility that a rare virus variant may be the only one to be
transferred to a susceptible host.
These observations do not indicate that dengue vaccines will
not be effective, but they do suggest that they may be aiming at
moving targets.
It has been only in the past few years that a dengue vaccine
has become a possibility. Both Hotta and Sabin failed in their
attempts to produce dengue vaccines.
30,37
Subsequent efforts by
the US Army met with little more success, with only a dengue
2 vaccine progressing to phase I clinical trials.
38
A tetravalent
vaccine developed at Mahidol University in Thailand using
viruses attenuated by in-vitro passage
32
and commercialised by
Aventis Pasteur showed initial promise, but is now being
reformulated. The US Army also has a classically attenuated
tetravalent vaccine which is entering trials. A tetravalent dengue
vaccine containing chimeric yellow feverdengue viruses also
has entered trials.
33
The viruses in this vaccine are composed of
a backbone of the core and non-structural protein genes of the
17D yellow fever virus vaccine, into which the pre-membrane
and envelope protein genes of each of the dengue virus
serotypes has been inserted. This results in a virus particle with
dengue virus proteins on its surface enclosing a chimeric yellow
feverdengue virus genome. This approach has the potential to
overcome the difficulties encountered with earlier tetravalent
dengue vaccines in which the four virus serotypes appeared to
replicate at different rates.
Dengue vaccine trials face some additional hurdles. The virus
record is incomplete because many of the countries in which
dengue occurs lack the facilities for systematic collection and
identification of dengue viruses, and some of those that do have
collections lack the resources to analyse the viruses in a timely
or systematic manner. Without information on the serotypes
and genotypes circulating in a region and the infection rates in
those areas, it will be extremely difficult to plan vaccine efficacy
trials.
The US Armed Forces Research Institute of Medical
Sciences study site at Kampong Phet, Thailand, is perhaps the
only site anywhere in the world for which sufficient data are
available to undertake a dengue vaccine efficacy study. The
US Army is attempting to overcome this problem by
3: Measures to control dengue among ADF
personnel deployed in dengue-endemic areas
Appropriate wearing of permethrin-treated uniform.
Applying DEET-based repellant to exposed skin.
Sleeping under bed-nets or in screened enclosures
wherever possible.
developing dengue virus preparations that will cause mild
disease in all who are infected with them. Such a virus
preparation could then be used safely in challenge tests of
dengue vaccinees. This would be much simpler than
undertaking large scale vaccine efficacy studies in populations
in which only a few per cent will develop clinical symptoms
of dengue each year.
Surveillance and control
In Asia, epidemics of dengue occur in cycles of 35 years,
probably due to the phenomena of enhancement of infection by
cross-reactive antibody produced in earlier infections,
39
so it
remains to be seen whether the remedial actions taken to reduce
exposure of ADF personnel to Aedes mosquito vectors
following the dengue outbreak in Timor in 19992000 (Box 3)
have been responsible for the subsequent reduction in the
number of cases.
Disease surveillance is a key component in disease
prevention: know your enemy. The ADF may have good
qualitative data about communicable diseases in areas where it
may be called on to operate, but it lacks quantitative data that
would help to prioritise disease risks. It may be impossible to
obtain these data before a deployment, but one of the best
disease surveillance systems available to peacekeeping forces,
once deployed, is the local civilian population. A case might be
made to develop the interfaces needed to be able to obtain
reliable, timely, public health data including that for dengue
from civilian populations in areas where the ADF operates.
Competing interests
The author participated in the development of commercial assays for the
diagnosis of dengue and receives a financial benefit from the sale of these assays.
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(Received 20 Jan 2003, accepted 5 Mar 2003)

Eelslnkl Unlverslty 3lomedlcul Llssertutlons No.

Dengue virus infection:
Diagnostics and molecular epidemiology

Lili Buhtamo

lnfectlon 3lology Reseurch Progrum
Lepurtment of vlrology
Euurtmun lnstltute, luculty of Medlclne
Unlverslty of Eelslnkl
llnlund

Acudemlc dlssertutlon
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1o be presented for publlc exumlnutlon wlth the permlsslon of the luculty of
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zcc ut z noon.

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Supervisors Professor Clll vupuluhtl
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Locent Eell Pllpurlnen
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Reviewers Locent Mlku Sulmlnen
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Luropeun Centre for Llseuse Preventlon und Control
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Nutlonul lnstltute for Eeulth und velfure
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Official opponent Professor Robert Lunclottl
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Centers for Llseuse Control und Preventlon
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Nornng n bology makes sense
excer n rne lgnr oj evoluron.
1heodoslus Lobzhunsky

Contents
LIS1 OI ORICINAL PU8LICA1IONS..............................................................................................................
A88RLvIA1IONS............................................................................................................................................6
A8S1RAC1....................................................................................................................................................... )
. RLvILW OI 1BL LI1LRA1URL ...................................................................................................................
. PRLlACL...................................................................................................................................................
.z lN1RCLUC1lCN 1C lLAvlvlRUSLS...............................................................................................................
.z.. 1axonomc classjcaron oj rne cenus |lavvrus..................................................................... o
.z.z |lavvruses as numan arnogens ...............................................................................................z
. LLNCUL vlRUS ........................................................................................................................................
.. Srrucrure and relcaron ............................................................................................................
..z. 1ransmsson .............................................................................................................................. zo
.. cenerc dversry and evoluron................................................................................................. zz
..| Ldemology .............................................................................................................................. z6
.. engue dsease ........................................................................................................................... z8
..6 engue n rravelers ....................................................................................................................
..; larnogeness ............................................................................................................................... |
..8 Laborarory dagnosrcs .............................................................................................................. ;
..p lrevenron................................................................................................................................... |
z. AIMS OI 1BL S1UDY ............................................................................................................................... q)
. MA1LRIALS AND ML1BODS ..................................................................................................................q
. S1ULY MA1LRlALS..................................................................................................................................
.. larenr samles ........................................................................................................................... |p
..z vruses ......................................................................................................................................... |p
.. cell lnes ....................................................................................................................................... o
..| Vonoclonal anrbodes............................................................................................................... o
.z ML1ECLS...............................................................................................................................................
.z. vrus solaron n cell culrure.......................................................................................................
.z.z NA exrracron and 1lc ........................................................................................................
.z. Sequencng and sequence analyss ...........................................................................................
.z.| l|A................................................................................................................................................ |
.z. LlA................................................................................................................................................ |
.z.6 lmmunonsrocnemsrry .............................................................................................................
.z.; Neurralzaron resr .....................................................................................................................
q. RLSUL1S AND DISCUSSION ................................................................................................................... 6
. LlACNCSlS Cl LLNCUL vlRUS lNlLC1lCN lN 1RAvLLLRS ............................................................................6
|.. vrologcal examnaron oj a jaral dengue case (l) ................................................................... ;
|..z evelomenr oj a new realrme 1lc mernod jor derecron oj LNv NA (ll) ................6o
|.. comarson oj rne early dagnosrc mernods (ll)..................................................................... 6|
.z. MCLLCULAR LPlLLMlCLCCY Cl LLNv S1RAlNS .................................................................................... 68
|.z. A global collecron oj LNv jrom |nnsn rravelers (lll)...........................................................68
|.z.z A collecron oj LNvz srrans jrom an endemc counrry,....................................................... ;z
venezuela (lv) ..................................................................................................................................... ;z
CONCLUDINC RLMARkS AND IU1URL PROSPLC1S .............................................................................. )6
ACkNOWLLDCLMLN1S .............................................................................................................................. )8
RLILRLNCLS.................................................................................................................................................8o
ORICINAL PU8LICA1IONS ..........................................................................................................................6

List of original publications

1hls thesls ls bused on the followlng orlglnul publlcutlons, whlch ure referred to ln
the text by thelr Romun numeruls.

I. Euhtumo L, vuorlnen S, Uzctegul NY, vupuluhtl C, Euupusulo E, Lumlo '
(zcc6). lutul dengue vlrus lnfectlon ln u llnnlsh truveler.
'ournul of Cllnlcul vlrology. Lec,,():z6.

II. Euhtumo L, Eusu L, Uzctegul NY, Lrru L, Nlkkurl S, luntele A, vupuluhtl C,
Pllpurlnen E (zcc). Lurly dlugnosls of dengue ln truvelers: Compurlson of u novel
reultlme R1PCR, NS untlgen detectlon und serology.
'ournul of Cllnlcul vlrology. 'un,,():.

III. Euhtumo L, Uzctegul NY, Sllkumkl E, Suurlnen A, Pllpurlnen E, vuherl A,
vupuluhtl C (zcc8). Moleculur epldemlology of dengue vlrus strulns from llnnlsh
truvelers. Lmerglng lnfectlous Llseuses. 'un,():8c.

Iv. Euhtumo L, Comuch C, Slerru C, Cumucho LL, Agulrre C, vupuluhtl C,
Uzctegul NY. Mulntenunce und genetlc dlverslflcutlon of the Amerlcun-Aslun
genotype of dengue vlrus type z ln venezuelu. Vanuscrr.

1he orlglnul urtlcles ure reprlnted wlth the permlsslon of thelr copyrlght holders.

6
Abbreviations
Arbovlrus urthropodborne vlrus
A1CC Amerlcun type culture collectlon
3EQ bluck hole quencher
3LAS1 buslc locul ullgnment tool
cLNA complementury LNA
CPL cytoputhlc effect
ct cycle threshold
LLNv dengue vlrus
Ll dengue fever
LEl dengue huemorrhuglc fever
LSS dengue shock syndrome
dsLNA doublestrunded LNA
LlA enzyme lmmunoussuy
lAM curboxyfluoresceln
ll1C fluoresceln lsothlocyunute
llA lmmunofluorescence ussuy
lgC lmmunoglobulln C
lgM lmmunoglobulln M
kLu kllodulton
MAb monoclonul untlbody
MLM mlnlmul essentlul medlum
NS nonstructurul proteln
CRl open reudlng frume
P3S phosphute buffered sullne
PCR polymeruse chuln reuctlon
preM premembrune proteln
PRN1 pluque reductlon neutrullzutlon test
RPMl Roswell Purk Memorlul lnstltute medlum
R1 reverse trunscrlptlon
Pfu pluque formlng unlt

,
Abstract

Lengue ls u mosqultoborne vlrul dlseuse cuused by the four dengue vlrus
serotypes (LLNv) und ls currently consldered us the most lmportunt
urthropodborne vlrul dlseuse ln the world. Neurly hulf of the humun populutlon
llves ln rlsk ureus, und ccc mllllon lnfectlons occur yeurly uccordlng to the
estlmutes by the vorld Eeulth Crgunlzutlon. 1he dlseuse cun vury from u mlld
febrlle dlseuse to severe huemorrhuglc fever und shock. A secondury lnfectlon
wlth heterologous serotype lncreuses the rlsk for severe dlseuse outcome. Lurlng
the lust three decudes the lmpuct of dengue hus drumutlcully lncreused ln the
endemlc ureus lncludlng the troplcs und subtroplcs of the world. 1he current
sltuutlon wlth musslve epldemlcs of severe dlseuse forms ln urbun envlronments
hus been ussocluted wlth socloecologlcul chunges thut huve lncreused the
trunsmlsslon und enubled the coclrculutlon of dlfferent serotypes. Consequently,
un lncreuse of dengue hus ulso been observed ln truvelers vlsltlng these ureus.
ln truvelers dengue ls rurely u llfe threutenlng dlseuse, however dlseuse
outcomes consldered utyplcul huve been reported. ln the current study, u futullty
cuused by u bruln hemorrhuge wus documented ln u young llnnlsh putlent
experlenclng u prolonged prlmury dengue lnfectlon. 1he lncreuslng lmportunce of
dengue ulso trlggered other studles thut ure complled ln thls thesls. 1he ulms
were to provlde lnformutlon of dengue vlrus strulns through vlrus
churucterlzutlon und moleculur epldemlologlcul studles of dengue vlrus lsolutes
from llnnlsh truvelers und from putlents from un endemlc country, venezuelu,
und to develop u novel dlugnostlc tool for the detectlon of the LLNv genome
from putlent sumples. 1o uccompllsh these objectlves clusslc vlrologlcul methods
lncludlng vlrus lsolutlon und dlfferent untlbodybused ussuys were used, ln
uddltlon to untlgen detectlon, R1PCRbused methods, sequenclng und sequence
unulysls.
lor lmprovlng the luborutory dlugnostlcs of dengue from eurly tlmepolnt
putlent sumples, u novel reultlme R1PCR method wus developed for the
detectlon of LLNv RNA bused on 1uqMun chemlstry. 1he method wus shown

8
to be sensltlve und speclflc for detectlng LLNv RNA und sultuble for dlugnostlc
use. 1he newly developed reultlme R1PCR wus compured to other uvulluble eurly
dlugnostlc methods lncludlng lgM und NS untlgen detectlon uslng u punel of
selected putlent sumples. 1he results suggest thut the best dlugnostlc rutes ure
uchleved by u comblnutlon of lgM wlth RNA or NS detectlon.
1he dengue vlrus strulns studled here lncluded LLNv strulns lsoluted
from serum sumples of llnnlsh truvelers collected ln zccczcc. 1he results of
sequence unulysls demonstruted thut the lsolutes presented u globul sumple of
LLNv strulns from dlfferent ureus lncludlng Aslu, Afrlcu und South Amerlcu. 1he
strulns lncluded ull four dengue vlrus serotypes und severul genotypes, whlch
munlfested us dengue fever ln llnnlsh truvelers. ln the present study sequence
unulysls wus ulso currled out for u collectlon of z novel LLNvz lsolutes from
venezuelun putlents ln zcc. A globul sumple of dlfferent LLNvz genotypes
und ull the uvulluble LLNvz sequences from venezuelu untll zcc8 were lncluded
ln the unulysls. 1he results showed thut unllke the puttern of severul other
countrles ln the reglon, venezuelun LLNvz excluslvely represented the Amerlcun
Aslun genotype, suggestlng thut no forelgn LLNvz llneuges huve recently been
lntroduced to the country. 1he results suggest thut the LLNvz vlruses huve been
mulntulned ln the ureu where they huve evolved lnto severul phylogenetlc groups
wlth churucterlstlc umlnoucld chunges ln thelr envelope genes.
Studylng the churucterlstlcs of the clrculutlng dengue vlruses ln the
endemlc ureus ls lmportunt for survelllunce of clrculutlng strulns und thelr
lnvolvement ln locul epldemlcs. 1he uctlve survelllunce ls not currently performed
ln muny countrles mulnly becuuse of the luck of flnunclul resources. lnfected
people ure pluylng u mujor role ln lntroduclng dengue vlrus to novel ureus, but
ulso provlde u wuy to study these vlruses from endemlc ureus. 1o our knowledge,
the LLNvz lsolute from u llnnlsh truveler returnlng from Chunu, Afrlcu, wus the
flrst documentutlon of dengue ln the country, demonstrutlng the potentlul of
truvelers us sentlnels for dengue ln the ureus lucklng survelllunce. 1he uptodute
lnformutlon of clrculutlng LLNv strulns comblned to the uvulluble epldemlologlcul
lnformutlon ulds ln better understundlng of the nuture of the epldemlcs ln the
endemlc ureus.
. evew oj rne lrerarure

. Review of the literature

. Prejoce

vlruses ure extremely smull blologlcul ugents thut shure severul propertles
wlth cellulur llfe. vlruses reproduce und huve genomes thut undergo evolutlon
enubllng them to udupt to chunges ln thelr envlronment. vlruses ure obllgutory
lntrucellulur purusltes thut use the host cell muchlnerles, components und energy
for productlon of progeny vlrlons. vlruses huve coevolved closely wlth thelr host
orgunlsms, und ull the three domulns of llfe, Lukuryu, Prokuryu und Archueu ure
purusltlzed by thelr speclflc vlruses ().

.z lntroducton to jlovvruses

Lengue vlrus belongs to the |lavvrdae fumlly, genus |lavvrus. 1he vlrus
fumlly |lavvrdae conslsts of smull (c6c nm ln dlumeter) enveloped unlmul
vlruses thut huve u genome of u slngle molecule of posltlve sense RNA. 1he fumlly
ls dlvlded lnto three generu: neacvrus, lesrvrus und |lavvrus. Cnly the genus
|lavvrus lncludes urthropodborne members (urbovlruses) (z). Arbovlruses
demonstrute blologlcul flexlblllty thut enubles them to lnfect und repllcute ln
dlfferent host specles, ln urthropod und vertebrute cells (). 1hls strutegy hus
been udvuntugeous by ensurlng effectlve trunsmlsslon, spreudlng, survlvul und
mulntenunce of urbovlruses ln nuture.
c

.z.. 1axonomic classification of the Cenus Ilavivirus

1he nume of the genus |lavvrus (referred to us fluvlvlruses) comes from
jlavus, the Lutln word for yellow. 1he type specles of the genus ls Yellow fever
vlrus und currently neurly ,c fluvlvlrus specles ure known. lluvlvlruses cun be
cutegorlzed bused on thelr vector ussoclutlons, vlruses trunsmltted by mosqultoes
( specles), vlruses trunsmltted by tlcks ( specles) und vlruses for whlch no
urthropod vector hus been ldentlfled, no known vector vlruses (6 specles) ().
ln uddltlon, severul tentutlve specles huve been ussocluted wlth the genus,
lncludlng genetlcully dlvergent llneuges lsoluted from buts, numed 1umunu but
vlrus (1A3v) () und from mosqulto cells, numed Cell fuslng Agent vlrus (ClAv)
(). lluvlvlruses cun be untlgenlcully clusslfled lnto 8 complexes thut correlute to
the dlfferences observed ln the llfe cycles und host runge (6). 1hese lnclude z
sepurute untlgenlc groups of tlckborne vlruses, ussocluted wlth rodent or seublrd
hosts. 1he clusters of mosqultoborne vlruses lnclude one untlgenlc group of
vlruses ussocluted wlth blrds und culex spp. mosqulto vectors und groups of
vlruses ussocluted wlth mummul hosts und Aedes spp. vectors. 1he noknown
vector vlruses huve been dlvlded lnto z untlgenlc groups, both lncludlng vlruses
lsoluted from buts und rodents (,). 1he host, vector und untlgenlc clusslflcutlons
ulso resemble the grouplng of these vlruses ln phylogenetlc trees (8) (llgure ).
Eowever, for some vlruses phylogenetlcully ussocluted wlth mosqultoborne
vlruses there ure no vectors currently known (Lntebbe but vlrus, Yokose vlrus)
und lt hus been hypotheslzed thut they huve lost thelr ublllty to be vector borne
().


llgure . Nelghborjolnlng phylogenetlc tree bused on complete open reudlng frume nucleotlde
sequences of fluvlvlrus specles lncludlng representutlves of mosqultoborne, tlckborne und no
known vector vlruses. *) Currently no vectors ure known for Lntebbe but und Yokose vlruses
desplte thelr phylogenetlc grouplng umong mosqultoborne fluvlvlruses. ccc bootstrup
repllcutlons were conducted, the support vulues ubove ,c ure lndlcuted. 3ur represents
nucleotlde substltutlonsjslte. vlrus specles: 'upunese encephulltls vlrus ('Lv), Usutu vlrus (USUv),
Alfuy vlrus (ALlv), vest Nlle vlrus (vNv), St Louls encephulltls vlrus (SLLv), Roclo vlrus (RCCv),
3uguzu vlrus (3ACv), lokoberu vlrus (lClv), 3ussuquuru vlrus (3SQv), lguupe vlrus (lCUv),
ledougou vlrus (lLLv), 2lku vlrus (2llv), dengue vlrus (LLNv), Lntebbe but vlrus (LN1v), Yokose
vlrus (YClv), Seplk vlrus (SLPv), yellow fever vlrus (Ylv), Rlo 3ruvo vlrus (R3v), Montunu myotls
leukoencephulltls vlrus (MMLv), Modoc vlrus (MCLv), Apol vlrus (APClv), Meubun vlrus (MLAv),
1yulenly vlrus (1YUv), lyusunur forest dlseuse vlrus (llLv), Lungut vlrus (LC1v), Louplng lll vlrus
(Llv), 1lckborne encephulltls vlrus (13Lv) und Cell fuslng ugent vlrus (ClAv).
z

.z.z Ilaviviruses as human pathogens

ln fluvlvlrus lnfectlons, the cllnlcully lll putlents represent the tlp of the
lceberg us most lnfectlons leud to subcllnlcul lnfectlon or to mlld dlseuse wlth
unspeclflc flullke symptoms. 1he cllnlcul symptoms cuused by fluvlvlruses lnclude
fever und rush, whlch ure ulso ussocluted wlth other urbovlrul dlseuses. Currently,
ut leust z specles of fluvlvlruses ure ussocluted wlth humun dlseuse (1uble )
(,z). 1he tlckborne fluvlvlruses ure known to cuuse both encephulltlc und
huemorrhuglc dlseuse. Eowever, for the mosqultoborne fluvlvlruses dlseuse
ussoclutlons seem to correlute wlth thelr vector mosqulto specles. 1he mosqulto
borne fluvlvlruses trunsmltted by culex spp. mosqultoes, such us vest Nlle vlrus,
ure generully ussocluted wlth the encephulltlc type of dlseuse whereus the Aedes
spp. trunsmltted vlruses lnclude cuusutlve ugents for febrlle dlseuses wlth
huemorrhuglc symptoms such us dengue und yellow fever vlruses (8).
ln terms of globul dlseuse burden the most lmportunt fluvlvlrul dlseuse ls
dengue, whlch ls cuused by four closely reluted mosqultoborne dengue vlruses
(LLNv) thut uccount for un estlmuted ccc mllllon lnfectlons euch yeur ln the
troplcs und subtroplcs of the world. 1he other lmportunt mosqultoborne
fluvlvlruses lnclude Yellow fever vlrus (Ylv) (endemlc ln South Amerlcu und
Afrlcu), cuuslng severe heputltls und huemorrhuglc dlseuse () und 'upunese
encephulltls vlrus ('Lv), the leudlng cuuse of vlrul encephulltls ln Aslu ().
Addltlonully, vest Nlle vlrus (vNv) ls u mosqultoborne fluvlvlrus thut hus spreud
from lts orlglns ln Afrlcu to Lurope (,6) und Aslu und recently ulso to the New
vorld (,,8) cuuslng febrlle or encephulltlc dlseuse (). Another fluvlvlrus reluted
to vNv, Usutu (USUv), hus ulso spreud from Afrlcu to Lurope (zc) ulthough there
ls llmlted lnformutlon on the puthogenlclty to humuns (z).
1lckborne encephulltls vlrus (13Lv) ls endemlc ln u lurge ureu spunnlng
from Lurope to Aslu, cuuslng encephulltlc dlseuse (zz). vucclnes ugulnst fluvlvlrus
lnfectlons ure currently uvulluble ugulnst Ylv, 'Lv und 13Lv (z). Putlents usuully
recover fully from fluvlvlrus lnfectlons wlth mlld symptoms, but permunent

purulysls, prolonged neurologlcul symptoms und perslstent lnfectlons huve ulso
been reported from lnfectlons cuused by 13Lv (z), vNv (z) und 'Lv (z).

1uble . lluvlvlruses cuuslng humun dlseuse, globully most lmportunt vlruses ure murked ln bold
(Modlfled from Could & Solomon, zcc8) (z).

Ilavivirus vector association Animal host
association
Ceographical
distribution
Buman disease
Alkhurmu 1lck Eumun, sheep,
cumels
Arublun Penlnsulu Euemorrhuglc fever
lyusunur lorest
Llseuse
1lck

Monkeys lndlu Euemorrhuglc fever
Lungut 1lck

Unknown Muluyslu, 1hullund,
Slberlu
Lncephulltls
Louplng lll 1lck Sheep, grouse,
hures
Ul, lrelund Lncephulltls
Cmsk huemorrhuglc
fever
1lck Muskruts,
rodents?
vestern Slberlu Euemorrhuglc fever
Powussun 1lck Smull mummuls Russlu, USA, Cunudu Lncephulltls
1ickborne
encephalitis
1lck Rodents Lurope, Aslu Lncephulltls
3uguzu Mosqulto (culex spp.) Unknown Afrlcu lever
3unzl Mosqulto (culex spp.) Unknown Afrlcu lever
3ussuquuru Mosqulto (culex spp.) Unknown 3ruzll lever
llheus Mosqulto (culex spp.?) 3lrds South und Centrul
Afrlcu
lever
1apanese
encephalitis
Mosqulto (culex trltu
enlorhynchus)
3lrds, plgs Aslu Lncephulltls
Murruy vulley
encephulltls
Mosqulto (culex
annulrosrrs)
3lrds Austrullu Lncephulltls
Roclo Mosqulto (culex spp.?) 3lrds 3ruzll Lncephulltls
St Louls encephulltls Mosqulto (culex spp.) 3lrds USA, South und
Centrul Amerlcu
Lncephulltls
West Nile Mosqulto (mulnly culex
spp.) und tlcks
3lrds, horses vorldwlde? Lncephulltls
Usutu vlrus Mosqulto (culex spp.) 3lrds Afrlcu, Centrul Lurope lever, rush

loutungo Unknown Rodents? Senegul lever, rush
Ntuyu Mosqulto Unknown Afrlcu, lever
Dengue q Mosqulto (Aedes spp.) Eumun, monkeys 1roplcs und
subtroplcs
lever, huemorrhuglc
fever
Yellow fever Mosqulto (Aedes spp.,
naemagogus spp.)
Eumun, monkeys Afrlcu, South Amerlcu Euemorrhuglc fever,
heputltls
Seplk Mosqulto Unknown New Culneu fever
2lku Mosqulto (Aedes spp.) Monkeys? Afrlcu, Aslu lever, rush
vesselsbron Mosqulto (Aedes spp.) Unknown Afrlcu, Aslu lever?
Spondwenl Mosqulto (Aedes
clrcumluteolus)
Unknown Afrlcu lever
Modoc Unknown Rodent? USA Lncephulltls
Lukur but Unknown 3uts? Afrlcu lever
Rlo 3ruvo Unknown 3uts USA, Mexlco lever
Apol Unknown Rodents? 'upun Lncephulltls


. Dengue vrus

A brief history of dengue

1he nume for dengue dlseuse comes from the Swuhlll lunguuge, meunlng u
dlseuse cuused by un evll splrlt. Although denguellke lllness wus reported from
Chlnu neurly ccc yeur ugo, the flrst mujor epldemlcs occurred ln the ,
th
century
ln dlfferent purts of the world. 1he globul shlpplng trude most llkely medluted the
lntroductlon und spreudlng of the prlnclpul vector of dengue, un Afrlcun orlgln
mosqulto Aedes aegyr, und ulso dengue vlruses. Lurlng the eurly ccs dengue
wus shown to be u mosqultoborne fllteruble ugent. Lengue vlrus strulns were
flrst lsoluted ln the cs, when reseurch focused on studylng the mujor cuuses of
lllness und morbldlty of the troops ln the Puclflc und Aslu (z6).

.. Structure and replication

Cenome

As for ull fluvlvlruses, the dengue vlrus genome ls u slngle RNA molecule of
posltlve polurlty (ssRNA). 1he genomlc RNA ls the only vlrul mRNA, contulnlng u
slngle open reudlng frume. 1he genomlc RNA us such ls lnfectlous und generutes
the productlon of vlrlons when trunsported lnto u sultuble host cell. 1he genome
ls upproxlmutely lb ln length und lt ls flunked by conserved untrunsluted
reglons (U1R) whlch form secondury structures thut medlute genome
clrculurlzutlon und huve lmportunt functlons ln genome repllcutlon (z,). 1he U1Rs
ure well conserved ln sequence und structure. 1he dengue vlrus ' U1R ls
upproxlmutely cc bp ln length und hus u type l cup (m,CpppAm) ut the ' end. lt
hus u zloop secondury structure where the lurger ' termlnul stemloop hurbors
the most conserved sequence reglons. 1hls loop ls sepuruted by u poly U
6
sequence from the smuller loop thut lncludes the AUC sturt codon. 1he ' U1R
follows the stop codon of NS gene und ls upprox 8c,c bp ln length, formlng
three domulns. Unllke the cellulur mRNAs, the fluvlvlrus genome does not huve u
poly A tull ut the ' end. 1he genome cycllzutlon through lnteructlons of ' und '
ends ure requlred for repllcutlon lnvolvlng severul elements of the genomlc RNA
(z8).

Structure

1he fluvlvlrus vlrlon conslsts of host cellderlved llpld blluyer, three vlrul
structurul protelns, cupsld, membrune und envelope proteln, und the RNA
genome (llgure z). 1he genome ls pucked lnto un lcosuhedrul cupsld composed of
vlrul cupsld proteln (C), whlch ls u smull buslc dlmerformlng proteln ulso found to
colocullze to the nucleus ln lnfected cells (z). ln lmmuture vlrlons found lnslde
the lnfected cells, the envelope glycoproteln (L) ls urrunged ln trlmers thut muke
the lmmuture vlrlon surfuce to huve u splky uppeurunce. ln thls form, the
precursor of the membrune proteln (preM) prevents the premuture fusogenlc
uctlvlty of the envelope proteln. Lurlng the vlrlon muturutlon, the prepurt ls
cleuved from M by furln, und the envelope proteln ls reurrunged from trlmers to
heudto tull dlmers thut lle flut ugulnst the membrune muklng the muture vlrlon
surfuce uppeur smooth (c,). ln fluvlvlrus lnfectlons, the muln untlbody
responses ure formed ugulnst the structurul protelns L und preM, und ln uddltlon
to these, the 1cell responses ure ulso developed ugulnst Cproteln (z).

,

virus encoded nonstructural proteins

ln fluvlvlrus lnfected cells, ln uddltlon to the structurul protelns, seven vlrus
encoded nonstructurul (NS) protelns cun be detected (NS, NSzA, NSz3, NS,
NSA, NS3 und NS). 1he functlons for ull the lndlvlduul NSprotelns ure not well
churucterlzed, however, they ure known to medlute the RNA repllcutlon und vlrul
polyproteln processlng () (llgure ). 1he NS proteln ls u multlmerformlng
glycoproteln, whlch ls secreted from lnfected cells. ln dengue vlrus lnfectlon,
putlents huve meusuruble levels of NS proteln ln the blood, whlch cun be utlllzed
us u dlugnostlc murker of the lnfectlon. NS proteln hus been shown to lnteruct
wlth the host lmmune system, but lt ulso hus functlons ln repllcutlon. 1he NSzA
und NSz3 ure smull membruneussocluted protelns, NSz3 functlonlng us u
cofuctor for the multlfunctlonul NS proteln, whlch hus trypslnllke serlne
proteuse und hellcuse uctlvltles. 1he structure of NS ls churucterlzed ln detull (
,). 1he NSA und NS3 ure smull membrune ussocluted protelns, NSA hus been
ussocluted wlth membrune ulterutlons (8) und NS3 hus been ussocluted wlth
membrune structures lnvolved ln the repllcutlon. 1he NS proteln ls the vlrul RNA
dependent RNA polymeruse medlutlng the genome repllcutlon. Also for NS, the
structure ls known (). Cf the nonstructurul protelns, NS ls known to rulse
untlbodyresponses. lt ls ulso known to evoke 1cell responses, ln uddltlon to NS,
NS3 und NS (z).
8

Life cycle in a cell

Currently, the host cell receptors und posslble coreceptors of dengue
vlruses ure not well churucterlzed, however, severul potentlul receptors or
components of the dengue receptor complex huve been ldentlfled lncludlng the
lumlnln receptor (z,), munnose receptor (), dendrltlccell receptor () ln
humun cells und heut shock protelns ln mosqulto cells (6).
Lengue vlrus blnds to the cellulur receptor vlu Lproteln, whlch medlutes
the receptormedluted uptuke of the vlrlon to the host cell by endocytosls. After
the lnternullzutlon the low pE of the endosome trlggers u conformutlonul chunge
ln the Lproteln, whlch reveuls u fuslon peptlde medlutlng the fuslon between the
vlrlon und endosome membrunes (,). As u result, the cupsld ls releused from
the vlrul envelope lnto the cell. 1he cupsld dlssoclutes und releuses the vlrul
genome lnto the host cell cytoplusm where lt ls trunsluted us u slngle lurge
polyproteln (llgure ). 1he polyproteln ls turgeted to the endoplusmlc retlculum
(LR) where lt ls orlented by lts slgnul sequences und membrune unchor domulns,
leuvlng lt purtly on the cytosollc und purtly to the LR lumenul slde. 1he processlng
by vlrus und host encoded proteuses leuds to the formutlon of lndlvlduul vlrul
protelns: three structurul protelns, cupsld (C), u precursor for the membrune
proteln (preM) und envelope proteln (L), und the seven nonstructurul protelns
thut huve functlons medlutlng the repllcutlon, polyproteln processlng und vlrlon
ussembly (c).
1he repllcutlon tukes pluce ln vlruslnduced veslculur membrune structures
ussocluted wlth the LR. 1he vlrul sense RNA genome ls repllcuted through u
negutlvesense lntermedlute, whlch ls ln turn used us u templute ln muklng more
genomlc posltlve strunds. 1hrough un unknown mechunlsm, the genomes ure
pucked lnto cupslds. 1he vlrlons bud lnto the LR lumen us lmmuture forms thut
huve preM und L protelns ln the surfuce (). 1he lmmuture vlrlons ure
trunsported through the trunsColgl network (z) where they ure mutured by u
cleuvuge of prjM creutlng lnfectlous, muture vlrlons thut ure trunsported out of
the cell by exocytosls (z).

llgure z. vlrlon structure. Arrungement of envelope protelns ln lmmuture vlrlon (trlmers) und ln
muture vlrlon (dlmers).

llgure . lluvlvlrus genome und polyproteln processlng. Modlfled from 3urtenschluger & Mlller
zcc8 (c).
zc
..z. 1ransmission

vector mosquitoes

All the known vectors of LLNv ure mosqultoes belonglng to Aedes genus.
1he specles known to huve the ublllty to become lnfected by LLNv, to repllcute lt
und trunsmlt lt lnclude Ae. aegyr, Ae. albocrus und Ae. olynenss of the
subgenus Sregomya. 1hese specles ure ut leust purtlully domestlc und
unthropophlllc. 1he llfe cycle of u mosqulto lncludes four sepurute stuges: egg,
lurvu, pupu und udult (llgure ), the flrst three stuges requlrlng un uqueous
envlronment. 1he durutlon of the developmentul stuges depend on the
envlronment's temperuture und uvullublllty of food ut the lurvul stuge. lor Ae.
aegyr lt tukes roughly 8c duys ut room temperuture to reuch the udult stuge
(z6).

llgure . Mosqulto llfe cycle.

z

Dengue virus transmission cycles in the wild and urban environments

1wo trunsmlsslon cycles ure known for dengue vlruses, one of them
lnvolvlng nonhumun prlmutes (monkeys) und jungle mosqultoes, referred to us
the sylvutlc cycle, und the second belng the humun to humun epldemlc
trunsmlsslon cycle occurrlng ln urbun envlronments (llgure .). 1he llfe cycle of
LLNv lnvolves u repllcutlon step ln both mosqulto und prlmute hosts. lollowlng
un lnfectlous blood meul from u vlremlc host, ufter u perlod of 8z duys the
mosqulto cun lnfect new prlmute hosts. ln both cycles, from un lnfected mosqulto
femule, the vlrus ls trunsmltted to the progeny mosqultoes through trunsovurlul
trunsmlsslon und posslbly ls ulso trunsmltted between mosqultoes sexuully (z6).
1he humunto humun trunsmlsslon presents u purtlculur uduptutlon of dengue
vlruses, us for most other urbovlruses humuns ure deud end hosts thut ure not
uble to produce sufflclent levels of vlremlu to lnfect new mosqultoes (z6). LLNv
cun be trunsmltted from humun to humun by lnfectlous blood of u vlremlc putlent
vlu needlestlck or mucocutuneous exposure (). ln uddltlon to LLNv, humun
to humun trunsmlsslon hus ulso been reported for Ylv und vNv (z).

llgure . Sylvutlc und urbun trunsmlsslon cycles of LLNv.
zz

.. Cenetic diversity and evolution

As for ull RNA vlruses, the LLNv RNAdependent RNA polymeruse mukes
errors durlng repllcutlon und creutes vurlublllty ln the vlrus genome.
Consequently, dengue vlruses exlst us populutlons of genetlc vurlunts, ulso culled
quuslspecles (6,,). Addltlonul vurlublllty ln LLNv genomes ls derlved from
recomblnutlon (8,). 1he relutlve proportlons of vurlunts ln vlrus populutlons
chunge over tlme due to the effects of genetlc drlft und selectlve pressures. 1he
selectlon turgets vlrus phenotype bused on lts lnteructlons wlth the envlronment.
1he phenotyplc propertles of LLNv uffectlng the host lnteructlons ln prlmutes und
ln mosqultoes, such us the lnfectlvlty, repllcutlon und trunsmlsslon efflclencles ure
cruclul. lt hus been reported thut most of the mututlons ln LLNv genomes ure
deleterlous thus hlghllghtlng the lmportunce of selectlon ln LLNv evolutlon
(6c,6).
Lengue vlruses ure hlghly udupted to thelr mosqultohosts und presumubly
huve thelr orlglns ln mosqultovlruses thut flrst udupted to sylvutlc llfe cycles
between jungle mosqultoes und nonhumun prlmutes. 3used on the phylogenetlc
unulysls, lt hus been suggested thut the LLNvs lnvolved ln humun epldemlcs
orlglnute from the sylvutlc LLNv strulns. As the sylvutlc trunsmlsslon cycles huve
been demonstruted for ull serotypes, lt ls consldered thut the four serotypes
huve emerged lndependently from thelr uncestrul sylvutlc progenltors. lrom
these sylvutlc cycles the jump to humuntohumun urbun cycle wus posslble vlu
mosqultoes feedlng ulso on humuns ln rurul ureus. 1he sylvutlc llfe cycles of LLNv
ure found only ln Afrlcu for LLNvz, whlle they ure found for ull LLNv types ln the
Muluy penlnsulu, suggestlng un Aslutlc orlgln for dengue vlruses (6z).
1he evolutlonury rutes reported for LLNvs ure upproxlmutely c
to c

substltutlonsjsltejyeur (668). 3used on these rutes, lt hus been estlmuted thut
dengue vlruses were sepuruted from thelr progenltors upproxlmutely ccc yeurs
ugo, und thut the zoonotlc trunsfer from the sylvutlc cycles to urbun epldemlc
cycles occurred between z und zc yeurs ugo (6). lt hus been estlmuted thut
z
the genetlc dlverslflcutlon of epldemlc strulns to the dlfferent genotypes occurred
wlthln the lust zcc yeurs, und colnclded wlth the beglnnlng of mujor humun
epldemlcs (6). 1he dlverslflcutlon wus llkely llnked to the vlrus uduptutlon to
humun to humun trunsmlsslon ln vurlous geogruphlcul reglons (6).
1he four dengue vlrus serotypes cun coclrculute ln the endemlc ureus
becuuse the lmmunlty to one serotype does not protect from the lnfectlon by u
heterologous serotype. 1hls ls llkely u result of selectlon thut wus drlven by the
restrlctlve effects of the crossprotectlve untlbodles rulsed ugulnst heterologous
serotypes. 1he dengue vlrus strulns, whlch were uble to escupe thls neutrullzutlon,
hud u slgnlflcunt competlng udvuntuge und becume the domlnunt llneuges. 1hls
evolutlonury uduptutlon not only enubled the coclrculutlon of the four serotypes
but ulso hud u greut lnfluence to thelr puthogenlclty for humuns (6z).

variety of dengue viruses: genotypes within serotypes

1he four dengue vlrus types (LLNv) form u phylogenetlc group thut ls
more closely reluted to one unother thun to other fluvlvlruses, ulso formlng un
untlgenlc complex of thelr own. Lesplte the close relutlonshlps between the four
serotypes, they ure consldered sepurute fluvlvlrus specles bused on thelr untlgenlc
und genetlc dlfferences (). 3ecuuse of thelr untlgenlc dlfferences, the four LLNv
types ure ulso culled dengue vlrus serotypes. Lengue vlruses ure dlverse, the four
LLNv serotypes dlffer ln nucleotlde sequence upproxlmutely z . lrom the
four LLNv serotypes, LLNv uppeurs to be the most dlvergent, followed by
LLNvz, wlth LLNv und LLNv belng most closely reluted to one unother.
Most of the sequence dutu uvulluble on dengue vlruses ln publlc dutubuses
conslsts of purtlul genomlc sequences of envelope, NS und NS genes, however,
the numbers of complete genome sequences huve lncreused durlng the lust
yeurs. Lengue vlruses of one serotype cun be further sepuruted lnto severul
genotypes (or subtypes) bused on thelr grouplng ln phylogenetlc unulysls (llgure
6). ln phylogenetlc unulysls, the most wldely used genomlc reglon ls the envelope
gene, however, sequences from vurlous genes huve been used ln LLNv genotype
z
determlnutlon. 1he phylogenetlc unulysls ulso supports the blologlcul sepurutlon
of dengue vlruses bused on the dlfferences ln thelr trunsmlsslon cycles. 1he
dengue vlrus strulns thut ure orlglnutlng from trunsmlsslon cycles between non
humun prlmutes und jungle mosqultoes belong to the sylvutlc genotype und ure
sepurute from the epldemlc strulns ussocluted wlth urbun humunto humun
trunsmlsslon. lor the epldemlc LLNvs, severul dlfferent wuys und styles of
numberlng or numlng huve been proposed dependlng on the uuthor, resultlng ln
vurlous numes for u glven genotype.
1he muln genotypes of the four LLNv serotypes ure shown ln llgure 6.
vlthln serotype vlruses (LLNv), muln genotypes ure sepuruted lncludlng one
sylvutlc und two epldemlc genotypes thut were orlglnully descrlbed from South
Lust Aslu (genotype l) und lndoneslu (genotype ll) (6,,,c). 1he LLNvz vlruses
huve been sepuruted lnto 6 genotypes. 1he sylvutlc genotype lncludes strulns
from SouthLust Aslu und Afrlcu. 1he flve epldemlc genotypes lnclude one wlth u
globul dlstrlbutlon (Cosmopolltun genotype [lv|) und one genotype orlglnully
descrlbed from Centrul und South Amerlcu (Amerlcun genotype [lv|). Addltlonully,
two llneuges of Aslutlc orlgln ure sepuruted (Aslun genotypes l und ll) (,) und one
genotype from the Amerlcus genetlcully ussocluted wlth the Aslutlc vlruses, ls
referred to us the AmerlcunAslun genotype (lll), whlch hus been ussocluted wlth
severe dlseuse (,z).
LLNvvlruses huve been sepuruted lnto llneuges found orlglnully from
the lndlun subcontlnent (lll), 1hullund (ll), the Amerlcus (lv), S.L. Aslu (v) und one
llneuge present ln South Lust Aslu und the South Puclflc (l) (,). No sylvutlc strulns
of LLNv huve been lsoluted or sequenced, however, evldence for thelr
exlstence hus been obtulned by serologlcul studles (6z). 1he LLNv vlruses ure
found to represent sepurute genotypes, one of them ls sylvutlc (lll). 1he
epldemlc genotypes huve orlglnuted from 1hullund (ll), other purts of Aslu (l), the
Amerlcus und Afrlcu (v) und the South Puclflc reglon (lv) (6z).

z

llgure 6. Rudlul nelghborjolnlng phylogenetlc tree bused on envelope genes of 8z dengue vlruses
deplctlng the genotypes wlthln the four dengue vlrus serotypes. LLNv genotypes lv, LLNvz
genotypes lv und sylvutlc genotype, LLNv genotypes lv, LLNv genotypes lll und sylvutlc
genotype.

z6

..q Lpidemiology

Current pandemic

1he dlseuse cuused by the four dengue vlrus serotypes ls currently
consldered the most lmportunt mosqulto borne vlrul dlseuse ln the world,
estlmuted to cuuse ccc mllllon lnfectlons yeurly (,,,). 1he lncldence hus
lncreused cfold ln the lust c yeurs (,). Lengue cuuses severe publlc heulth
problems ln endemlc ureus uround the troplcs und subtroplcs of the world, und no
vucclnes or speclflc treutment currently exlst. Prlor to 6cs less thun c countrles
reported dengue outbreuks. Lengue ls now endemlc ln over cc countrles ln ull
the subtroplcs und troplcs of the world. 1hese lnclude countrles ln Afrlcu und the
Amerlcus, ln uddltlon to SouthLust Aslu und the vestern Puclflc, whlch ure the
most serlously uffected. lt hus been estlmuted thut upproxlmutely c of the
whole humun populutlon llve ln rlsk ureus (,,,) (llgure ,).
Prlor to the Second vorld vur, dengue wus known us u nonfutul febrlle
dlseuse thut cuused selfllmlted epldemlcs wlth long lntervuls. A drumutlc chunge
ln the epldemlology und dlseuse severlty wus observed slnce the Second vorld
vur, whlch wus followed by musslve epldemlcs of dengue huemorrhuglc fever.
1hls wus flrst observed ln Aslu where the post wur tlme wus churucterlzed by
economlcul boom und urbunlzutlon. Prlor to the 8cs, severe dengue wus u rure
dlseuse ln the Amerlcus where the observed slgnlflcunt expunslon of dengue und
lncreused dlseuse severlty wus cleurly ussocluted wlth the lntroductlon of multlple
serotypes (z6).
1he globul cllmute chunge hus been blumed for the emergence of dengue
(,8) und models of dengue emergence huve been mude bused on the cllmute
chunge estlmutes (,). lt ls not cleur how cllmute chunge wlll uffect dengue
trunsmlsslon even though cllmutlc fuctors huve been llnked to LLNv
epldemlology. Chunges cuused by the Ll Nlnophenomenon, such us the umount
z,
of rulnfull und temperuture, however, huve been shown to uffect dengue
trunsmlsslon through chunges ln vector populutlons und trunsmlsslon efflclency
(8c).

1he current understundlng of the fuctors uffectlng the current dengue pundemlc
sltuutlon (6z,,) ure llsted below:

. Adaptations in both virus and vector to support the urban DLNv life cycle
vector speclullzutlon to feed on humuns und to breed on urtlflclul uqueous
envlronments provlded ln the urbun settlngs
vlrus uduptutlon to the urbun vectors und humuns, uduptutlon of LLNv
serotypes to one unother: escupe of the crossneutrullzutlon by heterologous
untlbodles

z. Increased urbanization affecting vector and host densities
Eumun populutlon growth und concentrutlon to cltles
Chunges ln the envlronment: urbun envlronments, especlully slums provldlng
vector breedlng hubltuts

. 1ravel related geographic spreading of vectors and viruses
1ruvellng of lnfected humuns, trunsport of muterluls contulnlng lnfected
mosqultoeggs und lurvue
Clobul lnvuslon of Aedes aegyr und Aedes albocrus

z8

llgure ,. Areus ut rlsk of dengue trunsmlsslon ln red (Modlfled from vEC, zcc) (,,).

.. Dengue disease

Lengue vlrus lnfectlon cun be usymptomutlc or u febrlle dlseuse thut muy
develop further to severe dlseuse forms lnvolvlng huemorrhuglc symptoms. 1he
lncubutlon perlod from the lnfectlous mosqultoblte to onset of symptoms cun
runge from to duys (8).

Development of humoral immune response to dengue virus

1he dengue vlrus speclflc untlbodles produced durlng lnfectlon ure
consldered to provlde protectlon through vurlous mechunlsms lncludlng vlrus
neutrullzutlon, uctlvutlon of the complement system und untlbodydependent cell
medluted cytotoxlclty. 1he protectlve effects huve been demonstruted ln pusslve
lmmunlzutlon experlments, where udmlnlstered dengue vlrus untlserum
protected mlce from lethul lnfectlon (z6,8z). 1he neutrullzlng untlbodles
z
produced durlng dengue vlrus lnfectlon lnclude vlrus serotype speclflc untlbodles
und crossreuctlve untlbodles thut ure reuctlve ugulnst ull serotypes. After the
ucute phuse of the dlseuse the speclflclty of the neutrullzlng untlbodles lncreuses
over tlme. 1he protectlve lmmunlty ugulnst the homologous serotype ls llfelong,
whereus the crossprotectlve lmmunlty ugulnst the other serotypes ls short llved,
lustlng only for u few months (6z).
Lue to the luck of sufflclent crossprotectlve lmmunlty between the
dlfferent dengue vlrus serotypes, sequentlul or secondury dengue vlrus lnfectlons
wlth heterologous serotypes ure posslble. 1he secondury lnfectlons ure
ussocluted wlth elevuted rlsks of severe dlseuse outcomes. ln these lnfectlons the
preexlstlng heterologous, nonneutrullzlng untlbodles huve been shown to
purtlclpute to the puthogenesls by enhunclng the lnfectlon. 1ertlury und even
quuternury dengue lnfectlons huve been documented ln endemlc settlngs but
thelr ussoclutlon wlth dlseuse severlty ls currently not known (8).
lndlvlduul vurlutlon occurs ln untlbody responses to dengue vlrus.
Eowever, the prlmury und secondury lnfectlons ure dlstlngulshuble bused on thelr
untlbody responses. A prlmury lnfectlon ls churucterlzed by uppeurunce of lgM
untlbodles eurly durlng the ucute phuse, reuchlng meusuruble levels on duy
ufter onset of fever (llgure 8). 1he lgM peuks for upproxlmutely z weeks und
decllnes to undetectuble levels ufter z months. 1he levels of lgMcluss
untlbodles ure hlgher thun those of lgC untlbodles, whlch ln prlmury lnfectlons
uppeur ufter lgM (llgure 8).
Lurlng ucute febrlle und eurly convulescent phuse of prlmury lnfectlons,
lgC untlbody tlters ure low. vhereus lgM domlnutes the humorul lmmune
response ln prlmury lnfectlon, the secondury lnfectlon ls domlnuted by lgC. ln
compurlson to the prlmury lnfectlons, ln secondury lnfectlons the klnetlcs of lgM
ure more vurled, und the response ln most putlents ls deluyed, lowered or even
ubsent (8). ln contrust, the lgC untlbodles reuch qulckly hlgh tlters und ure
detectuble ulreudy ln the ucute phuse of secondury lnfectlon (z6, 8).

c

llgure 8. 1lmlng of dlugnostlc murkers of prlmury dengue lnfectlon ln putlent serum (modlfled
from Eulsteud, zcc,) (8).

Clinical manifestations of dengue virus infection

3used on symptoms ulone, lt ls not eusy to dlstlngulsh dengue from other
troplcul dlseuses, such us Chlkungunyu vlrus lnfectlon or mulurlu, und relluble
dlugnosls requlres luborutory tests (8). 1he dengue dlseuse ls cutegorlzed
uccordlng to dlseuse severlty by the vorld Eeulth Crgunlzutlon lnto dengue fever
(Ll), dengue huemorrhuglc fever (LEl) und dengue shock syndrome (LSS) (8).
Eowever, the symptoms of dengue dlseuse cun vury greutly from one putlent to
unother.


Dengue fever (DI)

Lengue fever ls churucterlzed wlth sudden onset of fever, however the
spectrums of the symptoms vury dependlng on the putlent.
Llfferent types of uches und pulns ure common, often the heuduche ls retro
orbltul und ls uccompunled wlth e.g. rush, myulglu, loss of uppetlte, nuuseu,
vomltlng und ubdomlnul puln. Addltlonully symptoms muy lnclude chunges ln
tuste metulllc tuste und flushlng of the fuce.
ln uddltlon to fever, the cllnlcul deflnltlon of Ll lncludes two or more of
the followlng symptoms: heuduche, retroorbltul puln, muscle or jolnt puln, rush,
huemorrhuglc munlfestutlon or leucopenlu (8). 1he mlld huemorrhuglc
symptoms of skln, such us petechlue muy be observed. Addltlonully, other
symptoms lnclude spontuneous bleedlng, such us gum bleedlng, lncreused
menorrhuglc bleedlng, gustrolntestlnul bleedlng und hemuturlu.

Dengue haemorrhagic fever (DBI)

Approxlmutely of Ll develops lnto LEl, whlch ls dlfferentluted from Ll
by the lncreuse of vusculur permeublllty. 1hls ls seen ln huemutologlcul studles us
thrombocytopenlu, elevuted huemutocrlt und hypoprotelnuemlu. 1he cllnlcul
munlfestutlons lnclude hlgh fever (up to >C), whlch cun be blphuslc,
huemorrhuges, thrombocytopenlu und hemoconcentrutlon, heputomeguly und
slgns of clrculutory fullure. Accordlng to vEC, LEl lncludes fever,
thrombocytopenlu und elevuted huemutocrlt.

Dengue shock syndrome (DSS)

3oth LEl und LSS lnclude vusculur chunges, thrombocytopenlu und
cougulutlon dlsorders. ln uddltlon to the symptoms descrlbed for LEl, LSS
lnclude nurrow pulse pressure or hypotenslon. Approxlmutely one thlrd of LEl
z
putlents develop shock. Lengue fever or dengue huemorrhuglc fever cun develop
lnto u more severe condltlon usuully ufter , duys of the dlseuse, when the fever
dlsuppeurs. 3efore the onset of shock, putlents huve severe ubdomlnul puln. 1he
typlcul slgns of u clrculutory fullure cun be seen us skln becomlng cool und pulse
becomlng rupld und nurrow. 1he putlents ln shock ure ln dunger, und plusmu
volumereplucement therupy ls needed to prevent deuth. 1he deuth rutes of the
severe forms of dengue dlseuse, LEl und LSS, cun be slgnlflcuntly reduced by
correctly tlmed supportlve treutment. Eowever, wlthout treutment the LEl
futullty rutes cun be us hlgh us zc (,) und reuch ln LSS (86).

1reatment

Currently, no speclflc medlcutlon ls uvulluble for treutment of dengue.
Eowever, some medlclnes lncludlng cortlcosterolds (8,) huve been suggested to
uld dengue putlents wlth severe symptoms. ln selftreutment of Ll, restlng und
preventlon of dehydrutlon by drlnklng enough wuter ls recommended.
Purucetumol ls preferred us untlpyretlc. Asplrln (Acetylsullcyllc ucld) should be
uvolded due to the untlcougulunt propertles. 1he muln hospltulcure treutment of
LEl und LSS putlents ls the retulnlng of the lntruvenous volume by correctly
tlmed lntruvenous fluld replucement. lrequent monltorlng of plutelet und
hemutocrlt of LEljLSS putlents ls essentlul for tlmlng the treutment correctly.
Severul plusmu volume replucement solutlons huve been used, lncludlng plusmu
expunders und electrolyte solutlons. ln severe cuses, blood trunsfuslons huve ulso
been used us treutment (,6).

..6 Dengue in travelers

Slmllur to the populutlons ln endemlc ureus, most of the truvelers lnfected
wlth LLNv ure usymptomutlc. 3used on serologlcul surveys, untlbodles huve been
reported ln z.8 of truvelers returnlng from endemlc ureus (,6). 1he tlmlng of
the dlseuse onset und the known lncubutlon perlod from the lnfectlous mosqulto
blte to the onset of symptoms cun be used ln excludlng dengue ln truvelers. 1he
dlseuse ls not llkely to be dengue lf the symptoms sturt luter thun z weeks ufter
returnlng from the endemlc ureu.
ln muny Luropeun countrles lmported dengue ls not reported, however,
currently, the 1ropNetLurop (Luropeun Network on lmported lnfectlous Llseuse
Survelllunce) frumework ls collectlng the dutu on dengue ln Luropeun truvelers. A
cleur puttern of seusonullty of dengue ln Luropeun truvelers hus been observed,
followlng the mlgrutory hublts to wurmer cllmutes durlng wlnter (88). ln some
prevlous studles, mulurlu, gustroenterltls und fever of unknown etlology preceded
the number of dengue dlugnoses ln truvelers (8,c). Eowever, u globul network
of truvel und troplcul medlclne cllnlcs, the CeoSentlnel Survelllunce Network,
reported dengue us u more frequent lnfectlon thun mulurlu ln truvelers returnlng
from troplcs outslde Afrlcu ().
1he trend ln u globul scule resembles the sltuutlon observed ln Luropeun
studles, the truvelers obtulnlng the lnfectlon most frequently ln Aslu. ln contrury
to the sltuutlon ln Aslu, where dengue ls prlmurlly u dlseuse of young chlldren, ln
truvelers lt ls mulnly u dlseuse of young udults who ure uctlve ln truvellng (88,
).

..) Pathogenesis

Cell tropism

Lurlng the eurly phuse of the dlseuse, dengue vlrus cun be detected from
perlpherul blood for u relutlvely short perlod of tlme, the levels llkely to be
correspondlng to the quuntlty of the lnfected tlssues. 3used on uutopsy und n
vvo studles of LLNv lnfected humuns, the cell troplsm of LLNv lncludes cells of
the lmmune system, llver und endothellul cells of blood vessels. After belng
lnjected lnto the humun bloodstreum by u mosqulto, dengue vlrus hus been
shown to lnfect muny cell types. 1he dendrltlc cells of the epldermls (skln
Lungerhuns cells) () und kerutlnocytes () ure posslble flrst turgets. vhen the
vlrus reuches the lymph nodes, monocytes und mucrophuges ure ulso lnfected,
und become the mujor turgets of the lnfectlon dlssemlnutlng the vlrus through
the lymphutlc system to cells of mononucleur llneuge lncludlng monocytes,
myelold dendrltlc cells und mucrophuges ln llver und spleen (6).

Current knowledge of the pathogenic mechanisms of dengue

Severul vlrus und host speclflc fuctors huve been ussocluted wlth dlseuse
severlty und puthogenesls of dengue dlseuse. 1he study of dengue puthogenesls
currently lucks u good unlmul model, us slmllur cllnlcul munlfestutlons mlmlcklng
severe dengue ln humuns ure not seen ln other unlmuls. 1he current unlmul
models lnclude murlne models (,,8) generuted by experlmentul lnfectlon wlth
LLNv, whlch often develop u neurologlcul lnfectlon seen only rurely ln lnfected
humuns (z6). 1he nonhumun prlmutes experlmentully lnfected wlth LLNv
develop vlremlu, but the repllcutlon ls not us effectlve us ln humuns, resultlng ln
lower levels of vlremlu. Eowever, lf u hlgh dose of vlrus ls used, subcutunlc
huemorrhuglc munlfestutlons develop ulso ln Rhesus mucuques ().

Severul hypotheses und lnvolvement of vurlous fuctors huve been
proposed to expluln the puthogenesls of dengue, however, the necessury or
sufflclent fuctors ure not known und the knowledge of puthogenesls of dengue ln
humuns ls mulnly bused on the studled futul cuses. 1he fuctors ussocluted wlth
severe dlseuse outcomes lnclude host genetlc fuctors, uge, lmmunologlcul stutus,
chronlc dlseuses, und vlrus struln propertles (cc). 1he observed dlfferences ln
dlseuse putterns ln dlfferent reglons suggest lnvolvement of vurlous fuctors, us ln
SouthLust Aslu, LEl ls mulnly u dlseuse uffectlng chlldren, whlle lt uffects mostly
udults ln the Amerlcus (c). lt hus been suggested thut dlfferent LLNv serotypes
und llneuges muy huve dlfferences ln thelr blologlcul propertles, whlch cun be
llnked to the puthogenlclty (cz). Eowever, those ure not llkely to expluln the
observed puthogenlclty completely us ull LLNv types ure uble to cuuse LEljLSS
(cc).
As the vlrul loud ln humuns corresponds to the dlseuse severlty (c,c),
the lnublllty of lmmunologlcul responses to control the repllcutlon of LLNv und
the effects of the lnfected cells on other cells ln the body ure llkely to pluy u mujor
role. 1he hlgh vlrul loud ls consldered to result ln upoptosls und necrosls ln vurlous
tlssues, und trlgger the development of lmbulunce ln the proflles of soluble
medlutors und cytoklnes resultlng ln endothellul cell dysfunctlon und blood
cougulutlon ubnormulltles observed ln LEl und LSS (6).
1he lnvolvement of lmmune enhuncement ln dengue dlseuse wus
suspected bused on epldemlologlcul studles ussoclutlng LEl to secondury
lnfectlons (c). Accordlng to the untlbodyenhuncement (ALL) theory, the cross
reuctlve untlbodles, whlch cun be muternul untlbodles ln young chlldren or
untlbodles from un eurller lnfectlon wlth heterologous LLNv serotype, blnd to the
lnfectloncuuslng vlrus but ure unuble to neutrullze lt. 1hese untlbodyvlrus
complexes enhunce the lnfectlon of monocytemucrophuges through blndlng to
thelr lcreceptors. ln vrro studles showed thut, lndeed, the presence of cross
reuctlve nonneutrullzlng untlbodles lncreused LLNv lnfectlon ln cells of
mucrophuge llneuge (c6). Accordlng to ALL theory, the enhunced lnfectlon leuds
to the uctlvutlon of u chuln of reuctlons leudlng to lmmunoputhology medluted by
1cell uctlvutlon, lnterferon, complement und plutelet uctlvutlon, cytoklnes und
6
ultered functlons of endothellul und eplthellul cells. 1he enhunced lnfectlon rute ls
hypotheslzed to cuuse hlgher vlremlu ln putlents wlth secondury lnfectlons, whlch
eventuully leuds to plusmu leukuge. Eowever, the onset of plusmu leukuge occurs
ufter the peuk vlremlu, whlch suggests un lmmune-medluted mechunlsm. 1he
enhuncement wus thought to be cuused by crossreuctlve untlbodles known to
exlst ugulnst Lproteln (c,), however, ln u recent study they were shown to be
mulnly turgeted ugulnst preM proteln of LLNv (c8). vhereus the ALL theory
emphuslzes the lmportunce of the preexlstlng untlbody ln the puthogenesls of
dengue, lt does not expluln LEl ln prlmury lnfectlons.
1he denguevlrus speclflc 1 cell responses huve ulso been ussocluted wlth
severe dlseuse, und huve been suggested to purtlclpute ln cleurlng the lnfectlon
und ln lmmunoputhogenesls (6z). lrom putlents wlth secondury lnfectlons, the
recovered uctlvuted 1cells were found to show low ufflnlty to the lnfectlve
serotype. lnsteud, they hud hlgh ufflnlty to the heterologous serotype,
presumubly representlng the one encountered prevlously (c).
Moleculur mlmlcry hus ulso been proposed to pluy u role ln dengue
puthogenesls by trlggerlng un uutolmmunetype of response. ln the LLNv
envelope proteln, u zcumlno ucld resldue ureu wus found to resemble u proteln
fumlly of blood clottlng fuctors. 1he putlent untlbodles ugulnst Lproteln were
further experlmentully shown to crossreuct wlth plusmlnogen (c). Slmllurly,
untlbodles ugulnst the NS proteln huve been shown to blnd ulso blood clottlng
fuctors, lntegrlns und endothellul cells ().
1he mechunlsms of the complement system ln dengue puthogenesls ure
poorly churucterlzed, however, they ure consldered to pluy un lmportunt role. 1he
uctlvutlon of complement ls ussocluted wlth plusmu leukuge, whlch colncldes to
hlgh levels of Cu und Cu uctlvutlon products ln putlent plusmu. Addltlonully,
reduced umounts of complement components ure reported from putlents wlth
LSS. Lengue vlrus NS proteln hus been shown to lnteruct wlth complement
lnhlbltory fuctor clusterln (z) und the soluble NS proteln wus ulso found to
uctlvute complement, enhunced by untlNS proteln untlbodles ().

,

..8 Laboratory diagnostics

1he current luborutory dlugnosls of dengue ls bused on detectlon of
murkers of LLNv lnfectlon ln putlent serum. 1hese lnclude vlrul components und
untlbodles thut ure present ln the putlent serum ut dlfferent tlme polnts of the
lnfectlon (llgure 8). 1hls mukes the dlugnostlcs of ucute dengue lnfectlon
compllcuted, und often severul test types or pulred sumples ure needed for
relluble dlugnosls. lurthermore, lnformutlon of the tlmlng of sumpllng ln regurd to
the dlseuse onset ls needed for chooslng the udequute dlugnostlc method (8).
1he dlugnostlc tests used vury from country to country, und ln uddltlon to
commerclul test klts, lnhouse dlugnostlc methods ure ulso wldely used. ln Lurope,
the Luropeun Network for Llugnostlcs of lmported vlrul Llseuses (LNlvL)
provldes quullty control sumples for evuluutlon of the dlugnostlc tests (,).

1he luborutory crlterlu for conflrmutlon of dengue vlrus lnfectlon uccordlng to
vEC (8) lnclude demonstrutlon of ut leust one of the followlng ln putlent
sumple(s):

) Llve dengue vlrus by vlrus lsolutlon
z) lourfold or greuter chunge ln reclprocul lgC or lgM tlters to one or more
LLNv untlgens ln pulred sumples
) LLNv untlgen
) LLNv genome by R1PCR
8

Detection of antiDLNv antibodies

1he most wldely used trudltlonul dengue dlugnostlc methods ure bused on
detectlng untlbodles ugulnst dengue vlrus ln putlent serum. All members of the
genus fluvlvlrus shure structurul slmllurltles ln thelr envelope protelns, whlch ure
seen us serologlcul crossreuctlons ln serologlcul tests. 1he crossreuctlve
untlbodles from prevlous fluvlvlrus lnfectlons or vucclnutlons cun cuuse fulse
posltlve test results especlully ln lgC detectlon. Llugnostlcs bused on serology ure
not fluvlvlrus speclflc methods, und thus u posltlve test result should be
consldered us fluvlvlrus untlbody posltlve. 1he only serologlcul methods vulld for
typlng fluvlvlrus lnfectlons und dengue vlrus lnfectlons uccordlng to the lnfectlon
cuuslng serotype ure neutrullzutlon tests. 1hese methods ure not used ln routlne
dlugnostlcs us they ure tlme consumlng und requlre worklng wlth lnfectlous vlrus.
Relluble serodlugnosls ls bused on pulred sumples where u dlugnostlc rlse
ln untlbody tlters cun be observed between the ucute (<6 duys ufter onset) und
convulescent (6c duys ufter onset) sumples. ln prlmury lnfectlons, the lgM
untlbody tlters rulse to detectuble levels ln 8c of the putlents by duy ufter the
onset of fever (6,,). 1he lgC untlbodles become detectuble shortly ufter lgM
und by duy , most of the putlents huve detectuble levels.
1he most commonly used serologlcul method for untlLLNv untlbodles ls
cupture lgMenzyme lmmunoussuy (MucLlA) bused on lnuctlvuted vlrus untlgen
(8). ln uddltlon to LlAbused methods lmmunofluorescence ussuys (llA), luterul
flowjlmmunochromutogruphybused rupld tests und lmmunoblot methods
(8,) huve ulso been used ln detectlng untldengue lgM. 1he methodologles
used ln detectlon of lgC ure slmllur to those used ln lgM detectlon, uddltlonul
methods lnclude dotblot methods (zc) und llA (z). 1hese methods huve
lurgely repluced the prevlously used hemugglutlnutlon lnhlbltlon (El) tests, whlch
were prevlously used to meusure totul untlbodles (6).

Detection of DLNv genome

1he detectlon of LLNv genome ln putlent serum sumples enubles speclflc
dengue dlugnosls durlng the eurly stuge of the dlseuse when serologlcul methods
ure not relluble. 1he vlremlc phuse of the dlseuse colncldes wlth the fever, wlth lts
durutlon vurylng from z to c duys (cc). A posltlve test result enubles dlugnosls,
however, u negutlve test result does not exclude the posslblllty of dengue us
lndlvlduul vurlutlon ln vlremlu levels und tlmlng muy occur. 1he tlmlng of the
sumpllng ls cruclul for RNA detectlon, however, the storuge und sumple hundllng
ure ulso lmportunt for retulnlng vlrul RNA ln the sumples.
Compured to the other dlugnostlc methods, LLNv genome detectlon
requlres severul hundllng steps lncludlng sumple RNA extructlon und the ussuy
ltself. Unllke the serologlcul methods, detectlon of vlrul RNA ls vulneruble for
contumlnutlon, und requlres speclflc luborutory fucllltles und equlpment.
Eowever, unllke serologlcul methods, LLNv genome detectlon enubles speclflc
rupld LLNv dlugnosls from u slngle eurly phusesumple und enubles the typlng of
the lnfectlon cuuslng serotype, whlch ls lmportunt for the epldemlologlcul follow
up.
Llfferent types of reversetrunscrlptlon polymeruse chuln reuctlon (R1
PCR)bused methodologles huve been used ln detectlon of LLNv RNA ln putlent
sumples. ln prlnclple, vlrul RNA ls extructed from serum sumple, trunscrlbed to
cLNA ln u reverse trunscrlptlon (R1) reuctlon elther ln u sepurute reuctlon or ln u
one step formut und umpllfled by polymeruse chuln reuctlon (PCR). Lependlng on
the purpose, the umpllflcutlon turgets lnclude hlghly conserved reglons of the
LLNv genome, such us the NS und ' U1R, or ureus wlth more vurlublllty, such us
the CpreM und Lgene reglons.
c

Conventional R1PCR

1he umpllflcutlon products of conventlonul R1PCR ure detected vlsuully on
ugurose gels uslng ethldlum bromlde stulnlng. 1he umpllcons ure usuully severul
hundreds of buse pulrs long, und cun be utlllzed ln studylng the sequence of the
umpllfled PCR product by sltespeclflc restrlctlon enzyme unulysls (zz), nuclelc
ucld hybrldlzutlonbused methods (zz) or by sequenclng the PCR product
(z6). Runnlng severul rounds of umpllflcutlon cun enhunce the sensltlvlty of the
R1PCR ussuy. ln nested umpllflcutlon, the second umpllcon ls locuted wlthln the
flrst one. Conventlonul R1PCR methods huve u sepurute detectlon step ln
uddltlon to the umpllflcutlon ltself, muklng these methods slower ln compurlson
to newer reultlme methodologles comblnlng both steps. Eowever, conventlonul
methods huve not been totully repluced by the reultlme uppllcutlons, us they ure
robust, less expenslve und cun be currled out uslng buslc thermul cyclers und
reugents (z,c).

Realtime R1PCR

1wo prlnclpul methodologles of reultlme PCR huve been wldely used ln
vlrus nuclelc ucld detectlon. 1hese lnclude sequence speclflc detectlon of
umpllfled PCR products uslng fluorogenlcully lubeled probes, und methods thut
ure bused on uccumulutlon of u fluorescent dye bound to the doublestrunded
LNA umpllcon ln u sequence unspeclflc munner (). ln both wuys, the produced
fluorescence ls proportlonul to the umpllfled turget sequence und cun be
quuntlfled (z). 1he reultlme PCR umpllcons ure usuully shorter thun those used
ln conventlonul PCR. Addltlonully, the PCR lnstruments und reugents used ullow
very rupld PCR steps, detectlng the results slmultuneously thereby shortenlng the
tlme requlred for the ussuy. Severul types of lnstruments huve been developed by
dlfferent munufucturers, dlfferlng ln the technologles used to perform the
thermul cycllng, detectlng the fluorescence und unulyzlng the obtulned dutu.

Most of these enuble reultlme follow up of the uccumulutlon of the fluorescence
ln sumples whlle the protocol ls runnlng.

1aqMan chemistry

1he sequencespeclflc reultlme PCR methods ure bused on probes blndlng
speclflcully to the turget sequence, whlch ls umpllfled ln PCR. 1hese lnclude
vurlous probe types und detectlon prlnclples. Cne of the most wldely used probe
bused systems ln LLNv RNA detectlon utlllzes the ' nucleuse uctlvlty of 1uq LNA
polymeruse und duullubeled probes (ulso culled 1uqMun chemlstry). 1uqMun
probes huve u fluorescent dye ut the ' end, und u quencher dye ut the ' end.
vhen the probe ls lntuct, the quencher dye prevents the fluorescent reporter dye
from emlttlng fluorescence through fluorescence resonunce energy trunsfer,
whlch ls dependent on the dlstunce between the reporter und quencher
molecules. 1he probe ls deslgned downstreum of PCR prlmer slte ln the turget
sequence, und durlng the prlmer extenslon step of the PCR umpllflcutlon, the
probe ls cleuved by 1uq LNA polymeruse (). 1hls results ln sepurutlon of the
quencher und reporter from unother, und enubles the reporter to emlt
fluorescence thut cun be meusured (llgure ). 1hls does not occur unless the
probespeclflc sequence ls umpllfled, muklng the method hlghly speclflc. ln the
presence of u probemutchlng umpllflcutlon product, the reporter molecules ure
cleuved from the probes ln euch cycle of PCR creutlng un lncreuse ln fluorescence
lntenslty thut corresponds to the umount of umpllcon produced. Severul 1uqMun
bused R1PCR methods huve been used to detect und type LLNv RNA ().

z

llgure . Prlnclple of 1uqMun chemlstry.
. vhen the probe ls lntuct, the quencher molecule (Q) prevents emlsslon of fluorescence of the
reporter dye (R). ln the ubsence of probe speclflc turget sequence no fluorescent slgnul ls emltted.
z. Probe und prlmers blnd to thelr speclflc turget sequence. .1uq LNA polymeruse cleuves the
probe durlng PCR umpllflcutlon und reporter und quencher become sepuruted. Reporter dye emlts
fluorescence thut cun be detected und meusured by u reultlme PCR lnstrument.

SY8R Creen based methods

Unllke the sequencespeclflc methods thut utlllze probes, the SY3R Creen
bused methods meusure fluorescence produced by SY3R Creen l dye thut blnds
to uny doublestrunded LNA. ln un ussuy wlth sequence speclflc prlmers, the
lncreuse ln fluorescence ls proportlonul to the umount of the product produced.
1he speclflclty ls thus dependent on the prlmers used, but ullows the detectlon of
vurluble sequences, whlch cun be dlfferentluted bused on meltlng curve unulysls
of the umpllflcutlon products. 1hls unulysls shows the speclflc temperutures, ln
whlch the fluorescent dye dlssoclutes from the dsLNAPCR product. 1he meltlng
temperuture ls dependent on the umpllfled product sequence, us the proportlons
of CjC und Aj1 result ln dlfferent meltlng temperutures. 1hls hus been used ln reul
tlme detectlon of LLNv genome, glvlng u meltlng temperuture proflle thut cun be
sepuruted from those of other fluvlvlruses (cz). Eowever, us ull double
strunded LNA wlll produce fluorescent slgnuls, lncludlng prlmerdlmers und
unspeclflc umpllflcutlon products, the lnterpretutlon of the results of SY3R Creen
bused reultlme PCR ussuys cun be chullenglng, und depend on u cleurly
recognlzuble, unlque meltlng temperuture of the turget sequence.

Other RNA detection methods

ln uddltlon to the ubovementloned methods, R1PCR bused LNA
mlcrourruy detectlon () und umpllflcutlon by nuclelc ucld sequencebused
umpllflcutlon (NAS3A) huve ulso been used ln detectlon of LLNv RNA ().

Detection of viral antigens

1he dengue vlrus NS untlgen detectlon methods huve been shown to be
sultuble for eurly dlugnosls us the untlgen ls detectuble ln putlent serum prlor to
the uppeurunce of untlbodles (,). Commerclully uvulluble ussuys for
detectlon of LLNv untlgens ln putlent serum huve been uvulluble for u fulrly short
perlod of tlme (8). 1hese ure now uvulluble ln LlA und rupld
lmmunochromutogruphytest formuts, however, the LlA formut hus been
reported to be most sensltlve (). 1he current dutu on the NS untlgen testlng
hus showed thut the test muy huve dlfferent sensltlvltles for prlmury und
secondury lnfectlons, us preexlstlng untlNS untlbodles muy lnterfere wlth the
test ln secondury lnfectlons, competlng wlth the monoclonul untlbody used ln the
untlgencupture ussuy. lurthermore, lt hus been suspected thut the monoclonul
untlbodles used ln the tests muy prefer some LLNv types to others. Eowever,
more studles on the performunce ls needed for thls test, but the prellmlnury
results seem promlslng, us NS untlgen detectlon ls u LLNv speclflc method
sultuble for eurly dlugnosls. lurthermore, unllke R1PCR bused methods, ln u rupld
test formut the NS untlgen test does not requlre speclflc luborutory fucllltles to
curry out the test (c), und ls very eusy to use (). Eowever, further
lmprovements und more extenslve evuluutlon of the rupld test formut huve been
suggested necessury, lncludlng un lnternul posltlve control (z). ln uddltlon to
NS detectlon, commerclul klts ure ulso uvulluble for Lproteln untlgen detectlon,
but these huve been shown to be less sensltlve ln compurlson to NS detectlon
().

virus isolation

vlrus lsolutlon ls not commonly used ln routlne dlugnostlcs, but lt
constltutes deflnltlve proof of LLNv lnfectlon. As ln vlrus genome detectlon, the
sumpllng tlme und optlmul sumple storuge ure cruclul (,). Currently, the
most wldely used method of LLNv culture from putlent seru ls to use cultured
mosqulto cells (Aedes albocrus C6j6), but severul other mosqultocell llnes und
mummullun cell llnes commonly used ln vlrus cultures such us monkey kldney cells
ure ulso sultuble. 1he most sensltlve ulthough lmpructlcul method reported ls
lnoculutlon of llve mosqultoes (cc). 1he ublllty of dengue vlrus to cuuse
cytoputhlc effects (CPL) on lnfected cells vurles und ls llkely to be dependent on
vlrus struln propertles. lrom vlrus lsolutlon cells LLNv cun be detected und
serotype determlned uslng dengue vlrus speclflc R1PCR methods or monoclonul
untlbodles ln un lmmunofluorescence formut.

.. Prevention

vaccine and drug development

ln future, untlvlrul compounds ugulnst LLNv ure llkely to become
commerclully uvulluble. Compounds lnhlbltlng LLNv repllcutlon, enzymutlc
uctlvlty, receptor blndlng und fuslon ure currently studled. 1he ulm of these
studles ls to ldentlfy potentlul drugs thut could prevent Ll from developlng to
LEl or LSS (). Addltlonully, therupeutlc untlbodles ure currently studled for
thelr potentlul ln the treutment of dengue (6).
1he luck of un unlmul model und the poor understundlng of the
puthogenesls of dengue huve mude the development of un effectlve und sufe
dengue vucclne dlfflcult. An optlmul vucclne would rulse un equully effectlve
neutrullzlng untlbody response to the four serotypes slmultuneously, us otherwlse
6
the vucclne could predlspose to severe dlseuse. 1he current types of dengue
vucclnes ln development lnclude chlmerlc, llveuttenuuted, lnuctlvuted, repllcutlon
lncompetent und subunlt vucclnes. Muny of these huve not provlded sufflclent
protectlon or huve cuused sldeeffects (,). Cne of the leudlng cundldutes ls u
chlmerlc vucclne bused on u yellowfever llveuttenuuted vucclne buckbone,
where the preM und L genes huve been repluced by those of LLNv. 1hls
vucclne ls currently ln phuse z cllnlcul trluls, und hus glven promlslng results on
humun volunteers (8).

vector control

vector control ls currently the muln dengue preventlon method. 1he udult
mosqultoes do not requlre wuter und the mosqulto eggs cun tolerute deslccutlon
for months. Eowever, uquutlc hubltuts ure requlred for the lurvue und pupul
stuges. 1hese ure reudlly uvulluble ln urbun envlronments, lncludlng domestlc
freshwuter contulners und everythlng thut collects rulnwuter from trush cuns to
flower pots (z6). Restrlctlon of uqueous envlronments outdoors und preventlon
of mosqultoes both lndoors und outdoors by nets und lnsectlcldes ure the muln
wuys of vector control ln the endemlc ureus of the world. Ae. aegyr ls u duy
uctlve mosqulto, und truvelers to endemlc ureus should weur protectlve clothlng
und upply lnsect repellents on skln und clothlng ulso durlng duytlme und whlle
truvellng ln cltles (,6).

z. Ams oj rne srudy
,

z. Aims of the study

Lengue ls not u new dlseuse, however, the recent wldenlng of endemlc
ureus und lncreuse of severe dlseuse forms huve mude dengue one of the most
lmportunt mosqultoborne vlrul dlseuses of munklnd. 1he lnfectlon cuused by the
four dengue vlrus serotypes ls currently u mujor publlc heulth problem ln the
endemlc ureus of the subtroplcs und troplcs of the world. 1he lncreused lncldence
und prevulence of dengue ln the endemlc ureus ulso uffects the truvelers vlsltlng
these ureus. 1he lncreused lmportunce of dengue us u dlseuse of truvelers wus
ulso observed ln llnlund. vlthln the lust ten yeurs, the numbers of posltlve
dlugnoses huve rlsen steudlly, now reuchlng over c dlugnoses yeurly lncludlng
one conflrmed futullty. Severe dengue dlseuse und futulltles umong truvelers ure
rure, und relutlvely llttle lnformutlon ls uvulluble of these cuses muklng thelr
descrlptlon und lnvestlgutlon lmportunt. Lue to the rlsk for severe dlseuse
outcomes, the recognltlon of dengue lnfectlon ln truvelers ls lmportunt. 1he
dlugnostlcs of dengue vlrus lnfectlon ls compllcuted by the serologlcul cross
reuctlons wlth untlbodles from other fluvlvlrul lnfectlons. Llugnostlc methods thut
provlde u relluble und speclflc dlugnosls of dengue vlrus lnfectlon ln the eurly
stuge of the dlseuse ure needed.
1he globul dlstrlbutlon und composltlon of dengue vlrus sero und
genotypes clrculutlng ln dlfferent geogruphlcul reglons ls constuntly chunglng.
1he churucterlzutlon und phylogenetlc unulysls of dengue vlrus strulns from
truvelers provlde uptodute lnformutlon on the globul epldemlology lncludlng
ureus lucklng uctlve survelllunce. ln the countrles endemlc for dengue vlruses, the
followup of the clrculutlng dengue vlrus strulns provldes lnformutlon of the locul
epldemlologlcul sltuutlon und evolutlon of these vlruses. 1he epldemlology of
dengue vlrus type z wus eurller studled ln venezuelu ln the lute cs when
evldence of serotype z dlverslflcutlon und recomblnutlon were observed. A
colluborutlon study wlth venezuelun reseurchers ulmed ut updutlng the current
sltuutlon ln venezuelu.
z. Ams oj rne srudy
8

1he speclflc ulms of the study were to

Curry out u vlrologlcul lnvestlgutlon of the flrst futul dengue cuse ln llnlund
for conflrmutlon of the etlology of the lnfectlon und ulmlng ut
ldentlflcutlon of the lnfectlve vlrus serotype

Levelop u LLNv RNAdetectlon method to be uble to dlugnose dengue
from eurly tlmepolnt putlent sumples und to compure thls method to
other uvulluble methods lncludlng NS untlgen detectlon, vlrus lsolutlon
und serologlcul methods for chooslng the optlmul methodologles for
dlugnoslng dengue ln truvelers

lsolute und churucterlze LLNv strulns from llnnlsh truvelers for
ldentlflcutlon of the lnfectlve LLNv seround genotype und for studylng
thelr genetlc relutlonshlps to prevlously churucterlzed LLNv strulns

Anulyze u collectlon of z LLNvz lsolutes from un endemlc country,
venezuelu, for u followup of LLNvz epldemlology und evolutlon by
determlnlng und unulyzlng envelope gene sequences

. Varerals and mernods

. Materials and methods

. Study moterols

.. Patient samples

Lengue putlent serum sumples were obtulned from EUSLA3 depurtment of
vlrology, the vlrul zoonoses dlugnostlc unlt thut currently performs dengue
dlugnostlcs us the only luborutory ln llnlund. 1he seru were stored ut zcC,
ullquots for vlrus lsolutlon trluls und R1PCR were stored ut ,cC untll use.
lnformutlon on the truvel hlstory, onset und vurlety of symptoms of the putlents
wus collected by u questlonnulre from the cllnlcluns who treuted the putlents ln
hospltuls uround llnlund.
As lnformutlon on the onset of symptoms wus not uvulluble for ull the
putlents, the putlent seru were selected for vlrus lsolutlons und eurly tlmepolnt
dlugnostlc tests by chooslng the flrst uvulluble serum from u dlugnosed putlent
uslng u crlterlon of lgC tlter (llA) zc or less. Uslng these crlterlu, u punel of
sumples wus selected from putlent sumples collected ln zcc8.
Autopsy sumples lncludlng the puruffln embedded tlssue sumples (bruln,
lung, kldney) und bruln tlssue sumple used ln publlcutlon l were obtulned from
1umpere Unlverslty Eospltul.

..z viruses

1he reference dengue vlrus strulns used ln neutrullzutlon ussuys und us posltlve
controls ln llA und R1PCR lncluded LLNv (Euwull), LLNvz (New Culneu C),
LLNv (E8,) und LLNv (Ez) from the collectlons of Euurtmun lnstltute. 1he
c
venezuelun LLNvz strulns were obtulned from Reglonul Luborutory for Llugnosls
und Reseurch of Lengue und other vlrus dlseuses (Lurdldev) Aruguu Stute,
venezuelu, where the z LLNvz strulns were lsoluted from putlent seru collected
ln zcc ln Aedes albocrus C6j6 cells.

.. Cell lines

Cultured cell llnes used ln thls study were orlglnully obtulned from A1CC
collectlons lncludlng vero L6 (A1CC: CRL86) green monkey kldney cell llne
grown ln cell culture bottles ln MLM supplemented wlth fetul culf serum,
glutumlne und untlblotlcs (penlclllln, streptomycln) ut ,C ut CCz und C6j6
(A1CC:CRL66c) Aedes albocrus mosqultocell llne, grown ln Clbco Lelbovltz L
medlu (lnvltrogen, Curlsbud, Cullfornlu, USA) ut room temperuture or ut z, C
wlth closed llds.

..q Monoclonal antibodies

1he monoclonul untlbodles (MAbs) ugulnst dengue vlrus Lproteln were obtulned
us mouse hybrldomu cell llnes l (LLNv), E (LLNvz), L (LLNv), Ec
(LLNv) und 8 (fluvlvlrus crossreuctlve MAb), () were klndly provlded by
professor Lrnest Could from the Unlverslty of Cxford, Ul. 1he cell llnes were
cultured ln RPMlmedlu. 1he supernutunts from confluent cells were clurlfled by
centrlfugutlon und used us prlmury untlbodles ln lmmunofluorescence ussuys. 1he
fluvlvlrus crossreuctlve MAb E3z wus provlded by Slrkku vene from Swedlsh
lnstltute for lnfectlous Llseuse Control, Stockholm, Sweden.

.z Methods

All vlrus culture und lsolutlon experlments were currled out ln blosufetylevel
(3SL) luborutorles ln Euurtmun lnstltute, Unlverslty of Eelslnkl.

.z. virus isolation in cell culture

vlrus lsolutlons from putlent serum sumples were performed ln z cm
z
culture
bottles uslng vero L6 und C6j6 cells. 1he cells were flrst rlnsed wlth P3S wlth
udded untlblotlcs to remove the culture medlu, und lnfected wlth c l of the
serum sumple for hour. After hour, fresh medlu wus udded. 1he vero L6 cells
were grown ut , C ln u CC
z
lncubutor und C6j6 cells ut room temperuture. 1he
lnfected cells were observed for cytoputhlc effects und studled further for vlrul
untlgens ln llA und the culture medlu sumples for the presence of vlrul RNA by R1
PCR.

.z.z RNA extraction and R1PCR

RNA from vlrus lsolutlon culture supernutunts und humun serum sumples were
extructed uslng QluAmp vlrul RNA mlnl klt (Qlugen, Ellden, Cermuny) uccordlng to
the munufucturer's lnstructlons. RNA from tlssue sumples wus extructed uslng
1RlPURL lsolutlon reugent (Roche Applled Sclence, Munnhelm, Cermuny).

z
Conventional R1PCR

1he dengue vlrus typlng semlnested R1PCR wus performed uslng the prevlously
descrlbed prlmers turgetlng the CpreM reglon ln LLNv genome (z,). ln brlef, u
bp reglon of ull LLNv types wus umpllfled by R1PCR (prlmers L und Lz),
whlch wus used us u templute ln the second round wlth L prlmer und LLNv type
speclflc reverse prlmers (1S) produclng LLNv type speclflc umpllflcutlon
products (LLNv: 8z bp, LLNv: z bp, LLNv: zc bp, LLNvz: bp). ln
uddltlon to thls protocol, unother semlnested R1PCR wus ulso used, umpllfylng u
zzc bp conserved reglon of NS gene of most fluvlvlruses (6c). 1he LLNvz
envelope genesequences were umpllfled uslng prevlously descrlbed prlmers (8).

1he R1reuctlons were performed uslng Lxpund Reverse 1runscrlptuse (Roche
Applled Sclence, Munnhelm, Cermuny) elther by gene speclflc reverse prlmers or
rundom hexumers. 1he PCR steps were currled out uslng Recomblnunt 1uq LNA
polymeruse (lermentus, vllnlus, Llthuunlu) und 1uq Lxtender PCR uddltlve
(Strutugene, Lu 'ollu, Cullfornlu, USA) uccordlng to the munufucturer's
lnstructlons.

1aqMan realtime one step R1PCR

lor settlng up the 1uqMun ussuy, u few modlflcutlons of the prlmers were flrst
tested for thelr performunce ln PCR umpllflcutlon uslng SY3R Creen l. 1hese tests
were done on the posltlve control RNAs (LLNv) thut were flrst reverse
trunscrlbed to cLNA uslng Lxpund Reverse 1runscrlptuse (Roche Applled Sclence,
Munnhelm, Cermuny). 1he PCR wus done uslng Llght Cycler lust Sturt LNA
Muster SY3R Creen l Muster klt (Roche Applled Sclence, Munnhelm, Cermuny) on
Llght Cycler reultlme PCR lnstrument uslng u progrum of C mln followlng
umpllflcutlon c x u cs cycle of C, 6c C s und ,z C. 3used on the
umpllflcutlon results und meltlng curve unulysls, the best prlmers (forwurd '
CCAC1ACACC11ACACCACACCCC, reverse ' CACACACCACCA1C1C1CC1C) were

chosen for further testlng on 1uqMun reultlme R1PCR uslng u 6lAM
ACCA1A11CACCC1CCCAMC33EQ probe. 1he onestep test formut wus chosen
for uvoldlng contumlnutlons und for the euse of performunce. 1he test wus
optlmlzed on Qlugen Quuntl1ect Cnestep Probe R1PCR klt (Qlugen, Ellden,
Cermuny) ln u cl reuctlon volume uslng cc nmol of euch prlmer und zc nmol
of the probe, lncludlng l of templute RNA uslng u progrum of c C c mln
followed by x cycle of C s, 6cC mln ln Applled 3losystems optlcul 6
well plutes uslng A3l PRlSM ,,cc Sequence Letectlon Systemlnstrument. 1he
probe wus purchused from Applled 3losystems und prlmers from Cllgomer.

.z. Sequencing and sequence analysis

1he R1PCR products were enzymutlcully purlfled for sequenclng uslng LxoSAPl1
(US3 Corporutlon, Chlo, USA) und when necessury uslng gel extructlon by Qlugen
gel extructlon klt (Qlugen, Ellden, Cermuny). 1he sequenclng reuctlons were done
ut the sequenclng core fuclllty of Euurtmun lnstltute (Sequenclng unlt,
Euurtmunlnkutu , PLz, ccc, Unlverslty of Eelslnkl, Eelslnkl, llnlund). 1he
obtulned sequences were trlmmed und comblned to consensus sequences uslng
the progrum Peuks (uvulluble ut http:jjmekentosj.comjsclencejpeuksj), und
progrums of the Studen puckuge (uvulluble ut
http:jjmekentosj.comjsclencejpeuksj und http:jjstuden.sourceforge.netj).

1he multlple sequence ullgnments were done uslng Clustulvz or MUSCLL
progrums uvulluble onllne (http:jjwww.ebl.uc.ukj clustulw und
http:jjwww.ebl.uc.ukj1oolsjmusclejlndex.html). 1he phylogenetlc unulysls of
nucleotlde sequences wus performed uslng MLCA softwure (6).

.z.q IIA

1he lmmunofluorescence ussuy (llA) wus used ln thls study for two purposes, for
detectlon of untlbodles ugulnst u known LLNv untlgen und for screenlng for vlrul
untlgens uslng u known untlbody. 1he prlmury untlbody ln dlugnostlc procedures
wus u putlent lgC or lgM detected by u fluorescent (ll1Clubeled) secondury
untlbody, untlhumun lgC or lgM ('uckson lmmunoreseurch, vest Crove,
Pennsylvunlu, USA). ln detectlon of vlrul untlgens from vlrus lsolutlon cells, known
LLNvuntlbody posltlve humun seru or MAb wus used. 1he MAbs were detected
by ll1Clubeled untlmouse conjugute (LAlC Cytomutlon, Clostrup, Lenmurk).
llA slldes for dlugnostlc llA tests were mude by lnfectlng vero L6 cells for
, duys dependlng on the vlrus, detuchlng the cells by trypslnLL1A und growlng
the cells overnlght on presterlllzed cwell slldes ut ,C ln u molst envlronment.
1he next duy, the slldes were gently rlnsed ln P3S to remove the unuttuched cells.
llnully, the slldes were ulrdrled und flxed ln chllled ucetone for , mlnutes und ulr
drled uguln. 1he slldes were stored ut ,cC prlor to use.
1he llA slldes from vlrus lsolutlon trluls were mude slmllurly, wlth the
exceptlon thut the lnfected cells (vero L6 or C6j6) were drled dlrectly on slldes.

.z. LIA

Commerclully uvulluble enzyme lmmunoussuys were used for detectlon of untl
dengue vlrus lgM uslng u cuptureLlA klt (lCCUS 1echnologles, Cypress,
Cullfornlu, USA) und for detectlng dengue vlrus NS untlgen from putlent seru, un
untlgen cuptureLlA, 3loRud dengue NS ACLLlSA (3loRud, MurnesluCoquette,
lrunce) wus used uccordlng to the munufucturer's lnstructlons.


.z.6 Immunohistochemistry

lmmunohlstochemlstry (lEC) wus performed by ventunu Llscovery
lmmunohlstochemlstry Sllde Stulner (ventunu Medlcul Systems, 1ucson, Arlzonu,
USA) uslng purufflnembedded thln sectlons of the tlssues. 1hln sectlons of
purufflnembedded LLNv lnfected veroL6 cells were used us posltlve controls
ln the lEC stulnlngs. Slldes were lncubuted wlth prlmury untlbody (punfluvl cross
reuctlve MAbs und LLNv speclflc MAbs) und untlmouse conjugute und
detected uslng ventunu LA3 blotlnuvldln detectlon klt.

.z.) Neutralization test

Pluque reductlon neutrullzutlon (PRN1) tests were currled out ln vero L6 cells
uslng reference vlrus strulns udupted to thls cell llne, wlth u known tlter ln pluque
formlng unlts (PlU) ln 6well plutes. A vlrus dllutlon produclng euslly reuduble
umount of sepurute pluques ln u well (upproxlmutely c pfu) wus chosen us the
worklng umount of the vlrus. 1he neutrullzutlon cupublllty of u serum sumple wus
tested ln u dllutlon serles uslng u stundurd umount of vlrus. 1he putlent serum
sumples were flrst heutlnuctlvuted by lncubutlng them c mln ut 6c C prlor to
lncubutlon wlth vlrus ut ,C for hour. lreshly confluent monoluyers of vero L6
cells on 6well plutes were lnfected wlth the serumvlrus mlxture for hour, und
then the lnoculum wus removed. Cells were overluld wlth u : mlxture of zxMLM
und z ugurose. 1he pluques were ullowed to develop for , duys dependlng on
the vlrus und then the cells were flxed ln c formuldehyde solutlon. 1he ugurose
wus removed und the plutes were stulned uslng crystul vlolet solutlon for
vlsuullzutlon of the remulnlng monoluyer und the pluques. 1he result from putlent
sumples ln pfu's wus compured to those of the controls treuted slmllurly wlthout
the serum. A dllutlon of the putlent serum, whlch cuused un 8c reductlon ln the
pluque formutlon of u glven vlrus, wus consldered us the PRN1
8c
tlter of u serum.

|. esulrs and dscusson
6
q. Results and discussion

{. Dognoss oj dengue vrus njecton n trovelers

1he number of dlugnosed dengue cuses ln llnnlsh truvelers hus trlpled ln u
short perlod of tlme (llgure c), now reuchlng over c dlugnosed cuses euch yeur
(Euhtumo et ul., unpubllshed). 1hls muy be purtly due to lncreused recognltlon of
the dlseuse, but ls ulso uffected by the lncreuse of people truvellng to endemlc
ureus (6z), und the extent of dengue trunsmlsslon ln these ureus. An unulysls of
the stutlstlcs of the llnnlsh truvel destlnutlons show thut 1hullund ls the most
populur slngle longdlstunce truvel destlnutlon. Slnce zcc6, upproxlmutely
cc ccc trlps were mude yeurly from llnlund to 1hullund (6).

llgure c. Number of trlps by llnnlsh truvelers uged , to ureus endemlc und potentlully
endemlc to dengue vlrus lncludlng truvel destlnutlons ln Centrulund South Amerlcu, Aslu, Cceunlu
und Afrlcu (obtulned from Stutlstlcs llnlund,
http:jjpxwebz.stut.fljLutubusejStutllnjllljsmutjsmut_fl.usp) und number of posltlve dengue
dlugnosls ln llnlund zccczcc bused on serologlcul testlng (EUSLA3) (Euhtumo et ul.,
unpubllshed).
,

q.. virological examination of a fatal dengue case (I)

ln truvelers, dengue ls usuully u prlmury lnfectlon wlth relutlvely mlld
symptoms. 1he sudden deuth of u prevlously heulthy young llnnlsh womun who
wus recoverlng from u severe dengue vlrus lnfectlon rulsed u lot of questlons und
trlggered u thorough exumlnutlon of the cuse. 1he putlent fell lll durlng her
journey ln South Lust Aslu ln Muy zccz. 3used on her truvel hlstory, the lnfectlon
most llkely orlglnuted from Muluyslu, where she truveled durlng the weeks prlor
to onset of the dlseuse.
1he dlseuse sturted us u typlcul Ll lncludlng hlgh fever, heuduche und rush.
lrom thls stuge the symptoms gruduully becume more severe, und the putlent
wus hospltullzed wlth nuuseu, vomltlng und cough, ln uddltlon to leukocytosls,
thrombocytopenlu und reduced blood pressure. 1he fever subslded on duy ufter
the onset, she developed edemu on the fuce und llmbs und luborutory tests
reveuled ubnormul blood clottlng. Cne week ufter the onset of fever, the
putlent's condltlon developed to LEl. She becume unconsclous, hud renul fullure
und developed huemorrhuglc symptoms lncludlng nose bleedlng und petechlue
on the skln. Addltlonully, she hud pulmonury edemu und pleurul effuslons.
1he symptoms were prolonged und on the fourth week the putlent wus
trunsferred to llnlund. 1he putlent stlll hud pleurul und ubdomlnul effuslons, renul
fullure, llver dumuge wlth lcterus und bleedlng from lntruvenous llnes. 1he renul
fullure wus treuted by hemodlulysls, und the prolonged sepsls syndrome by
methylprednlsolone. 1he putlent wus recoverlng, however she hud u sudden bruln
hemorrhuge ufter u routlnely performed dlulysls. 1he putlent dled duys luter,
und the uutopsy reveuled dlffuse hemorrhuges ln bruln, menlnges, endocurdlum,
puncreus und ovurles.
1he dlugnostlc tests of the eurly stuge sumples were performed ut
hospltuls ln Aslu und the lute stuge sumples were studled ln llnlund. Lurlng the
ucute phuse of the dlseuse, lgM but no lgC wus detected churucterlzlng the
dlseuse us u prlmury dengue lnfectlon. As no evldence of vlrus wus detected from
8
the exumlnutlon of the lute stuge serum or the uutopsy sumples studled by vlrus
lsolutlon, R1PCR or lmmunohlstochemlstry, the putlents prolonged symptoms of
renul fullure, ubnormul blood clottlng und hemorrhuges llkely were cuused by
lndlrect effects of the lnfectlon. Cortlcosterolds huve been used ln treutlng sepsls
syndrome (6) und were ulso udmlnlstered to the putlent descrlbed here wlth
observed lmprovement ln the putlent's condltlon.
1he ucute phuse sumples tuken ln Aslun hospltuls were not uvulluble for
further studles, such us vlrus lsolutlon or R1PCR. 1he untlbody responses of the
putlent were found to be crossreuctlve, und nontypuble by the pluque reductlon
neutrullzutlon (PRN1) tests. 1hese, however, provlded evldence thut the lnfectlon
wus cuused by LLNv, us the PRN1 tlters were hlgher to LLNv thun to 'Lv. 1he
lgMllA tlters were found to be hlgher ugulnst LLNv und LLNvz (:6c) thun
ugulnst the rest of the dengue vlrus types (:c:6c) suggestlng thut these LLNv
types muy huve been lnvolved (6). 1he reported concurrent clrculutlon of LLNv
und z ln Muluyslu zccz (66) would flt the untlbody flndlngs of the putlent,
suggestlng the posslblllty of u multlple serotype lnfectlon, whlch ure known to
occur ln ureus where LLNv types coclrculute (6,,68). 1he slgnlflcunce of the
lgMllA result ulone, however, remulns uncleur us those were not supported by
the PRN1.
vhlle the severe dengue dlseuse ls often ussocluted wlth secondury
lnfectlons, explulned by the ALLtheory, relutlvely llttle ls known ubout the rlsk
fuctors uffectlng the severe dlseuse outcomes of prlmury lnfectlons, whlch ulso
lnclude LEl und futulltles (6,,c). Severul host speclflc fuctors huve been
suggested to uffect the dlseuse outcome lncludlng ruce, sex und nutrltlonul
stutus. 1he lnfectlng vlrus type und lts puthogenlclty huve ulso been suggested to
lnfluence dlseuse outcome. ln u study of LEl of prlmury lnfectlons ln 1hullund
lnvolvlng mulnly young putlents, un ussoclutlon of severe dlseuse forms to LLNv
serotypes und wus observed (,). vurlous symptoms consldered unusuul ln
dengue huve been reported from truvelers such us skln tenderness, puncreutltls,
menlngltls (z) suburuchnoldul huemorrhuge (,z) und encephulltls (,).
Addltlonully, symptoms mlmlcklng typhold fever (,), compllcutlons durlng
pregnuncy (,) und futulltles (,6) huve been recently reported. ln clusslflcutlon

of dengue dlseuse severlty ln truvelers, the vEC clusslflcutlons huve been
questloned us the dlseuse ln truvelers cun be severe und yet not fulflll the current
LEl clusslflcutlon (, ,,). ln concluslon, our lnvestlgutlon conflrmed the
serologlcul dlugnosls of dengue vlrus lnfectlon ln the putlent by neutrullzutlon
tests but wus not uble to ldentlfy the lnfectlve LLNv serotype or serotypes
lnvolved. 1he results suggested thut our putlent experlenced u prolonged prlmury
dengue vlrus lnfectlon thut wus the underlylng cuuse of the futul bruln
hemorrhuge.
6c

q..z Development of a new realtime R1PCR method for detection of
DLNv RNA (II)

Assay design

1he requlrements for u dlugnostlc reultlme R1PCR test lncluded speclflclty
for LLNv RNA und u sufflclent sensltlvlty to detect LLNv RNA from putlent serum.
lrom the dlugnostlc luborutory's polnt of vlew, relluble quulltutlve results ln u
slmple und ufforduble formut preceded the needs of quuntlflcutlon und typlng of
dengue vlruses ln putlent sumples. 1o meet these crlterlu, we ulmed to deslgn un
ussuy bused on u slngle 1uqMun probe slmultuneously detectlng the four dengue
vlrus types. 1he sultuble genomlc reglons thut lncluded short hlghly conserved
sequences were seurched vlsuully from LLNv complete genome ullgnments of
the four LLNv serotypes. 3used on the ullgnments, the ldentlfled conserved
reglons were further exumlned by uslng them us query sequences ln seurches
ugulnst Cen3unk uslng the 3lustN ulgorlthm (uvulluble ut
http:jjblust.ncbl.nlm.nlh.govj3lust). 3used on the results, the 'U1R reglon wus
selected us u turget for the PCR umpllflcutlon. 1he prlmers und probe were
deslgned uslng Prlmer Lxpress Softwure verslon .c (Applled 3losystems) uslng
the settlngs for mlnor groove blnder (MC3) -probe. 1he use of u probe wlth
uttuched MC3 molecule, u conjuguted hulrplnllgund lncreuslng the meltlng
temperuture (1m) of the probe, enubled us to use u short probe sequence thut
sulted our turget sequence.

6

Determination of assay sensitivity and specificity

lor the ussuy sensltlvlty estlmutlon, the numbers of vlrul genomes ln the
posltlve control vlrus supernutunts were estlmuted bused on the meusured RNA
concentrutlon uslng u spectrophotometer. 1he RNA wus extructed wlthout currler
RNA thut would lnfluence the spectrophotometrlcul meusurement und the
meusured RNA quuntlty wus consldered to contuln only vlrul RNA. 1he obtulned
vulues were used ln the culculutlon of number of vlrul genome coples ln the RNA
extruct, whlch wus further tested ln reultlme R1PCR. A cfold dllutlon serles of
the RNA sumples wus run ln dupllcutes wlth the ussuy. 1hls sensltlvlty estlmutlon
resulted ln detectlon of less thun ccc genome coples for ull LLNv types per
reuctlon.
1he speclflclty of the test for LLNv RNA wus evuluuted by testlng RNA
extructs of other fluvlvlruses lncludlng Ylv, 'Lv, 13Lv, USUv und vNv. 1estlng
demonstruted thut none of these templutes guve posltlve results. Addltlonully,
putlent serum nuclelc uclds (n=z) from rundomly selected unreluted putlent
muterlul wus tested negutlve. 1hese results demonstruted thut the deslgned
prlmers und probe were speclflc for LLNv RNA. 1he test wus further evuluuted
uslng two externul control punels from the Luropeun Network for lmported vlrul
Llseuses (LNlvL) (). 1he flrst control punel wus provlded for the ussuy testlng
und lncluded the sumple ldentlty lnformutlon. 1he sumples lncluded z lyophlllzed
serum sumples thut were resuspended lnto cc l of PCR grude E
z
C for RNA
extructlon. 1he sumples lncluded seven sumples of LLNv RNA, 13Lv und Ylv
RNA und one negutlve sumple. 1he results of the LLNvRNA contulnlng sumples
were found to be posltlve und the nondengue und negutlve sumples negutlve.
ln zcc we purtlclputed ln the LNlvL Lxternul Quullty Assurunce round for
LLNv RNA detectlon. 1he z sumples provlded were tested wlthout uny sumple
lnformutlon und the results were sent to LNlvL. 1he results of the control
sumples were provlded ufter the results were collected from Luropeun
luborutorles unonymously, lncludlng lnformutlon of the method used. 1he results
of euch purtlclpunt were provlded confldentlully, lncludlng the ldentlty of euch
6z
sumple tested. 1he sumples lncluded LLNv sumples (LLNv), z unreluted
fluvlvlrul RNA contulnlng sumples und one negutlve sumple. Cnly two luborutorles
got the correct results ln detectlng the presence or ubsence of LLNv RNA, our
luborutory belng one of them (LNlvL, unpubllshed). 1he sumple lnformutlon
provlded by LNlvL showed thut the lowest umount of vlrul RNA ln the controls
wus zc genomesjml, correspondlng to z genome coples ln the cc l sumple
whlch wus used ln the RNA extructlon, eluted lnto c l, und u totul of l wus
used ln u c l reuctlon volume ln our ussuy. ln our ussuy, thls sumple repeutedly
guve slmllur results, one of the dupllcutes turnlng out posltlve on lute cycles, und
the other resultlng negutlve. 1he sumple wus consldered posltlve, the result,
however, suggested thut thls level of vlrul RNA (upproxlmutely z,
genomesjreuctlon) ln the sumple wus ut the detectlon llmlt of the ussuy. 1he
orlglnul sensltlvlty estlmutlons bused on culculutlons of spectrophotometrlcully
meusured totul RNA concentrutlons of the vlrus supernutunts guve results thut
lndlcuted lower ussuy sensltlvlty thun whut wus concluded bused on the externul
controls provlded by LNlvL. Cne reuson for thls could be the posslble truces of
cellulur RNA ln the RNA extructs thut could huve glven fulsely hlgh RNA
concentrutlons ln relutlon to the uctuul vlrul RNA concentrutlon.
1he sensltlvltles of dlfferent reultlme PCR bused methods ure reported us
detectlon levels estlmuted bused on dlfferent methods thut cunnot be dlrectly
compured. 1hese lnclude n vrro trunscrlbed RNA controls, numbers of pfu's und
culculuted numbers of genomes ln purlfled vlrul RNA. 3used on RNA trunscrlpts,
the detectlon sensltlvltles reported huve been ln the runge of hundreds or
thousunds genomecoplesjreuctlon (,) und bused on pfu's c, c,ccz pfu per
reuctlon (6). 1he levels reported ln putlent serum huve been reported us hlgh us
c
RNA coplesjml (,8) und to vury from c,cczccccc pfujml (,6). ln

regurd to these vulues, the estlmuted level of our ussuy sensltlvlty, under c
genomlc coples per reuctlon, cun be consldered hlghly sensltlve.

6

Lvaluation of the assay using patient samples

1he developed ussuy wus further evuluuted uslng u punel of serum
sumples from serologlcully dlugnosed dengue putlents. Cnly the sumples thut
guve cleur umpllflcutlon plots (llgure ) were consldered posltlve. Cut of 8
selected eurly sumples, j8 guve u posltlve reul tlme R1PCR result. Cf these,
were conflrmed by vlrus lsolutlon und z by u conventlonul R1PCR umpllflcutlon
und subsequent sequenclng. 1he results demonstruted thut ull LLNv serotypes
were detected wlth the ussuy, lncludlng dlfferent genotypes. ln of the reultlme
R1PCR posltlve sumples, the result could not be conflrmed by vlrus lsolutlon or
other R1PCR methods, llkely due to low umount of RNA or loss of lnfectlvlty
durlng the storuge. 1he results from the test evuluutlons demonstrute the
developed LLNv RNA detectlon method ls both sensltlve und speclflc for
detectlon of LLNv RNA und therefore sultuble for dlugnostlc use.

llgure . Ampllflcutlon plots of three LLNvRNA posltlve putlent sumples (ln dupllcutes) und the
negutlve controls ln 1uqMun reultlme onestep R1PCR.
6

q.. Comparison of the early diagnostic methods (II)

1he dlugnosls of dengue lnfectlon durlng the eurly phuse of the dlseuse ls
compllcuted, und often pulred sumples und severul methods ure requlred. 1hese
methods lnclude lgM detectlon, whlch ls vulneruble to crossreuctlons, und
dengue vlrus speclflc tests detectlng dengue vlrus RNA und untlgens. Lurlng the
flrst duys of the dlseuse, the lgM test often glves negutlve results, whereus ln
most putlents vlrul RNA or NS untlgen cun be detected (,).
1he NS detectlon methods huve become commerclully uvulluble relutlvely
recently, und compurutlve lnformutlon of LLNv NS und RNA detectlon methods
remulns scurce (8c,8). As the performunces of dlfferent 'lnhouse' LLNv RNA
detectlon methods vury, we wunted to compure u commerclul NS untlgen
detectlon method (3lCRAL Plutellu Lengue NS AC LlA) to our own 'lnhouse'
1uqMun reultlme onestep R1PCR. 1he compurlson of the uvulluble methods wus
currled out uslng u punel of selected serologlcully dlugnosed putlent serum
sumples. 1he ulm wus to study whether the performunces of RNA und NS
detectlon methods dlffered from one unother ln regurd to lgMLlA. Addltlonully,
the sumples were tested by vlrus lsolutlon und for lgCuntlbodles ln llA.
vhen conslderlng the results slmply bused on whether the dlugnosls wus
obtulned from u slngle sumple, regurdless of the lnformutlon of the sumpllng
tlme, the lgMLlA test uppeured to be the best method. A posltlve lgM result wus
obtulned ln upproxlmutely 8c of the sumples followed by NS detectlon
(upproxlmutely 6,) und RNA detectlon (upproxlmutely 6c). 1hls result ls llkely
to be uffected by the tlmedlstrlbutlon of the study muterlul, whlch lncluded
mostly sumples tuken ufter duy followlng onset of symptoms, whlch fuvors the
lgM detectlon. Cur study muterlul lncluded ultogether sumples thut were found
to be lgM negutlve. 3used on the lnformutlon of the tlmlng of the sumpllng ufter
onset of symptoms, these were tuken wlthln upproxlmutely one week ufter the
onset, wlth u few exceptlons (1uble z). All of these sumples were posltlve ln reul
6
tlme R1PCR, und 6 were posltlve ln the NS untlgen test, demonstrutlng thut
RNA detectlon performed sllghtly better thun NS detectlon ln our test muterlul.
1he RNA und NS detectlon methods huve been shown to be sultuble for
eurly dlugnosls of dengue lnfectlon durlng the tlme when the lgM responses ure
not yet detectuble. Eowever, secondury lnfectlons huve been shown to be
problemutlc for both lgM und NS untlgen detectlon, us lgM responses cun be
very low und preexlstlng untlNS untlbodles muy uffect NS detectlon sensltlvlty.
ln two of the NS negutlve sumples (no. z und no., ln tuble z), u secondury
lnfectlon could huve explulned the obtulned test results. Eowever, secondury
lnfectlon could ulso be suspected ln sumple , whlch hud lgC but no lgM on duy
ufter onset, but the reultlme R1PCR und NS tests were posltlve.
Cur results were ln ugreement wlth eurller reports ln showlng thut ln
generul the tlme spun when NS cun be detected ln putlent serum ls longer thun
thut of LLNv RNA. 1he tlme for NS untlgenemlu ln putlent serum ulso colnclded
wlth lgM for u longer perlod of tlme thun the vlrul RNA. 1hls would suggest thut
NS detectlon would be the method of cholce over RNA detectlon, us lt ls ulso u
very eusy method to perform, und currently ulso uvulluble us u rupld test formut
where the result cun be reud ln mlnutes wlthout speclul luborutory equlpment.
Eowever, the few NS negutlve results ln sumples thut were found posltlve for
LLNv RNA rulse questlons of the sensltlvlty und performunce of the NS detectlon
ussuy, und of the klnetlcs of NS ln putlent serum. 1he NS untlgen detectlon rutes
ln prlmury und secondury lnfectlons huve been vurluble ln dlfferent studles,
however, ulso the detectlon sensltlvltles of NS protelns from dlfferent vlrus sero
und genotypes should be evuluuted. 1he thorough study of lndlvlduul vurlutlon
und klnetlcs of LLNv NS und RNA ln putlents requlres lurger sumple punels, und
preferubly serlul sumples from euch putlent. Cur study muterlul wus smull, und
selected bused on u prlor serologlcul dlugnosls thut muy huve lnfluenced the
obtulned results. 1he study muterlul lncluded z sumples thut were negutlve ln
LLNv speclflc tests, und thus the orlglnul serologlcul test results could be cross
reuctlons. lurthermore, the rellublllty of the lnformutlon uvulluble to us regurdlng
the onset of the dlseuse ln some putlents, where the dlugnostlc test results
suggested eurller phuse of the dlseuse thun reported, could be questloned.
66
lt cun be concluded thut comblnutlons of methods ure needed for
obtulnlng the most relluble results. ln our sumple muterlul the dlugnostlc rutes
were hlgher when lgM results were comblned to RNA or NS detectlon, yleldlng
sensltlvlty for comblned RNA und lgM, und neurly 6 sensltlvlty for the lgM
und NS comblnutlon.
1he questlon of whlch one of the two LLNv speclflc tests, NS or RNA
detectlon, would be u better method to be comblned wlth lgM detectlon remulns
to be thoroughly studled wlth lurger sumple punels. Addltlonul studles ulmed ut
provldlng lnformutlon of the klnetlcs of vlrul RNA und NS ln putlent sumples
would be lmportunt und mlght provlde lnslghts to the course of LLNv lnfectlon ln
humun body. Ns ls u vlrul nonstructurul proteln thut ls secreted out of lnfected
cells, thus lt ls not surprlslng thut NS cun be detected durlng vlremlu. Eowever, lt
ls lnterestlng thut lt cun sometlmes ulso be detected post vlremlu us deflned by
vlrus lsolutlon und R1PCR. 1he reusons for thls ure not known, however, us the
vlremlu und elevuted NS levels huve been ussocluted wlth lncreused dlseuse
severlty, NS klnetlcs muy be llnked to the course of lnfectlon und puthogenesls.
6,

1uble z. 1est results of the lgM negutlve putlent sumples.

IgG
titer
Virus
isolation
Real-time
RT-PCR
NS1
Ag
Days after
onset
1 <10 + + + 3
2 80 - + - 4
3 <10 + + + 2
4 <10 + + + 2
5 <10 + + + 1
6 <10 + + + ?
7 <10 + + + 4
8 <10 - + + 8
9 <10 + + + ?
10 <10 - + + 14
11 <10 + + + 1
12 <10 + + + 4
13 80 + + + 3
14 <10 + + + 20
15 <10 + + + 2
16 20 + + + 2
17 320 - + - 7
18 <10 - + - 5
19 <10 + + + 5

68

{.z. Moleculor epdemology oj DLNv strons

q.z. A global collection of DLNv from Iinnish travelers (III)

Prlor thls study, no lnformutlon exlsted on the sero or genotypes of
dengue vlruses lnfectlng llnnlsh truvelers. A vlrus lsolutlon upprouch wus tuken
wlth the ulm of lsolutlng und churucterlzlng these vlruses. 1he vlrus lsolutlon wus
currled out ln purullel ln cultured mosqulto (C6j6) und mummullun cells (vero
L6), both of whlch ulso could support the growth of fluvlvlruses other thun LLNv,
thut could be the posslble cuuse for the observed seroposltlvlty of the putlents.
A totul of c serum sumples were selected from putlent sumples collected
ln zcc for the lsolutlon trlul, uslng u crlterlon of un lgC tlter less or equul to
zc, und the flrst uvulluble sumple from u glven putlent. 1he vlrus lsolutlon trlul
cells were observed for cytoputhlc effects, und hurvested when necessury. ln
ubsence of u CPL, the cells were pussuged on duy , und hurvested on duy z. 1he
cells from duys , und z post lnfectlon were studled for the presence of vlrul
untlgen ln lmmunofluorescence ussuy (llA) uslng LLNv type speclflc MAbs ().
lrom the llA posltlve culture supernutunts, RNA wus extructed und umpllfled ln
LLNv typlng R1PCR (z,). 3used on the typlng R1PCR und MAb llA, u totul of
LLNv were lsoluted lncludlng LLNv, z LLNvz, LLNv und z LLNv (1uble
). 1he vlrus lsolutlon posltlve sumples were obtulned from putlents uged zz6,
lncludlng 6 femules und mules.
1he vlrus strulns dlffered ln thelr growth puttern ln the two cell llnes, of
the lsolutes only grew ln mosqulto cells, und bused on the low umount of posltlve
cells seen ln llA, the serum sumples elther contulned u low umount of vlrus, or the
vlrus strulns grew poorly n vrro. 6 of the lsolutes grew ln both cell llnes. 3used on
the proportlon of posltlve cells observed ln llA, only one struln grew equully well
ln both cell llnes, und the rest of the duul troplc vlruses uppeured to grow better
ln mummullun thun ln mosqulto cells, us deflned by the umount of posltlve cells
6
observed ln llA. Eowever, even lf some strulns seemed to prefer the mummullun
cells, none of the strulns fulled to grow ln C6j6 mosqulto cells. Cur overull
lsolutlon results conflrm thut C6j6 mosqulto cells ure more sensltlve thun
mummullun cells ln lsolutlon of LLNv from putlent sumples (8). 1he culture
propertles of some strulns preferrlng mummullun cells ls lnterestlng, und merlts
further lnvestlgutlon us the culture propertles could be reluted to the uduptutlon
to prlmute hosts und puthogenlclty for humuns.
1he R1PCR product umpllfled ln dengue vlrus typlng R1PCR (z,) from the
CpreM reglon ( bp) wus sequenced und used for the phylogenetlc unulysls.
1he results showed the strulns to represent ull four serotypes und vurlous
genotypes (llgure ), mostly flttlng to the uvulluble truvel lnformutlon of the
putlents (llgure z). lour LLNv vlruses were lsoluted from truvelers returnlng
from Aslutlc orlglns, representlng two dlfferent genotypes, the Aslun genotype l
und the AmerlcunAfrlcun genotype v (ulso referred to us genotype lll) (8z). 1he
two LLNvz lsolutes were obtulned from truvelers returnlng from Chunu und Srl
Lunku, represented by Cosmopolltun und Aslun genotypes. 1he three LLNv
vlruses orlglnuted from Cubu, 3ruzll und Srl Lunku, ull representlng genotype lll.
1he two LLNv lsolutes from Srl Lunku und lndoneslu represented genotypes l
und ll respectlvely. Uptodute lnformutlon on dengue epldemlology ln Afrlcu ls
currently lncomplete, however, recent reports show lncreuslng recognltlon of
LLNv ln severul Afrlcun countrles (88). lnformutlon obtulned from LLNv
lnfected truvelers cun provlde uptodute lnformutlon on epldemlcs (86) und
novel vlrus types (8,). 1he flrst lsolutlon of LLNvz from Chunu demonstrutes the
potentlul of truvelers to uct us sentlnels for LLNv ln ureus thut do not huve
nutlonul survelllunce systems.
,c

1uble . Lengue vlrus lsolutes from llnnlsh truvelers.

Cell culture virus isolation

vero L6

C6j6
Patient
sex and
age
Isolate
no.
Serotype Origin
country
Year
Days post
infection
IIA
result
Days post
infection
IIA
result
ljz LLNv 1hullund zccz , +++ , +++
lj LLNv Muluysluj
1hullund
zccz ,, z z +
lj6 8 LLNv 1hullund zcc ,, z z +
Mj LLNv lndlu zcc +++ +++
Mj 6 LLNvz Srl Lunku zcc ,, z z +
ljzz LLNvz Chunu zcc , +++ , +
Mj z LLNv Cubu zccz , + z +
Mjz6 LLNv 3ruzll zcc ,, z +
lj , LLNv Srl Lunku zcc +++ z +
ljz LLNv Srl Lunku zccc c +++ c +
Mj, c LLNv lndoneslu zcc ,, z z +

llgure z. Crlglns of LLNv lsolutes from llnnlsh truvelers ln zccczcc. 1he ureus endemlc for
dengue vlruses ln zcc ure shuded ln grey (modlfled from CLC, zcc).
,

llgure . Nelghborjolnlng phylogenetlc tree bused on CpreM reglon ( bp) nucleotlde
sequences of ,c dengue vlrus strulns. ccc bootstrup repllcutlons were conducted, the support
vulues ubove c ure lndlcuted. lsolutes from llnnlsh truvelers ure murked ln grey.
,z

q.z.z A collection of DLNvz strains from an endemic country, venezuela
(Iv)

Cf ull the countrles ln Lutln Amerlcu, venezuelu reports the hlghest
number of LEl cuses (88). 1he mujor epldemlcs wlth LEl huve occurred ln
venezuelu slnce the 8cs, und huve been ussocluted wlth the lntroductlon of
Aslutlcorlgln LLNvz. Slmllur to other countrles ln the reglon, ln venezuelu the
Aslutlc LLNvz vlrtuully repluced the serotype thut clrculuted ln the ureu
prevlously, deslgnuted us the Amerlcun genotype LLNvz (8,c), whlch wus
not ussocluted wlth severe dlseuse (). Slnce the lntroductlon of the llneuge of
Aslutlc orlgln, referred to us AmerlcunAslun genotype (z), lt hus been shown to
dlverslfy lnto dlfferent llneuges ln venezuelu. Addltlonully, recomblnunt vlrus
between the Aslutlc und AmerlcunAslun genotypes hus been detected from
venezuelu (8).
A colluborutlon wlth venezuelun reseurchers provlded u collectlon of
LLNvz lsolutes for thls study from Aruguu Stute, venezuelu (llgure ) whlch
ulmed ut updutlng the epldemlologlcul sltuutlon of thls serotype ln venezuelu.
venezuelu ls currently u hyperendemlc reglon for the four LLNv types (). At
the tlme of the prevlous phylogenetlc study of LLNvz ln venezuelu, lncludlng
lsolutes from ,zccc, LLNvz wus one of the predomlnunt serotypes detected
ln epldemlcs und ussocluted wlth LEl (8). Lurlng the lust decude, u chunge ln
LLNv serotype domlnunce hus been observed ln venezuelu, und LLNv und
LLNv huve surpussed LLNvz us the most prevulent genotypes. Eowever, LLNv
z ls stlll constuntly detected ln putlents. ln thls study, the envelope (L) gene
sequences were determlned for z novel LLNvz strulns lsoluted ln zcc ln
Aruguu Stute (llgure ). 1he sequence unulysls lncluded ull the uvulluble LLNvz
strulns from venezuelu lncludlng recent sequences untll zcc8. Addltlonully, u
globul collectlon lncludlng representutlves of ull 6 known genotypes of LLNvz
were lncluded ln the unulysls.
,
1he results of the sequence unulysls suggest thut LLNvz evolutlon hus
contlnued ln venezuelu, und ull the lsolutes represented the AslunAmerlcun
genotype. ln compurlson to prevlously publlshed sequences und thelr unulysls,
genetlc dlverslflcutlon wus detectuble, us the venezuelun lsolutes were now
sepuruted lnto sepurute llneuges (llgure ). 1he envelope proteln sequence
compurlsons reveuled severul umlnoucld chunges, some of them found
excluslvely ln venezuelun lsolutes. 1he ldentlfled vlrus llneuges could not be llnked
wlth dlseuse severlty, us ull llneuges lncluded lsolutes from both Ll und LEl
cuses. All the LLNvz lsolutes studled here represented the sume genotype, whlch
wus ulreudy known to clrculute ln venezuelu. Cur results bused on lsolutes from
Aruguu stute suggests, thut unllke ln severul other countrles ln the South
Amerlcun reglon, no recent lntroductlon of forelgn LLNvz llneuges huve occurred
ln venezuelu. 3used on the prevlous musslve LLNvz epldemlcs ln venezuelu, lt
could be speculuted thut the mujorlty of the udult populutlon ls llkely to be
lmmune to thls serotype, leuvlng only the younger generutlons vulneruble to
LLNvz lnfectlon. 1he populutlon lmmunlty could restrlct the vlrus trunsmlsslon
und leud to serotype replucement. 1he mujorlty of the vlrus lsolutes studled here
orlglnuted from young putlents uged less thun zc yeurs, however, lsolutes were
ulso obtulned from putents representlng older uge groups (1uble ). 1he
epldemlologlcul fuctors enubllng the sustulned mulntenunce of severul llneuges of
LLNvz us mlnor serotype ln the coclrculutlon of the four serotypes ln venezuelu
ure not currently known. 1hose could lnclude mechunlsms lndependent from
humun populutlon lmmunlty, such us the mulntenunce of the vlrus ln mosqulto
populutlons.
,

1uble . venezuelun LLNvz strulns.
Isolate Strain name MonthjYear Crading Sex Age
Lzdcc LARL6c c8jzccc Ll M 8
Ldc LARL68 c6jzcc LEl l 6
L6d LARLc, j Ll l
L,dc LARL8 cjzcc Ll l
L8dc LARLz68 c,jzcc Ll l
Ldc LARL86 czjzcc Ll l
Lcdc LARLz6c6z cjzcc LEl M
Ldc LARLz zjzcc Ll l
Lzdc LARLz,c, c8jzcc Ll l 6
Ldc LARLz8c cjzcc Ll M z
Ldc LARLzz6 c8jzcc Ll l ,
L6dc LARLz6688 c,jzcc LEl M
L,dcc LARL cjzccc Ll M
L8dc LARLc cjzcc Ll M z
Lzcdc LARLz6c8 cjzcc Ll M 6
Lzdc LARL8z czjzcc Ll l 6
Lzzdc LARL8 czjzcc Ll M
Lzdc LARL68 cjzcc Ll l z
Lzdc LARLz,, cjzcc Ll M
Lzd LARLz zj LEl M 8,
Lz6d LARLz86 cj Ll l
Lz8dc LARLzzccc zjzcc Ll l z
Lzd LARLc zj Ll M c

llgure . Mup of the geogruphlcul locutlon of Aruguu Stute, venezuelu.
,

llgure . Nelghborjolnlng phylogenetlc tree bused on envelope gene sequences of 6c LLNvz.
ccc bootstrup repllcutlons were conducted, the support vulues ubove c ure lndlcuted. 1he z
venezuelun LLNvz lsolutes sequenced ln thls study ure murked ln grey.
concludng remarks and jurure rosecrs
,6
Concluding remarks and future prospects

1he lust decudes huve shown thut even lf dengue ls not u new dlseuse, ln u
relutlvely short tlme, lt hus become u mujor vectorborne vlrul dlseuse of munklnd.
Currently, no effectlve und sufe vucclnes ure uvulluble ugulnst dengue. 1he
geogruphlcul spreudlng of dengue vlruses requlres constunt monltorlng ln the rlsk
ureus thut huve sultuble mosqulto vectors (Ae. aegyr, Ae. albocrus) reudlly
uvulluble, lncludlng purts of Lurope.
1he effects of dengue vlrus lnfectlon reuch fur beyond the endemlc ureus,
us dengue ls ulso lncreuslngly uffectlng truvelers vlsltlng these ureus. 1he
populurlty of SouthLust Aslun tourlst uttructlons umong llnnlsh truvelers huve
cleurly lnfluenced the observed rlse ln dlugnosed cuses. Eowever, thls study hus
shown the orlglns und cuusutlve ugents of dengue lnfectlon ln llnnlsh truvelers to
lnclude vurlous endemlc ureus und ull LLNv serotypes. vlthln thls study, u novel
LLNv RNA detectlon method wus developed for routlne dlugnostlc use und
compured to other eurly dlugnostlc tests. vlth the truveler sumples, lt wus
evldent thut the best dlugnostlc rutes were obtulned by comblnlng lgM detectlon
to vlrul NS untlgen or RNA detectlon.
lortunutely, the mujorlty of the lnfectlons of truvelers ure mlld und severe
dlseuse und futulltles ure extremely rure. Eowever, lt seems llkely thut the
repeuted truvellng to endemlc ureus wlll lncreuse the occurrence of secondury
lnfectlons, whlch muy cuuse more severe dlseuse outcomes ln truvelers ln the
future. Currently, no speclflc drug ugulnst dengue ls uvulluble, however, lt ls
lmportunt to dlugnose dengue ln truvelers, us the dlugnosls ls u prerequlslte for
the monltorlng und hospltullzutlon of the putlent ln the cuse thut the dlseuse
turns worse. 1herefore, ln the future, rupld und relluble dlugnosls for dengue wlll
lncreuslngly be ln demund. Addltlonully, when lnformed ubout the potentlul rlsks
of secondury lnfectlons, the putlent muy tuke the mosqulto preventlon meusures
serlously the next tlme when truvellng ln the endemlc ureus.
1he greutest effects of dengue to publlc heulth ure seen ln the ureus thut
ure endemlc for these vlruses. luctors such us the populutlon lmmunlty stutus und
concludng remarks and jurure rosecrs
,,
vector competence ure llkely to uffect LLNv serotype prevulence und thelr co
clrculutlon, however, thelr effects ure not well known. 1he study of dengue vlrus
strulns from the endemlc ureus, both from truvelers or locul putlents, ls therefore
lmportunt und provldes lnformutlon thut could prove useful ln the future
preventlon of the dlseuse. 1he mupplng of the endemlc ureus und the knowledge
of the dlverslty of the clrculutlng dengue vlruses ure needed for plunnlng vector
control meusures und for developlng vucclnes und drugs. A dengue vucclne ls
llkely to become uvulluble ln the future, however, lt muy turn out too expenslve
for the people who llve ln the endemlc ureus. vector control ls un effectlve
meusure ugulnst dengue, und requlres more knowledge thun money. 1ruvel
ugencles huve udvertlsed thelr efforts ln tuklng cure of envlronmentul lssues
cuused by tourlsm ln the truvel destlnutlons. 1hls could be well extended to
vector control efforts ln dengue endemlc ureus, us these meusures would prevent
dengue und other mosqultotrunsmltted dlseuses ln both truvelers und resldents
ln the ureu.
1he recent developments on dengue dlugnosls, such us the NS untlgen
detectlon ln u rupld test formut huve mude the ussuy ltself eusy, but stlll u serum
sumple ls needed us u test muterlul requlrlng speclul equlpment. 1he mujorlty of
descrlbed dlugnostlc methods for dengue use putlent serum us the sumple
muterlul, ulthough evldence of detectuble umounts of vlrul components und
untlbodles hus been shown from other sumple types, such us sullvu. Luslly
collectuble, nonlnvuslve sumple muterluls merlt further studles on thelr potentlul
us dlugnostlc sumple muterlul wlth novel methodologles, such us NS untlgen und
RNA detectlon.

Acknowledgemenrs
,8

Acknowledgements

1hls study wus currled out ut Lepurtment of vlrology ln Euurtmun
lnstltute, Unlverslty of Eelslnkl. l wurmly thunk my thesls supervlsors, Professor
Olli vapalahti und Locent Beli Piiparinen for guldlng me through my thesls work.
l thunk Professor Clll vupuluhtl for ull the dlscusslons, support und everlustlng
optlmlsm. l um gruteful for the trust und freedom l huve hud to curry out
experlments und to follow my own puth. l thunk Locent Eell Pllpurlnen for
putlently provldlng unother polnt of vlew to my projects und for encouruglng me
to prlorltlze projects for completlng the thesls. l grutefully thunk Lr. Nathalie
Uzctegui for the essentlul role ln my thesls work, l cunnot thunk you enough for
the lnsplrutlon, guldunce, help und frlendshlp.
l wunt to thunk professor emerltus Antti vaheri for the support und udvlce
durlng these yeurs, und the heud of the Lepurtment, professor kalle Saksela for
provldlng the reseurch fucllltles to conduct thls work. 1he thesls commlttee
members, Locent Mai[a Lappalainen und Locent Mer[a Roivainen ure thunked for
the dlscusslons und thesls commlttee meetlngs. Locent Merju Rolvulnen ulso
revlewed thls thesls together wlth Locent Mika Salminen, both of whom l wlsh to
thunk for thelr vuluuble comments und suggestlons. Lr. Christopher Carroll ls
thunked for revlslng the thesls lunguuge.
l thunk my cllnlcul colluborutors Locent Anu kantele, Lr. Beli Siikamki
und Lr. Llina Lrra for the jolnt efforts ln collectlng putlent dutu und Sirkka vene
und Professor Lrnest Could for klndly provldlng reseurch muterluls und protocols.
1he couuthors Sakari vuorinen, Bannu Baapasalo, 1ukka Lumio, Simo Nikkari,
Cuillermo Comach, Cloria Sierra, Dara Camacho und Omar Aguirre ure thunked
for contrlbutlons ln the thesls urtlcles.
l um gruteful to the people of the vlrul 2oonoses lub for thelr help und
support. l wunt to thunk especlully 1ytti Manni for the guldunce und frlendshlp.
You showed me ln pructlce how excltlng lt ls to work wlth cells und vlruses, und
tuught me lmportunt clusslc vlrologlcul technlques.
Acknowledgemenrs
,
l wunt to thunk ulso Auli Saarinen, Irina Suomalainen, Pir[o Sar[akivi,
Leena kostamovaara und the ludles ut EUSLA3 2oonosls unlt, Ir[a Luoto, kirsti
Rih und Minna Ulmanen for ull the help und skllled technlcul usslstunce durlng
these yeurs.
l thunk ull the student fellows of the vlrul zoonosls group for muklng the
long duys ln the lub, on fleld und on the conference trlps so much more fun.
Neurly uny reuson wus enough for u llttle spontuneous purty wlth spurkllng wlne.
l wunt to especlully thunk Lssi Basu for the frlendshlp und the efforts ln our jolnt
projects. Support und coffee wus ulwuys served ulong wlth endless gosslps, ldeus
or solutlons to problems ut the
th
floor coffee room wlth the compuny of Suvi
kuivanen, 1ar[a Sironen, Anne 1skelinen, Satu kurkela, Niina Putkuri, Liina
voutilainen, Anu 1skelinen, Anna katz und Niina kivi. ln uddltlon, l huve hud
greut support durlng these yeurs ulso from the rest of the present und pust
members of the vlrul zoonoses reseurch group (und beyond). l wunt to thunk Lr.
kati Rsnen for the shured thesls lncubutorperlod und my fellow students
from the undergruduute yeurs, Sanna kuningas und 1erhi Peuralinna for thelr
understundlng, frlendshlp und cosupport.
Above ull, l thunk deurly my support teum outslde the lub, my fumlly und
frlends, for thelr slncere cure und love. 1hls work would not huve been posslble
wlthout you.

1hls study wus flnunclully supported by Eelslnkl 3lomedlcul Cruduute School, Crlon
lurmos Reseurch loundutlon, llnnlsh loundutlon lor Reseurch Cn vlrul Llseuses, llnnlsh
Soclety for Study of lnfectlous Llseuses, 3lomedlcum Eelslnkl loundutlon, Centre for
3lothreut Prepuredness, 1ekes und Acudemy of llnlund.

Eelslnkl, September zcc,

Llll Euhtumo
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ejerences
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ejerences

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dengue lnfectlon on u Puclflc lslund. Am.'.1rop.Med.Eyg. , Muy,z():c6.
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huemorrhuglc fever ln 1hullund from to zccz. Lpldemlol.lnfect. zcc 'un,():c
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)z. 'ensenlus M, 3erlld L, Crmuusen v, Muehlen ', Llndegren C, lulk ll. lutul
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from 1hullund. Scund.'.lnfect.Lls. zcc,,(c):,,6,,8.
)q. Cunhu 3A, 'ohnson L, McLermott 3. Atyplcul dengue fever mlmlcklng typhold fever
ln u college student truveler. Am.'.Med. zcc Apr,zz():e.
). 3errlngton vR, Elttl ', Cusper C. A cuse report of dengue vlrus lnfectlon und
uculculous cholecystltls ln u pregnunt returnlng truveler. 1ruvel Med.lnfect.Lls. zcc,
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)6. 3omers Ml, Lettlngu lL, vun Corp LC, Murtlnu 3L, Peerbooms PC, veenstru '.
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)8. Sudlro 1M, 2lvny ', lshlko E, Creen S, vuughn Lv, luluyunurooj S, et ul. Anulysls of
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). lumurusumy v, Chuu Sl, Eussun 2, vuhub AE, Chem Yl, Mohumud M, et ul.
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dlugnosls of ucute dengue vlrus lnfectlon. Slngupore Med.'. zcc, 'ul,8(,):666,.
8o. Eung v1, Nguyet NM, 1rung L1, 1rlcou v, Yoksun S, Lung NM, et ul. Llugnostlc
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8. Esleh C', Chen M'. 1he commerclul dengue NS untlgencupture LLlSA muy be
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8z. ANuegoonplput A, 3erllozArthuud A, Chow v, Lndy 1, Lowry l, Mul le Q, et ul.
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Crgnal ublcarons
6

Original publications

ARTICLES
THE LANCET Vol 355 March 25, 2000 1053
Summary
Background Severe forms of dengue, the most important
arboviral infection of man, are associated with
haemorrhagic disease and a generalised vascular leak
syndrome. The importance of dengue as a cause of
neurological disease is uncertain.
Methods During 1995, all patients with suspected CNS
infections admitted to a referral hospital in southern
Vietnam were investigated by culture, PCR, and antibody
measurement in serum and CSF for dengue and other
viruses.
Findings Of 378 patients, 16 (42%) were infected with
dengue viruses, compared with four (14%) of 286 hospital
controls (odds ratio [95% CI] 31 [1758]). Five additional
dengue positive patients with CNS abnormalities were
studied subsequently. No other cause of CNS infection was
identified. Seven infections were primary dengue, 13
secondary, and one was not classified. Ten patients had
dengue viruses isolated or detected by PCR, and three had
dengue antibody in the CSF. 12 of the 21 had no
characteristic features of dengue on admission. The most
frequent neurological manifestations were reduced
consciousness and convulsions. Nine patients had
encephalitis. No patient died, but six had neurological
sequelae at discharge. Phylogenetic analysis of the four
DEN-2 strains isolated mapped them with a DEN-2 strain
isolated from a patient with dengue haemorhagic fever, and
with other strains previously isolated in southern Vietnam.
Interpretation In dengue endemic areas patients with
encephalitis and encephalopathy should be investigated for
this infection, whether or not they have other features of
the disease.
Lancet 2000; 355: 105359
Introduction
Dengue is the most important arboviral infection of
man, with an estimated 100 million cases per year and
25 billion people at risk.
1,2
Infection presents classically
as dengue fever, a self-limiting but severe influenza-like
illness, or dengue haemorrhagic fever (DHF). In
southeast Asia, this is a disease predominantly of
children and characterised by increased vascular
permeability, plasma leakage, haemorrhagic
manifestations, and thrombocytopenia.
2
Epidemiological
evidence suggests that DHF is most likely when
infection with one dengue serotype is followed by a
secondary infection with a different serotype. Because of
controversy over whether dengue viruses cause
neurological disease,
36
we investigated prospectively the
role of dengue in acute nervous system infections in
southern Vietnam.
Patients and methods
Patients
The study was conducted on the intensive-care units at the
Centre for Tropical Diseases, Ho Chi Minh City, an infectious
disease referral hospital for much of southern Vietnam, a region
where dengue and Japanese encephalitis are endemic. The
study protocol was approved by the hospitals scientific and
ethical committee, and consent was obtained from the patient
or accompanying relative.
From Jan 1 to Dec 31, 1995, all children (under 15) and
adults with a suspected CNS infection were studied. CNS
infections were suspected in patients with a fever or history of
fever, and at least one of the following: reduced level of
consciousness (Glasgow coma score 14, or for children below
6 years, Blantyre coma score 4);
7
severe headache; neck
stiffness; focal neurological signs; tense fontanelle; or
convulsions. Patients with slide-positive cerebral malaria or
clinical features of tetanus were admitted to specialised wards
and were not included in this series. Nor were children between
6 months and 5 years of age with a simple febrile convulsion
(defined as a single convulsion lasting less than 15 min with
recovery of consciousness within 60 min).
A full history included details of drugs and other potential
CNS toxins (alcohol, recreational drugs). A detailed clinical
examination, including full neurolgical examination, was done
every day by a member of the study team until the patients
discharge. At lumbar puncture opening pressures were
measured, and CSF was taken for cell count and differential,
protein, glucose, Gram stain, and bacterial and viral culture.
Blood was taken for haematocrit, examination for malaria
parasites, platelet count, differential white cell count, blood
cultures, biochemical screen, and viral serology.
8
Patients with reduced consciousness were defined clinically
as having encephalitis if there was no metabolic abnormality or
other apparent explanation, and if they had any of the
following: CSF pleocytosis (corrected white cell count >5/L),
focal neurological signs, or convulsions other than simple febrile
convulsions.
9
If they had none of these features they were
considered to have an acute encephalopathy.
9
Convulsions were
treated with intravenous diazepam; repeated convulsions were
treated with intravenous phenobarbitone. Patients with clinical
signs of raised intracranial pressure were treated with mannitol.
Neurological manifestations of dengue infection
Tom Solomon, Nguyen Minh Dung, David W Vaughn, Rachel Kneen, Le Thi Thu Thao, Boonyos Raengsakulrach,
Ha Thi Loan, Nicholas P J Day, Jeremy Farrar, Khin S A Myint, Mary J Warrell, William S James, Amanda Nisalak,
Nicholas J White
Wellcome Trust Clinical Research Unit (T Solomon MRCP,
R Kneen MRCP, N P J Day MRCP, J Farrar MRCP, Prof N J White FRCP)
and Centre for Tropical Diseases (N M Dung MD, L T T Thao MD,
H T Loan MD), Cho Quan Hospital, Ho Chi Minh City, Vietnam;
Department of Virology, US Army Medical Component, Armed
Forces Research Institute of Medical Sciences, Bangkok,
Thailand (D W Vaughn MD, B Raengsakulrach PhD, K S A Myint MD,
A Nisalak MD), Sir William Dunn School of Pathology, University
of Oxford, UK (M J Warrell MRCPath, W S James DPhil); and Centre
for Tropical Medicine and Infectious Diseases, Nuffield
Department of Clinical Medicine, John Radcliffe Hospital,
Oxford, UK (T Solomon, R Kneen, N P J Day, J Farrar, N J White)
Correspondence to: Dr Tom Solomon, Department of Neurological
Science, University of Liverpool, Walton Centre for Neurology and
Neurosurgery, Lower Lane, Fazakerley, Liverpool L9 7LJ, UK
(e-mail: tom.solomon@virgin.net)
http://users.path.ox.ac.uk/~wjames/Solomon%20et%20al%20Lancet.pdf
Suspected septicaemia was treated with a third-generation
cephalospoin and gentamicin.
Patients admitted with DHF and no CNS abnormalities were
graded IIV using WHO criteria, modified to allow for manual
platelet counting. Patients with dengue shock syndrome (DHF
grades III and IV) were given Ringers lactate and dextran 40.
2
Following the one year study, when computer tomography
became available, five further patients with neurological
manifestations of dengue were investigated, and are included in
this report.
Virological and serological studies
IgM and IgG antibodies to dengue and to Japanese encephalitis
virus were measured in acute and convalescent sera and in CSF
using a double sandwich capture ELISA.
10
For single serum
samples 40 units of IgM to dengue (with dengue IgM greater
than Japanese encephalitits virus IgM), or for paired samples a
rise from less than 15 to more than 30 units was considered
evidence of acute dengue infection.
10
An IgM/IgG ratio of 18/1
or more was considered evidence for a primary dengue infection,
whilst a ratio below 18 was considered evidence for a secondary
infection.
10
IgG >100 units with IgM between 20 and 40 units
was considered evidence of recent secondary dengue infection.
For serological studies we recruited as controls 120 children
with diphtheria and 166 adults with typhoid sequentially
admitted to specialised wards. At this hospital both diseases
have geographical distributions and referral patterns similar to
those for CNS infections.
To type the dengue infections, virus isolation and nucleic
acid amplification were performed on sera and CSF. About 15
live Toxorrhyncites splendens mosquitoes were injected with 034
L of undiluted sample. After 14 days about 10 surviving
mosquitoes were tested for flavivirus antigen by indirect
fluorescent antibody assay of the head.
11
Virus positive
mosquitoes were used to infect Aedes albopictus C6/36 cell
cultures for identification of virus type using a panel of
monoclonal antibodies against dengue and Japanese
encephalitis virus in an ELISA.
12
Dengue virus RNA was
amplified by reverse transcriptase nested PCR of CSF and
serum.
13
Isolation of or PCR detection of dengue viruses in CSF
or a CSF anti-dengue IgM titre above 30 units were considered
diagnostic of CNS infection.
14,,15
Culture and PCR were not
done on controls.
To exclude other viral causes of CNS infection, CSF was
inoculated into Vero cells and rhesus monkey kidney (LLC-
MK2) cells, and examined by nested PCR for evidence of
enteroviruses, herpes, measles, mumps, or Epstein-Barr virus,
and cytomegalovirus.
16
CSF and acute serum were also
examined for Leptospira by PCR.
17
Paired sera were assayed for
antibodies to enteroviruses, herpes, measles, and mumps virus,
cytomegalovirus, and Mycoplasma pneumoniae using
complement fixation tests; for leptospirosis using the
microagglutination test; and for Salmonella typhi using the
Widal test. Single serum samples were assayed for Epstein-Barr
virus and HIV by IgM ELISAs.
Sera from patients with raised bilirubin and liver
transaminases were tested for hepatitis A virus IgM; IgM to
hepatitis B virus core and surface antigens; and hepatitis C virus
total immunoglobulins using enzyme immunoassays (HAVAB-
M EIA; Corzyme-M, and AUSYME monoclonal assays; HCV
EA 2nd Generation [Abbott Laboratories]).
Phylogenetic analysis
To investigate whether DEN-2 isolates in this study were likely
to represent strains circulating in southern Vietnam or new
imported strains, we contructed phylogenetic trees for isolates
of DEN-2 viruses cultured from the CSF and serum of two
patients. A 240 base-pair fragment encoding the junction
between the envelope and NS1 (non-structural) protein genes
was sequenced
18
from published primer pairs.
19
Fragments were
compared with those of a DEN-2 isolate from a child with
classical DHF, and with 181 homologous sequences of DEN-2
viruses in the GenBank database using the program ClustalX.
Phylogenetic trees were displayed using NJPlot.
Statistics
The odds ratio (OR) with 95% confidence interval (CI) was
used to express the strength of the association between a
neurological presentation and the dengue result. Diferences
between proportions were tested by Fishers exact test (Statview
4.02; Abacus Concepts).
Results
Epidemiology
378 patients (228 adults 150 children) with suspected
CNS infections were admitted to the hospital. 16 (42%;
nine adults and seven children) were infected with
dengue viruses compared with four (14%, all children
with diphtheria) of 286 matched hospital controls (OR
ARTICLES
1054 THE LANCET Vol 355 March 25, 2000
Patient Anti-dengue ELISA (units)* Virus isolation Dengue PCR Dengue summary
Acute serum Convalescent serum Interval CSF
IgM IgG IGM IgG (days) IgM IgG
1 67 17 53 19 7 0 0 Neg Neg Acute primary
2 3 5 0 1 Den-2, serum & CSF Neg Acute Den-2
3 11 8 74 286 7 0 0 Den-2, serum & CSF Den-3, CSF Acute secondary Den-2
4 65 399 12 258 21 Neg Neg Acute secondary
5 27 47 116 268 6 Den-2, serum Neg Acute secondary Den-2
6 50 304 4 212 9 33 327 Neg Neg Acute secondary
7 116 216 0 9 Neg Neg Acute secondary
8 116 279 86 224 19 11 372 Neg Den-3, serum Acute secondary Den-3
9 2 0 198 351 11 0 13 Den-3, serum Neg Acute secondary Den-3
10 48 10 34 3 Neg Neg Acute primary
11 25 133 11 132 13 22 134 Recent secondary
12 164 325 82 307 12 Neg Den-3, serum Acute secondary Den-3
13 51 216 20 223 Neg Neg Acute secondary
14 0 0 176 43 11 4 0 Den-1, serum Den-1, serum Acute primary Den-1
15 213 5 Acute primary
16 210 50 0 0 Neg Neg Acute primary
17 1 4 115 331 6 40 15 Neg Den-2, serum & CSF Acute secondary Den-2
18 25 258 23 225 7 8 310 Neg Neg Recent secondary
19 2 0 249 31 7 0 10 Den-3, serum Den-3, serum & CSF Acute primary Den-3
20 41 127 42 250 7 0 8 Neg Neg Acute secondary
21 166 27 Den-3, serum Neg Acute primary Den-3
*40 or more, or rise from <15 to >30 is diagnostic of dengue infection.
IgM: IgG ratio >18 defines primary infection; <18 defines secondary infection.
Table 1: Virological studies on 21 patients with neurological manifestations of dengue infection
31 [1758], p=0039). CNS patients with other
diagnoses have been reported previously.
8,20
During the
same period 1675 patients (1405 children) were
admitted with clinically diagnosed DHF of whom 296
(18%, 277 children) had dengue shock syndrome,
including 10 (06%, 9 children) with DHF grade IV.
Thus a neurological presentation occurred in 16 (1%) of
1691 patients admitted with suspected dengue infection.
Virology and serology
The 21 patients (16 from the one year study and the five
subsequent patients) are summarised in table 1. DEN-3
was isolated from the serum of three patients, DEN-2
from the serum of one and from serum and CSF of two
patients, and DEN-1 from the serum of one patient. In
six cases dengue virus RNA was detected by PCR. The
CSF samples positive for dengue virus were not bloody
ARTICLES
Patient Presenting symptoms and signs Coma score Admission Progress and outcome Neurological diagnosis
(see text) dengue grade
1 (M/19) Fever (385C) 3 days; headache; delirium 14/15 None Recovered over 9 days Encephalopathy
2 (M/6 mo) Fever (39C) for 3 days; vomiting; diarrhoea; 4/5 None Recovered and discharged within 48h Encephalopathy
drowsy; bulging fontanelle
3 (M/18) Fever (39C) for 2 days; headache; vomiting; 15/15 DF Recovered over 7 days Meningism
neck stiffness; petechial rash
4 (F/20) Fever (395C) for 5 days; headache; vomiting; 10/15 DHF III Developed arm dyskinesias; lip smacking; Hepatic encephalopathy
neck stiffness; coma; rash; hypotensive; pleural effusions; ascites; jaundice; bleeding
haemoconcentration diathesis; haemoglobinuria, anaemia requiring 2
unit blood transfusion; recovered over 15 days
5 (M/4) Fever (402C) for 6 days; anorexia; vomiting; 4/5 DHF III Recovered; discharged after 13 days Encephalopathy
diarrhoea; restless; narrow pulse pressure;
mottled skin; irritable; high pitch scream; jaw
jerk; pout reflex; hepatomegaly;
haemoconcentration, petechiae, bruising
6 (F/14) Fever (378C) for 4 days; muscle pain; no spinal 15/15 None Improved, but still mild spastic paraparesis at Transverse myelitis
tenderness; spastic paraparesis; power 1/5 on discharge, day 18
right, 3/5 on left; sensory level at T10; acute
retention of urine
7 (F/23) Fever (387C) for 5 days; vomiting; diarrhoea; 9/15 DHF III Anaemia required 2 unit blood transfusion; Hepatic encephalopathy
abdominal pain; hypotensive; coma; recovered over 6 days; discharged day 23
hepatosplenomegaly; jaundice; pleural effusions;
petechiae; bruising
8 (F/19) Fever (395C) for eight days; abdominal pain; 13/15 DHF III Coma score deteriorated to 5; developed Hepatic encephalopathy
diarrhoea; hypotension; petechial rash; bleeding downward deviation of gaze; DIC; pleural
from venepuncture sites; delirium effusions; peripheral oedema; acute renal failure
(Cr 80) treated with peritoneal dialysis; slow
recovery; at discharge (day 33) abnormal affect
and changed personality
9 (M/6) Fever (39C) for 2 days; anorexia; confused; 10/15 None Improved over 9 days; residual leg spasticity; Encephalitis
many generalised convulsions; coma; extensor dengue recovery rash
plantars
10 (M/21) Fever (39C) for 5 days; rigors; headache; 15/15 None Power recovered over 7 days, but brisk reflexes Transverse myelitis
muscle pains; spastic paraparesis, no spinal and clonus remained
tenderness
11 (F/25) Fever for 5 days; headache; stiff neck; confused 10/15 None Recovered over 20 days Encephalopathy
then mute
12 (F/39) Fever (38C) for 6 days; rigors; headache; 4/15 DHF III Further generalised convulsions; rigidity spasms; Hepatic encephalopathy
vomiting; neck stiffness; petechial rash; skin recovered over 15 days
haemorrhage; hypotension; jaundice;
haematemesis; coma; many generalised
convulsions
13 (M/20) Fever (395C) for 8 days; headache; vomiting; 8/15 DF Remained confused at discharge (day 7) Encephalitis
neck stiffness; several generalised convulsions;
coma; bruising; bleeding from venepuncture sites
14 (M/1) Fever (395C) for 1 day; cough; coryza; 3 4/5 None Recovered over 24 h; dengue recovery rash Encephalitis
generalised convulsions; drowsy
15 (M/9 mo) Fever (388C) for 7 days; cough; coryza; 1 4/5 DHF II Recovered over 48 h Encephalitis
generalised convulsion; drowsy; fixed flexion of
arms; haemoconcentration; petechial
rash/purpura
16 (M/3 mo) Fever (395C) for 4 days; cough; several R 1/5 None Recovered over 5 days Encephalitis
sided focal convulsions; coma; fixed flexion of
arms
17 (F/11) Fever (385C) for 4 days; headache; anorexia; 11/15 None Developed DHF III after 24 h; recovered over Encephalitis
coma; hepatomegaly; extensor plantars 5 days
18 (M/7) Fever (395C) for 4 days; headache; anorexia; 12/15 None Recovered over 5 days Encephalitis
neck stiffness; 1 generalised convulsion; coma;
spastic arms and legs; extensor plantars;
intermittent tremors
19 (M/12) Fever (403C) for 3 days; headache; anorexia; 14/15 None Recovered over 48 h; dengue recovery rash Encephalitis
vomiting; confusion; 2 generalised convulsions;
frontal release signs (jaw jerk, grasp reflex)
20 (M/30) Fever (40C) for 4 days; headache; anorexia; 7/15 None Improved, but poor short term memory, Encephalitis
dizziness; coma; brisk leg relexes; left plantar personality change, and brisk leg reflexes at
extensor discharge (day 15)
21 (M/8 mo) Fever (40C) for 3 days; diarrhoea; shock; 4/5 DHF IV Recovered over 14 days Hepatic encephalopathy
petechiae; jaundice; hepatomegaly; peripheral
oedema; obtunded
Table 2: Clinical features
except for one sample which contained 2 red cells/L.
20 of the 21 patients had serological evidence of dengue
infection and six had evidence of CNS infection by
dengue viruses. Seven infections were classed as primary
and 13 as secondary; one patient could not be classified
serologically. Six patients seroconverted during their first
week in hospital. No patient had Japanese encephalitis
virus cultured from CSF or serum. Patient 12 had
HBsAg and patient 4 had antibodies to HBsAg in serum.
Patient 6 had Leptospira detected in the serum by PCR
but not in the CSF. No other viruses or bacteria were
isolated or detected, serologically or by PCR.
Clinical and demographic features
Eight patients were from Ho Chi Minh City; 16 patients
were admitted during the rainy season (May to October)
(table 2). One child (patient 14) had had a convulsion
in the past. Five patients had received antibiotics
(cephalosporins) and one had received artesunate. All 21
patients were febrile on admission. 18 patients had a
reduced level of consciousness; three were fully
conscious, one with a severe headache, meningism, and
vomiting, and two with a spastic paraparesis.
In seven patients neurological manifestations
coincided with clinical features of DHF: one patient had
haemoconcentration (haematocrit 38% on admission,
335% at discharge) and a petechial rash, five had
bleeding or petechiae and a narrow pulse pressure, and
one had grade IV dengue shock. Two patients had
manifestations of dengue fever without significant
vascular leak (one with petechial rash, the other with
bruising). In patient 10 a spastic paraparesis followed a
dengue-like illness by 2 weeks.
In 12 patients there were no characteristic features of
dengue on admission. Seven of these were children, who
presented typically with a short febrile prodrome of
headache, vomiting, cough, and coryza, followed by a
reduced level of consciousness, often heralded by
convulsions. Three adults with abnormal behaviour were
initially thought to have hysteria. Eight patients had
generalised convulsions, and one had right-sided focal
convulsions of the arm and leg. Eight of the unconscious
patients had focal neurological signs on admission.
Laboratory findings
Three patients had CSF opening pressures above 20 cm;
three had CSF pleocytosis (>5/L) and seven had CSF
protein above 45 mg/dL. CSF to plasma glucose ratios
were normal. Three patients had a peripheral
leukocytosis (>1110
9
/mL), and two had leukopenia
(<410
9
/mL). No patient had hypoglycaemia. Five
encephalopathic patients had liver transaminases more
than 10 times normal with raised total bilirubin and a
bleeding diathesis. Prothrombin time was measured in
two of these, and was prolonged (30 s and 34 s). One of
these also had moderate hyponatraemia (128 mmol/L).
Five further patients had mild hyponatraemia (130135
mol/L). Acute CT scans were possible on three patients.
Patients 13 and 21 had normal scans. Patient 20 had
diffuse brain swelling; and an EEG on admission was
abnormal, with high-amplitude periodic slow wave
complexes (23 Hz) on a featureless background.
Outcome
Three patients with severe DHF deteriorated. Patient 4
developed arm dyskinesias and lipsmacking, pleural
effusions, and a bleeding diathesis. She became anaemic
and required 2 units of blood before eventually
recovering fully. Patient 8 developed disseminated
intravascular coagulation and oliguric acute renal failure,
requiring peritoneal dialysis. Her coma score
deteriorated to 5, with tremors of the right arm and
downward deviation of the eyes (figure 1). Patient 12
had further generalised convulsions and developed
rigidity spasms. Of the 12 patients with purely
neurological presentations, one subsequently developed
DHF grade III, and three developed a dengue recovery
rash just before discharge (figure 2), but in the other
eight patients there were no features of dengue at any
time in the illness.
The median coma recovery time for those admitted
with a reduced level of consciousness was 35 days
(range 145) days. No patient died. At discharge 15
patients had fully recovered but six had neurological
sequelae. Patients 6 and 10, with transverse myelitis, had
a mild spastic paraparesis, but could walk
independently; patient 9, who had presented with
encephalitis, had residual spasticity; patient 13 remained
confused; patients 8 and 20 had abnormal affect and
altered personality. Six patients were followed up 224
months later (median 205 months). Patients 13 and 17
were completely normal. Patients 8 and 20 still had
altered personality with labile mood; patient 20 also had
poor short-term memory but his follow-up EEG was
normal. Two children (patients 14 and 15) had had
ARTICLES
Figure 1: Downward deviation of eyes in a comatose 19-year-
old woman with acute secondary dengue 3 infection
Figure 2: Fine maculopapular dengue recovery rash; only
clinical feature of dengue in a 12-year-old boy with
encephalitis and primary dengue 3 infection
further convulsions associated with febrile illness, but
follow-up EEGs were normal. Developmental
assessments were normal in all children at follow up.
Nine patients met the case definition of encephalitis;
nine were diagnosed as acute encephalopathy and two as
transverse myelitis; one with meningism but no CSF
pleocytosis (patient 3) did not fit into any case
definition.
Dengue virus genotypes
The four DEN-2 virus sequences obtained from patients
2 and 3 grouped together with the sequence from the
patient with DHF, within genotype IIIb of DEN-2
21
(figure 3). Within this genotype the isolates from CNS
patients were most closely related to the DHF isolate,
two Vietnamese sequences isolated in 1987
18
and a
Chinese isolate of 1985.
Discussion
Dengue viruses now affect almost every country between
the tropics of Capricorn and Cancer. The expansion of
this flavivirus infection has been linked to resurgence of
the mosquito vector Aedes aegypti, to overcrowding, and
increasing travel.
22
Following massive epidemics of DHF
in Thailand in the 1950s and 1960s, the WHO adopted
criteria for diagnosing and treating dengue fever and
DHF.
23
Neurological manifestations received little
attention initially, but in the last twenty years there has
been increasing recognition of their possible importance.
Neurological findings reported in association with
dengue include mononeuropathies, polyneuropathies,
and Guillain-Barr syndrome.
24
Two patients in our
study had transverse myelitis. In one, the history of a
dengue-like illness 2 weeks previously suggested a post-
infectious aetiology. However the high fever, rigors,
headache and muscle pain during the acute presentation
are consistent with an acute dengue, and this was
supported by the serology. The fact that both patients
improved spontaneously without antibiotics or other
therapy supports a diagnosis of para-infectious
transverse myelitis.
Whilst few doubt that dengue infection can be
associated with clouding of consciousness, until now it
was not clear whether this represents CNS invasion by
the virus, a non-specific complication of severe dengue
disease, or even coincident infection with another,
unidentified arbovirus. Most published data consist of
case-reports or reviews of patients admitted to dengue
wards with classical features of dengue infection,
6,25,26
and
there has been no previous attempt to assess
prospectively dengue as a cause of neurological disease.
We found that 4% of patients with suspected CNS
infecions admitted to a referral centre were infected with
dengue viruses. Although these accounted for only 1% of
all dengue admissions to our referral hospital there were
more patients with neurological manifestations of
dengue than patients with DHF grade IV. Serological
evidence of dengue infection may persist for some weeks
but dengue infection is unlikely to be fortuitous in all
cases. CNS patients had three times the risk of dengue
infection compared with hospital controls. Ten patients
had virus detected or isolated, and six seroconverted in
hospital. Other causes of CNS syndromes were excluded
except that one encephalopathic patient was positive for
hepatitis B. One other patient with a spastic paraparesis
was seropositive for leptospirosis but, as far as we are
aware, acute transverse myelitis has not been described
among the neurological complications of leptospirosis.
27
Complications of severe dengue implicated as possible
causes of dengue encephalopathy include hypotension,
cerebral oedema,
24
microvascular or frank
haemorrhage,
28
hyponatraemia
29
and fulminant hepatic
failure
30
which may be part of a Reye-like syndrome.
31
Liver-function tests may be abnormal in 90% of dengue
infected patients.
32
Five of our encephalopathic patients
with severe DHF had greatly raised transaminases.
Although we were unable to investigate fully every
patient, the findings were consistent with fulminant
hepatic failure. In the remaining two DHF patients
raised CSF opening pressures may have reflected
cerebral oedema. Intracranial haemorrhage has been
postulated as a cause of coma in DF patients bleeding
ARTICLES
DENESN30 Thailand 83
U87321 Thailand 80
U87325 Thailand 86
AF119661 China 85
BD48 serum Vietnam 96 DHF control
Patient 2
Patient 3
CNS 504 serum Vietnam 95
CNS 504 CSF Vietnam 95
CNS 36 serum Vietnam 95
CNS 36 CSF Vietnam 95
DENESN25 Vietnam 87
U87341 Thailand 82
DENESN31 Vietnam 87
Figure 3: Phylogenetic analysis of DEN-2 strains described
FIgure represents the portion of derived tree corresponding to genotype IIIb. Genetic distance indicated with bar. Strains are represented by GenBank
sequence filename and country and year of isolation. Strains not identified (fuller versions of tree obtainable from author) are from Central and South
America. Genbank accession numbers for the five new sequences (boxed) are: for BD48, AJ272016; for CNS 504, AJ272015 (serum) and AJ272018
(CSF); and for CNS 36, AJ272014 (serum) and AJ272017 (CSF).
from other sites.
6
However, in patient 13 a normal CT
scan excluded this; and in patient 3 intracranial
haemorrhage seems unlikely given the presenting
features, normal CSF, and full recovery.
In ten encephalopathic patients there were no
characteristic features of dengue fever or DHF, and no
metabolic explanations for the coma. Should such
patients be considered to have dengue encephalitis? In
our study it is hard to explain the detection of dengue
viruses and IgM antibody in the CSF in some patients
other than by viral invasion across the blood-brain
barrier. The CSF cell counts indicate that none of the
lumbar punctures was traumatic. Strictly speaking,
encephalitis should only be diagnosed with histological
confirmation.
3
However, brain biopsy and necropsy is
not possible in many areas where dengue occurs. Most
published post-mortem series are on patients who died
from DHF, rather than encephalitis, and lesions in the
brain have been non-specific (oedema, vascular
congestion, and focal haemorrhages).
28,33
However,
perivenous encephalitis was seen in one patient.
34
Because of the difficulties in obtaining a pathological
diagnosis we used a clinical case definition based on the
surrogate indicators of CSF pleocytosis or focal
neurological signs.
35
Although three patients had a CSF
pleocytosis, the CSF can be acellular in a variety of viral
encephalitides.
35
Dengue first appeared in southern Vietnam in the
1960s and there are major epidemics every 34 years. All
four serotypes are endemic but DEN-1 and DEN-2, and
DEN-3 have been most frequently isolated in recent
years.
36
Dual infection, as seen in one of our patients,
had been reported previously.
37
DEN-3 has been
associated with neurological presentations most
frequently and in our study was implicated in six of 10
cases where the serotype was known. Whether this
represents a higher transmission rate, easier isolation, or
different virulence is not known. The phylogenetic data
suggest that the viruses associated with CNS disease
described here were drawn from the contemporary pool
of locally circulating viruses causing dengue fever.
The neurological manifestations of dengue infection
were similar to Japanese encephalitis. Many areas that
are endemic for dengue viruses are also endemic for
Japanese encephalitis virus. Antibodies to the two viruses
are cross-reactive, and encephalopathic patients with
anti-flavivirus antibody in the CSF were assumed to
have Japanese encephalitis. However diagnostic tests
that separate these two viruses have now been adapted
for field use,
38
and neurological manifestations of dengue
infection are likely to be recognised more often. In a
study in Thailand, four of 44 patients with suspected
Japanese encephalitis were shown to have dengue
infection by IgM capture ELISA.
30
In summary, we found a variety of pathophysiological
processes may interact to cause coma in some dengue
patients but in others no toxic, metabolic,
pathophysiological, or infectious cause of coma could be
identified, other than dengue virus itself. Although it
remains possible that an as yet unidentified viral agent is
causing encephalitits in patients who are also infected
with dengue, our findings suggest that dengue viruses
can cause encephalitis. In endemic areas dengue should
be considered in patients who present with the clinical
features of encephalitis, whether or not classical
manifestations of dengue are present. The WHO
adoption of standard definitions for dengue
encephalopathy and encephalitis would help clarify the
importance world-wide.
Contributors
Tom Solomon, Nguyen Minh Dung, Rachel Kneen, Le Thi Thu Thao,
Ha Thi Loan, Nicholas Day, Jeremy Farrar and Nicholas White
designed the clinical study and collected the data and the samples. Tom
Solomon, Boonyos Raengsakulrach, Khin S A Myint, Ananda Nisalak,
and David Vaughn were responsible for the virological and
immunological studies, and their interpretation. Mary Warrell,
William James, and Tom Solomon performed the phylogenetic analysis.
All authors contributed to the overall data analysis and intrepretation,
and writing the paper.
Acknowledgments
We thank the director and staff of the Centre for Tropical Diseases for
their support, in particular Tran Tinh Hien and the doctors and nurses
of the adult and paediatric intensive care units, Delia Bethell, Mary
Gainsborough, Bridget Wills, Deborah House, Christopher Parry, and
John Wain; Tim Endy, Jane Cardosa, Fenella Kirkham, Bruce Innis,
and John Newsom-Davis for helpful discussions; Philippe Perolat, Panor
Srisongkram, Ann Taylor, Annie Siemieniuk, Steven Read, Jamal
Ibrahim, Abdessamad Tahiri-Alaoui, Tipawan Kungvanrattana,
Naowayubol Nutkumhaeng, Somkiat Changnak, Somsak Imlarp,
Chonticha Klungthong, Vipa Thirawuth for laboratory support; Shelagh
Smith for neurophysiological advice. This work was funded by The
Wellcome Trust of Great Britain.
References
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2 World Health Organization. Dengue haemorrhagic fever: diagnosis
treatment and control, 2nd ed. Geneva: WHO, 1997.
3 Solomon T. Viral encephalitis in Southeast Asia. Neurol Infect
Epidemiol 1997; 2: 19199.
4 Sumarmo, Wulur H, Jahja E, Gubler DJ, Sutomenggolo TS,
Sulianti Saroso J. Encephalopathy associated with dengue infection.
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5 Kho LK, Sumarmo, Wulur H, Jahja E, Gubler DJ. Dengue
haemorrhagic fever accompanied by encephalopathy in Jakarta.
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6 Nimmannitaya S, Thisyakorn U, Hemsrichart V. Dengue
haemorrhagic fever with unusual manifestations. Southeast Asian
J Trop Med Pub Health 1987; 18: 398406.
7 Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features
and prognostic indicators in paediatric cerebral malaria: a study of
131 comatose Malawian children. Q J Med 1989; 71: 44159.
8 Solomon T, Kneen R, Dung NM, et al. Poliomyelitis-like illness due
to Japanese encephalitis virus. Lancet 1998; 351: 109497.
9 Plum F, Posner JB. Multifocal, diffuse and metabolic brain diseases
causing stupor or coma. In: Plum F, Posner JB, eds. Diagnosis of
stupor and coma. Philadelphia; F A Davis, 1982: 177303.
10 Innis BL, Nisalak A, Nimmannitya S, et al. An enzyme-linked
immunosorbent assay to characterize dengue infections where
dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg
1989; 40: 41827.
11 Kuberski TT, Rosen L. A simple technique for the detection of
dengue antigen in mosquitoes by immunofluorescence. Am J Trop
Med Hyg 1977; 26: 53337.
12 Kuno G, Gubler DJ. Santiago de Weil NS. Antigen capture ELISA for
the identification of dengue viruses. J Virol Methods 1985; 12: 93103.
13 Lanciotti RS, Calisher CH, Gubler DJ, Chang G-J, Vorndam AV.
Rapid detection and typing of dengue viruses from clinical samples
by using reverse transcriptase-polymerase chain reaction. J Clin
Microbiol 1992; 30: 54551.
14 Chen WJ, Huang KP, Fang AH. Detection of IgM antibodies from
cerebrospinal fluid and sera of dengue fever patients. Southeast Asian
J Trop Med Publ Health 1991; 22: 65963.
15 Lum LCS, Lam SK, Choy S, George R, Harun F. Dengue
encephalitis: a true entity? Am J Trop Med Hyg 1996; 54: 25659.
16 Read SJ, Jeffery KTM, Bangham CRM. Asceptic meningitis and
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17 Letocart M, Baranton G, Perolat P. Rapid Identification of
pathogenic Leptospira species (Leptospira interrogans, L borgpetersenii,
and L hirshneri) with species-specific DNA probes produced by
abritray primed PCR. J Clin Microbiol 1997; 35: 24853.
18 Rico-Hesse R. Molecular evolution and distribution of dengue
viruses type 1 and 2 in nature. Virology 1990; 174: 47993.
19 Fong M-Y, Koh C-L, Lam S-K. Molecular epidemiology of
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in 50 patients. In: 7th International Congress for Infectious Diseases
Hong Kong, 1996: 132.
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Med Hyg 1993; 48: 793802.
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children with fatal dengue: reflections on the pathogenesis of the
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1989; 140: 35160.
26 Row D, Weinstein P, Murray-Smith S. Dengue fever with
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27 Coyle PK. Leptospirosis. In: Gilman S, Goldstein GW, Waxman
SG, eds. Neurobase. 3rd ed. San Diego: Arbor Publishing, 1999.
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30 Innis BL, Myint KSA, Nisalak A, et al. Acute liver failure is one
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necrosis factor in Reyes syndrome associated with dengue virus.
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37 Gubler DJ, Kuno G, Sather GE, Waterman H. A case of natural
concurrent human infection with two dengue viruses. Am J Trop
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ARTICLES
The Dengue Virus Genome
Dengue virus is a small virus that carries a sin-
gle strand of RNA as its genome. The genome
encodes only ten proteins. Three of these are
structural proteins that form the coat of the
virus and deliver the RNA to target cells, and
seven of them are nonstructural proteins that
orchestrate the production of new viruses once
the virus gets inside the cell. The outermost
structural protein, termed the envelope pro-
tein, is shown here from PDB entry 1k4r
1
.
The virus is enveloped with a lipid membrane,
and 180 identical copies of the envelope pro-
tein are attached to the surface of the mem-
brane by a short transmembrane segment. The
job of the envelope protein is to attach to a cell
surface and begin the process of infection.
A Deadly Switch
In the infectious form of the virus, the enve-
lope protein lays flat on the surface of the
virus, forming a smooth coat with icosahedral
symmetry. However, when the virus is carried
into the cell and into lysozomes, the acidic
environment causes the protein to snap into a
different shape, assembling into trimeric
spike, as shown above from PDB entry 1ok8
2
.
Several hydrophobic amino acids at the tip of
this spike, colored bright red here, insert into
the lysozomal membrane and cause the virus
membrane to fuse with lysozome. This releas-
es the RNA into the cell and infection starts.
The hemagglutinin protein on the surface of
influenza virus plays a similar role, but the
two proteins use entirely different mecha-
nisms to perform a similar task.
The Hunt for a Dengue
Vaccine
A dengue vaccine has proven difficult to
develop, in part because there are four major
subtypes of dengue virus, each with slightly
different viral proteins. Many researchers cur-
rently believe that the deadly dengue hemor-
rhagic disease is caused when a person is
infected with one subtype, and then infected
later by a second subtype. The antibodies, and
immunity, gained from the first infection
appear to assist with the infection by the second
subtype, instead of providing a general immu-
nity to all subtypes. This means that an effec-
tive vaccine will have to stimulate protective
antibodies against all four types at once, a feat
that has not yet been achieved.
Building New Viruses
Dengue virus also makes several proteins that
create new viruses once it is inside a cell. Two
of the major ones are shown on the reverse.
Both are multifunctional proteins with several
enzymes strung together. The one on the left,
NS5 from PDB entries 1l9k
3
and 2j7w
4
, con-
tains a methyltransferase and a polymerase,
and the one on the right, NS3 from PDB
entry 2vbc
5
, contains a protease and a heli-
Dengue virus is a major
threat to health in
tropical countries around
the world. It is limited
primarily to the tropics
because it is transmitted
by a tropical mosquito,
but even with this limita-
tion, 50-100 million
people are infected each
year. Most infected
people experience dengue
fever, with terrible
headaches and fever and
rashes that last a week or
two. In some cases,
however, the virus
weakens the circulatory
system and can lead to
deadly hemorrhaging.
Researchers are now
actively studying the virus
to try to develop drugs to
cure infection, and
vaccines to block
infection before
it starts.
www.pdb.org
info@rcsb.org
MOLECULE OF THE MONTH:
DENGUE VIRUS
doi: 10.2210/rcsb_pdb/mom_2008_7
1ok8
1k4r
About the
RCSB PDB Molecule of the Month
Using selected molecules from the PDB
archive, each feature includes an
introduction to the structure and func-
tion of the molecule, a discussion of its
relevance to human health and welfare,
and suggestions for viewing and
accessing further details.
The RCSB PDB Molecule of the Month
is read by students, teachers, and scien-
tists worldwide at www.pdb.org.
This July 2008 edition was written and
illustrated by David S. Goodsell
(RCSB PDB and The Scripps
Research Institute).
RCSB Protein Data Bank
The Protein Data Bank (PDB) is the
single worldwide repository for the
processing and distribution of 3D
structure data of large molecules of
proteins and nucleic acids. The RCSB
PDB is operated by Rutgers, The State
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of California, San Diego two members
of the Research Collaboratory for
Structural Bioinformatics (RCSB).
It is supported by funds from the
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Sciences, the Office of Science,
Department of Energy, the National
Library of Medicine, the National
Cancer Institute, the National Center
for Research Resources, the National
Institute of Biomedical Imaging and
Bioengineering, the National Institute
of Neurological Disorders and Stroke
and the National Institute of Diabetes
& Digestive & Kidney Diseases.
The RCSB PDB is a member of
the worldwide PDB
(wwPDB; www.wwpdb.org).
Cryoelectron microscopy has been used to
study many aspects of the life cycle of the
dengue virus. In these structures, a low resolu-
tion image of virus, not quite detailed enough
to see atoms, is obtained by the electron
microscope, and then atomic structures of the
individual pieces are fit into the image to gen-
erate the final model. The one shown here,
from PDB entry 2r6p
6
, shows the envelope
protein on the surface of the virus (in white)
with many antibody Fab fragments (in blue)
bound to the viral proteins. By looking care-
fully at this structure, researchers have discov-
ered that the antibodies distort the arrange-
ment of the envelope proteins, blocking their
normal action in infection. Other dengue
virus structures in the PDB include immature
forms of the virus (for instance, in PDB entry
1n6g
7
) and structures that include the mem-
brane-spanning portions of the viral coat
(PDB entry 1p58
8
).
References:
1. 1k4r: Kuhn, R.J., Zhang, W., Rossmann, M.G., Pletnev, S.V.,
Corver, J., Lenches, E., Jones, C.T., Mukhopadhyay, S.,
Chipman, P.R., Strauss, E.G., Baker, T.S., Strauss, J.H. (2002)
Structure of dengue virus: implications for flavivirus organization,
maturation, and fusion. Cell 108: 717-725
2. 1ok8: Modis, Y., Ogata, S., Clements, D., Harrison, S.C.
(2004) Structure of the dengue virus envelope protein after mem-
brane fusion. Nature 427: 313-319
3. 1l9k: Egloff, M.P., Benarroch, D., Selisko, B., Romette, J.L.,
Canard, B. (2002) An RNA cap (nucleoside-2'-O-) methyltrans-
ferase in the flavivirus RNA polymerase NS5: crystal structure
and functional characterization Embo J. 21: 2757-2768
4. 1j7w: Yap, T.L., Xu, T., Chen, Y.L., Malet, H., Egloff, M.P.,
Canard, B., Vasudevan, S.G., Lescar, J. (2007) Crystal Structure
of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic
Domain at 1.85 Angstrom Resolution. J.Virol. 81: 4753
5. 2vbc: Luo, D.H., Xu, T., Hunke, C., Gruber, G., Vasudevan,
S.G., Lescar, J. (2008) Crystal Structure of the Ns3 Protease-
Helicase from Dengue Virus. J.Virol. 82: 173
6. 2r6p: Lok, S.M., Kostyuchenko, V., Nybakken, G.E.,
Holdaway, H.A., Battisti, A.J., Sukupolvi-Petty, S., Sedlak, D.,
Fremont, D.H., Chipman, P.R., Roehrig, J.T., Diamond, M.S.,
Kuhn, R.J., Rossmann, R.G. Binding of a neutralizing antibody
to dengue virus resulted in an altered arrangement of the surface
glycoproteins To Be Published
7. 1n6g: Zhang, Y., Corver, J., Chipman, P.R., Zhang, W.,
Pletnev, S.V., Sedlak, D., Baker, T.S., Strauss, J.H., Kuhn, R.J.,
Rossmann, M.G. (2003) Structures of Immature flavivirus parti-
cles EMBO J. 22: 2604-2613
8. 1p58: Zhang, W., Chipman, P.R., Corver, J., Johnson, P.R.,
Zhang, Y., Mukhopadhyay, S., Baker, T.S., Strauss, J.H.,
Rossmann, M.G., Kuhn, R.J. (2003) Visualization of membrane
protein domains by cryo-electron microscopy of dengue virus
Nat.Struct.Biol. 10: 907-912
DENGUE VIRUS
case. Each of these enzymes performs a differ-
ent part of the life cycle. The polymerase
builds new RNA strands based on the viral
RNA, the helicase helps to separate these
strands, and the methyltransferase adds
methyl groups to the end of them, protecting
the RNA strands and coaxing the cell's ribo-
somes to create viral proteins based on them.
The viral proteins are created in one long
polyprotein chain, which is finally clipped
into the functional units by the protease. The
little chain colored blue is a portion of anoth-
er viral protein, NS2B, that assists with the
protease activity.
Additional Reading:
1. Mukhopadhyay, S., Kuhn R.J., Rossmann M.G. (2005) A
structural perspective of the Flavivirus life cycle. Nature Reviews
Microbiology 3: 13-22.
2. Whitehead, S.S., Blaney, J.E., Durbin, A.P., and Murphy, B.R.
(2007) Prospects for a dengue virus vaccine. Nature Reviews
Microbiology 5: 518-528.
3. Halstead, S.B. (2007) Dengue. Lancet 370: 1644-1652.
4. Qi, R.-F., Zhang, L. and Chi, C.-W. (2008) Biological charac-
teristics of dengue virus and potential targets for drug design. Acta
Biochimica et Biophysica Sinica 40: 91-101.
1l9k
2j7w
methyltransferase
polymerase
protease
helicase
2vbc
Topics for Further
Exploration
1) The dengue virus is surrounded by 180
copies of the envelope protein. Many other
viruses are surrounded by capsids com-
posed of many identical proteins, and these
often appear in multiples of 60, such as
180, 240 or 420 copies. What is significant
about these numbers? Can you find exam-
ples of each in the PDB?
2) Dengue virus is a member of a family of
flaviviruses that are spread by ticks and mos-
quitoes. Other examples include yellow fever
virus and West Nile virus. Looking at the
structures in the PDB, can you see similari-
ties in the proteins made by these viruses?
3) Dengue virus replicates in the
cytoplasm of infected cells, without enter-
ing the nucleus. Can you think of any prob-
lems this might cause, and how the dengue
virus solves them with its ten viral proteins?
Exploring the Structure

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RNA coplesjml (,8) und to vury from c,cczccccc pfujml (,6). ln

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