Involvement of the liver in dengue infections

Duncan R. Smith# and Atefeh Khakpoor

Molecular Pathology Laboratory, Institute of Molecular Biosciences,
Mahidol University, Thailand

Abstract
Dengue virus, the causative agent of dengue fever (DF) and more severe forms of the disease dengue
haemorrhagic fever (DHF) and dengue shock syndrome (DSS) can infect a number of different types of
cells. While monocytes and macrophages are considered to be primary target cells driving the pathology
of the disease, numerous studies have implicated the liver as a site of dengue virus replication and both
clinical observations and experimental data support a role of the liver in dengue disease. This review
aims to provide a brief overview of the data supporting a role of the liver in the pathology of dengue
disease.

Keywords: Liver; dengue virus; dengue fever; dengue haemorrhagic fever; dengue shock syndrome.

Introduction forms of the disease dengue haemorrhagic

With an estimated 75500 million infections
per year and 3.6 billion people living at
the risk of infection,[1] dengue is one of the
most important public health problems in
most tropical and subtropical countries. The
causative agent of dengue, the dengue viruses
(DEN-V), are transmitted to humans by the bite
of infected female mosquitoes belonging to the
Aedes family, most commonly Aedes aegypti
and Aedes albopticus.[2]
Infection with DENV can be asymptomatic
or can result in a wide spectrum of disease,
varying from a mild, non-specific viral illness
to dengue fever (DF) to the more severe
fever (DHF) and dengue shock syndrome
(DSS). DF usually starts with a high fever and
is often accompanied by a rash, headache
and abdominal pains lasting 27 days. Frank
haemorrhage is uncommon during DF but
gastrointestinal bleeding, gingival bleeding
and petechiae have been reported in some
patients.[3] DHF can occur in primary infections
but is more common in secondary infections
and also starts with a high fever that is not
significantly different from that observed in
DF patients. Haemorrhagic phenomenon
may be minimal, but around the time of
defervescence, patients develop the hallmark
of DHF, namely, significant plasma leakage,
which in DSS is further characterized by

#
E-mail: duncan_r_smith@hotmail.com; Tel.: (662) 800 3624; Fax (662) 441 9906

Dengue Bulletin Volume 33, 2009 75

Involvement of the liver in dengue infections
tachycardia and hypotension.[4] If the plasma
leakage is severe and remains untreated,
patients may develop shock, which can be
fatal.[4,5]
While the main manifestations of severe
dengue disease are primarily haemorrhagic in
nature, a significant body of work has been
accumulated which implicates the liver as a
critical part of the disease pathology.
Hepatomegaly
Perhaps the most obvious sign of the
involvement of the liver in dengue infections
is the high proportion of dengue cases with
liver enlargement. In one of the earliest
large-scale series of clinical investigations into
dengue, Halstead and colleagues observed
that the frequency of liver enlargement was
similar in both primary and secondary dengue
infections, and the authors proposed that a
moderate liver enlargement may be a part
of the normal pathological response to
dengue infection.[6] More recent studies have
been somewhat divided, with some reports
suggesting that hepatomegaly is present
at between 50100% of cases [7-14] while
others document a significantly lower rate of
hepatomegaly.[15-17] In addition, some studies
support a higher rate of hepatomegaly in DHF/
DSS cases as opposed to DF cases[13,14] although
this may not reach statistical significance.[14] On
balance, the studies tend to support a high level
of hepatomegaly in dengue cases, with perhaps
a slightly higher rate in the more severe cases,
although this may depend somewhat upon the
exact case definitions used.
Liver enzymes
Upon injury to the liver, the enzymes,
aspartate aminotransferase (AST) and alanine
aminotransferase (ALT), are released into the
76 Dengue Bulletin Volume 33, 2009
bloodstream, and as a consequence these
enzymes are believed to be sensitive indicators
of liver damage. Perhaps, unsurprisingly, these
enzymes are frequently elevated in dengue
patients, as has been shown in numerous
studies.[7,9,12,14,16,18-22] In one large series of
patients examined for both AST and ALT levels,
Kuo and colleagues evaluated 240 dengue
patients from the 19871988 outbreak in
Taiwan.[20] Elevated levels of AST and ALT
were found in 93.3% and 82.2% of cases
respectively. While the majority of patients had
only mildly or moderately elevated levels of
these transaminases, some 10% (11% and 7%
for AST and ALT respectively) of patients had
levels elevated by 10-fold or greater. Somewhat
lower levels of liver enzyme disorder were
noted by de Souza and colleagues in their
study of 1585 dengue patients, and they
observed alterations of AST and ALT levels
in 63% and 45% of patients respectively.[22]
Interestingly, however, the authors noted that
the average levels of AST and ALT were
significantly higher in DHF patients than in DF
patients, an observation supported by other
studies.[9,14] Several authors have noted that
the levels of serum AST are greater than serum
ALT,[18,20,21] which is in contrast to the normal
finding with viral hepatitis.[23] Some evidence
has suggested that there is a greater degree
of involvement of the liver in infections with
DENV-3 and DENV-4.[24] Overall, the studies
are consistent with elevated levels of liver
enzymes being a common characteristic of
dengue disease, and as such possibly represent
a discriminating factor in differentiating
dengue from other febrile diseases,[19] but is of
less use in differentiating DF from DHF.
Liver specimen studies
While both hepatomegaly and alterations in
liver enzymes point to the involvement of
the liver in the disease, they are unable to
distinguish between the result of a bystander

or secondary effect.[25] As such, a number
of studies have sought to provide direct
evidence for the involvement of the liver in the
disease process. The earliest of these studies
undertook direct histological investigations
of specimens from the livers of fatal cases
of dengue infection.[26-28] The predominant
findings in these studies were microvesicular
steatosis and small foci of hepatocellular
necrosis in addition to the presence of
councilman bodies, Kupffer cell hyperplasia
and mononuclear cell infiltrates at the portal
tract.[26-28] In this respect, the liver damage seen
in fatal dengue cases is significantly less severe
than that seen in fatal cases of yellow fever
virus infection.[29] While relatively uncommon,
cases of fulminant hepatitis have also been
documented.[30-32]
Several studies have used an
immunohistochemical approach to detect
the presence of dengue antigens in
liver specimens. [33-40] These studies have
predominantly used antibodies directed
against dengue E protein,[34-40] although one
recent study used an antibody directed against
dengue NS3 protein,[33] which gives a greater
degree of certainty that infected cells are
undergoing viral replication and do not reflect
the presence of endocytosed or phagocytosed
virus particles without viral replication. The
majority of the studies detect, to a greater
or lesser extent, the presence of dengue
antigen in hepatocytes, the major cell type
composing the liver.[33-38,40] Interestingly, while
some studies have suggested that 8090%
of hepatocytes show immunoreactivity, [36]
other studies fail to detect dengue antigen in
hepatocytes at all.[39] Whether this extreme
difference is due to methodological or sample-
preparation differences, or truly reflects a
different tissue tropism of some dengue virus
lineages remains unclear at this point. While
some studies have detected the presence of
dengue antigen in Kupffer cells,[36,38,39] the
Dengue Bulletin Volume 33, 2009 77
Involvement of the liver in dengue infections
study by Balsitis and colleagues did not detect
the presence of immunoreactive NS3 in these
cells,[33] suggesting thereby that Kupffer cells do
not support replication of the dengue virus,
and immunoreactivity to dengue structural
proteins noted by others[36,38,39] may reflect
phagocytosed virus. This would be consistent
with the studies by Marianneau and colleagues
who showed that dengue was efficiently taken
up by isolated primary human Kupffer cells,
but that the infection was non-productive.[41]
Dengue-specific RT-PCR and in situ
hybridization has been employed in several
studies to detect the presence of the dengue
genome.[36,38,39,42-44] As with the studies utilizing
immunohistochemistry, while the majority of
the studies detect the presence of the dengue
genome in liver samples, and more specifically
in hepatocytes and to a lesser extent in Kupffer
cells,[36,38,43,44] other studies do not detect the
presence of the dengue genome in either
hepatocytes or Kupffer cells.[39] Again, this may
well reflect either methodological differences
or inherent differences in virus tropism.
Several studies have recovered infectious
dengue virus from liver specimens[27,38,45,46] and,
in particular, Rosen and colleagues recovered
infectious dengue serotype 2 or 3 from 5 out of
17 liver specimens from fatal cases of dengue
infection through the mosquito inoculation
technique[46]. However, these studies do not
use defined populations of cells from the liver
and as such, no information as to the cell types
involved in the disease process is available.
Mouse model studies
A number of studies have utilized various mouse
model systems to understand the pathogenesis
of dengue infections. [33,47-56] Perhaps the
simplest model is the intraperitoneal and
intravenous injection of DENV-2 into BALB/c
mice.[49,55,56] Histopathological analysis of these
Involvement of the liver in dengue infections
animals showed severe liver injury, including
hepatocyte swelling and vacuolization, necrosis
and steatosis. Liver damage was more severe,
and occurred earlier in the intravenous injection
system as compared to the intraperitoneal
injection system.[49,55,56] Ultrastructural studies
showed the accumulation of cytoplasmic lipid
droplets as well as mitochondrial swelling in
hepatocytes, possibly related to the induction
of apoptosis.[55,56] Evaluation of liver enzymes
showed a peak of both AST and ALT at seven
days post-infection, which was correlated to
the extent of liver injury.[55,56] Dengue virus
antigens were detected not only in hepatocytes
but also in Kupffer cells.[55,56]
While SCID mice are apparently resistant
to dengue infection,[54] SCID mice transplanted
with human liver cell lines HepG2 or Huh-7[47,57]
or with K562 (a erythroleukemia cell line)
cells[54] and intraperitoneally injected with
DENV-2[47,54] or DENV-4[57] have all been used
as model systems, with somewhat conflicting
results. For example, while An and colleagues
report high levels of the virus in the liver of
SCID mice with transplanted HepG2 cells[47]
in the early stage post-infection, Lin and
colleagues report little or no involvement of
the liver in the same SCID mice engrafted with
human K562 cells.[54]
Immunocompetent C57BL/6 mice
injected with a high titre of DENV-2[51] showed
elevated levels of AST and ALT on days 3,
5 and 7 post-infection, which was elevated
further after a second inoculation. Dengue
RNA was detected in the liver of infected
mice and a strong correlation was found
between T cell activation and hepatic cellular
infiltration. Dengue infection of interferon
receptor-deficient (AG129) mice is uniformly
lethal,[58] and the presence of immunoreactive
NS3 was detected in hepatocytes, implying
dengue virus replication in these cells [33]
78 Dengue Bulletin Volume 33, 2009
being consistent with reports of dengue virus
infection of human primary hepatocytes.[59]
While some of these studies failed to
detect the involvement of the liver,[51,54] other
studies have demonstrated a clear evidence of
the involvement of the liver and of hepatocytes
specifically.[33,55,56] Where liver involvement
is seen in the model systems, the changes
observed reflect the pathology of human
disease.
Isolated primary liver cells
To date, only two studies have investigated the
susceptibility to dengue infection of primary
(untransformed) human liver cells.[41,59] The
first study isolated Kupffer cells from liver
sections obtained during partial hepatectomy
for liver cancer[41] and demonstrated that
while dengue virus entered into Kupffer cells
efficiently, no viral progeny were produced
and that infected cells underwent cell death
by apoptosis. The second study[59] utilized
commercially obtained hepatocytes purified
from liver transplantation cut-downs and
demonstrated productive infection with
DENV-2 strain 16681 as assessed by plaque
assay and cellular expression of both structural
and non-structural proteins (E and NS1). A
significant cytokine response was observed
which included the up-regulation of TRAIL,
MIP-1, MIP-1, IFN-, IL-8 and RANTES.
The profile of cytokine induction was similar,
but not identical to the profile generated by
DENV-2-infected HepG2 cells undertaken
in parallel with the infection of the primary
hepatocytes. Although not formally evaluated,
evidence of the induction of apoptosis in
response to DENV-2 infection was observed
in the infected primary hepatocytes which
showed extensive nuclear fragmentation.[59]
Infected primary hepatocytes additionally

showed the up-regulation of TRAIL,[59] a type-
II transmembrane protein that has significant
proapoptotic effect in liver cells,[60] and that
has been proposed to be the primary mediator
of apoptosis induction in dengue-infected
transformed hepatocytes.[61]
Studies on transformed liver cells
Transformed liver cells are broadly permissive
to dengue infection,[62,63] and a large number
of studies have investigated cellular responses
to dengue infection in transformed liver
cells. These studies have investigated
virus attachment, entry, replication and
the consequent liver cell death. Heparin
sulphate has been implicated in dengue virus
attachment to liver cells, either as a direct virus
receptor[64] or, more probably, as either a low
affinity-binding molecule[65] or receptor co-
factor.[66] Two cell surface-expressed proteins
have been implicated as liver cell-expressed
dengue virus receptors, and it has been
proposed that these proteins act in a serotype-
specific manner.[66,67]
The first protein implicated, GRP78 or
BiP, was proposed to act as a receptor for
DENV-2[67] while the 37/67kda high-affinity
laminin receptor protein has been implicated as
a DENV-1 receptor.[66] A critical role for GRP78
as a chaperone protein in the replication of
dengue has also been suggested.[68,69] The
37/67kda high-affinity laminin receptor
protein has additionally been implicated as a
receptor for Sindbis virus,[70] adeno-associated
virus,[71] tick-borne encephalitis virus, and
Venezuelan equine encephalitis virus, [72]
while GRP78 has been implicated as a co-
receptor for coxsackie virus A9.[73] The issue
of serotype-specific entry of the dengue virus
into liver cells is somewhat controversial, as
other identified dengue virus receptors such
Dengue Bulletin Volume 33, 2009 79
Involvement of the liver in dengue infections
as DC-SIGN[74]and Hsp79/90[75] in different cell
types do not show such a specificity, although
in some cases, not all serotypes have been
investigated.[75] Dengue virus entry to liver cells
has been suggested to occur predominantly,
but not solely, by clathrin-coated pit-mediated
endocytosis and it has been proposed that as
much as 20% of virus entry to liver cells may
occur through alternate pathways.[76] While
somewhat controversial, support for the
concept of multiple entry pathways has also
been presented in studies on dengue virus
entry into Vero (monkey kidney) cells.[77] One
study has suggested that dengue virus entry
into HepG2 cells is modulated at least in part
by the cell cycle,[78] as has been found with
dengue entry into insect cells.[79]
Two studies have investigated global gene
expression changes in HepG2 cells by either
microarray analysis[80] or cDNA-AFLP.[81] Both
studies estimate that some 500 gene transcripts
belonging to a number of different pathways
are differentially regulated in response to
dengue infection. The microarray analysis
identified a number of genes involved in the
innate immune response and, in particular,
pattern recognition genes such as TLR3,
TLR8, RIG-1 and MDA5 were identified as
being up-regulated.[80] Some genes identified
such as IL-6, IL-8, RANTES and IFN-[80] were
consistent with data generated from analysis
of dengue-infected primary hepatocytes.[59]
Perhaps significantly, the microarray analysis
of dengue virus-infected HepG2 cells also
detected the up-regulation of caspases 8 and
10 (see next section).
In contrast to the data which suggests that
some 500 genes have altered transcriptional
regulation,[80,81] proteomic analysis of dengue-
infected HepG2 cells have only identified
some 17 proteins as being differentially
expressed, and these proteins were primarily
Involvement of the liver in dengue infections
involved in the regulation of transcription
and translation.[82] Interestingly, a secretome
analysis of dengue-infected HepG2 cells
identified significantly more proteins as
being differentially regulated by dengue
virus infection, with 35 proteins being down-
regulated and 24 proteins being up-regulated
in infected HepG2 cells.[83] Several proteins,
including -enolase, superoxide dismutase
(SOD), peptidyl-prolyl isomerases A and B
(cyclophilins A and B), tissue inhibitor of
metalloproteinases 1 and 2 (TIMP-1 and 2)
and macrophage migration inhibitory factor
(MIF) identified in the secreteome of dengue
virus-infected HepG2 cells (as opposed to
the secretome of uninfected HepG2 cells),
have been previously identified in other virus
infections or inflammation situations.[83]
Two groups have shown that autophagy,
the lysosomal degradation pathway, is activated
in response to dengue virus infection of liver
cells[84-86] and it has been suggested that in liver
cells, autophagic vesicles act as sites for dengue
virus replication.[86] Again, as with dengue virus
receptor usage,[66,67,87] it has been proposed that
interactions between the dengue virus and the
autophagy pathway are serotype-specific.[84,86]
Given that the endocytosis pathway interacts
with the autophagic pathway, a model has
been proposed to link dengue virus entry and
replication in liver cells in terms of a continuing
interaction with membranes of an endosomal-
autophagosomal lineage.[88]
Apoptosis
Cellular apoptosis in the liver upon dengue
virus infection has been reported both in vivo
and in vitro.[35,40,63,89,90] Histological examination
of the livers of fatal cases of dengue virus
infection note the presence of councilman
80 Dengue Bulletin Volume 33, 2009
bodies which are believed to be the remains
of cells undergoing apoptosis.[38] Dengue virus
infection of primary cultures of human Kupffer
cells and hepatoma cell lines induces apoptosis
as evidenced by DNA laddering,[63,89-91] and
infection of primary human hepatocytes
produces morphological changes characteristic
of apoptosis.[59] The mechanism by which the
dengue virus induces apoptosis remains to
be clearly elucidated. While some authors
have proposed that the apoptosis of liver cells
occurs by a p53-independent mechanism,
based in part on the high level of apoptosis
in the p53-null cell line Hep3B,[63] other
authors have proposed that apoptosis occurs
by a p53-dependent mechanism.[92] In 2005,
Matsuda and colleagues[61] presented evidence
that Apo2L or TRAIL (tumour necrosis factor-
related apoptosis-inducing ligand) is induced
by dengue infection and that this interacts
with the Apo2L/TRAIL receptor DR5/TRAIL-R2
expressed on the surface of liver cells[61]
inducing apoptosis. This would suggest that
apoptosis was induced primarily through
an extrinsic apoptosis pathway. In contrast
Nasirudeen and Liu, who have suggested
apoptosis primarily results through an
intrinsic, mitochondrially-mediated pathway.
[92]
However, more recently, Nasirudeen and
Liu also suggested that apoptosis in liver cells
may be mediated through caspase 1.[93] Other
authors have proposed that the critical event
in triggering apoptosis in dengue-infected
hepatocytes is the interaction between the
dengue virus capsid protein and the human
death domain-associated protein Daxx,[94] and
that nuclear localization of the dengue capsid
protein is essential for the interaction with
Daxx and subsequent apoptosis.[95] Further
research is required to integrate the various
suggested models of induction of apoptosis in
liver cells as a consequence of dengue virus
infection.

Alternate mechanisms
A number of alternatives to direct infection of
liver cells have been suggested to account for
the involvement of the liver in dengue disease.
These mechanisms include damage induced
by the infiltration of activated lymphocytes,
especially CD8+ T cells,[51] bystander lysis
mediated by CD4+ cytotoxic T cells after
activation by dengue-infected Kupffer cells,[96]
as well as the action of antibodies against
dengue proteins cross-reacting with host cell
proteins.[97] Overall, the theories of an alternate
mechanism of liver damage in dengue infection
are somewhat less well supported than models
proposing direct infection of cells. However,
there is no evidence ruling out either model,
and it is possible that both mechanisms, direct
and indirect liver involvement, may occur
together in dengue infections.
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Involvement of the liver in dengue infections
Conclusions
A large body of clinical and experimental
evidence points to the involvement of the liver
in the pathobiology of dengue virus infections
of humans. The balance of evidence suggests
that hepatocytes are directly involved in
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forms of the disease.
Acknowledgement
DRS is supported by the Thailand Research
Fund and Mahidol University.
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