Download Introduction Dengue viruses are RNA viruses belong to the family

Introduction
Dengue viruses are RNA viruses belong to the family Flaviviridae . It is the most common cause
of arboviral diseases worldwide (Yasmashiro, 2004). It consists of phylogenetically and
antigenically distinct four serotypes of dengue viruses , DENV 1, DENV 2, DENV 3 and DENV
4.These serotypes were classified by Albert Sabin in 1944, and infection with one type confers
long-term immunity, it is to that type only and not to the other three. ( Humaira Zafar et al)
Although outbreaks of disease clinically consistent with dengue have been reported for centuries,
it was not until 1943 in Japan and 1945 in Hawaii that the first two dengue viruses were isolated
(named DENV1 and DENV2, respectively) .In the latter half of the 20th century, DENV
transmission followed the spread of its principal mosquito vector, Aedes aegypti , and was likely
accelerated by urbanization and globalization. Spatial patterns in concurrent and sequential
circulation of DENV1–4 should be considered along with virus and host genetics as potentially
important population-level risk factors for severe dengue illness because secondary infection
with a heterologous DENV type may increase the probability of severe disease . Although
international transmission of disease is not new, it is notable that airline passenger numbers have
increased by 9% annually since 1960, enabling infected human hosts to move the viruses long
distances more quickly . Increased urbanization along with substandard housing, unreliable water
supply, and poor sanitation provide an environment for Ae. aegypti proliferation in close
proximity to human hosts. (Jane P. Messina et al)
The WHO and US centers for disease control has considered dengue fever a major health threat
for Brazil, Pakistan and India (CDC Report, 2013).The epidemiology of DF has been drastically
changed, resulting in the increased incidence of disease worldwide along with the worsening of
clinical symptoms. The course of epidemic is dependent upon the rate of contact between the
host, the infecting vector and the threshold theory. This theory states that the introduction of few
infected individuals in the community will not give rise to an outbreak unless the density of
vector exceeds a certain critical level (CDC Report, 1980).
Dengue viral infection is becoming a growing threat to the population of developing world
(Senanayake, 2006). DF is a terrible viral disease. It has involved many tropical regions of the
world. It has been estimated that about 40% of world population is at the risk for this infection
(Durand, 2003).
The substandard housing, crowding, and deterioration in water, sewer, and waste management
systems associated with unplanned urbanization have created ideal conditions for increased
transmission of mosquito-borne diseases in tropical urban centers. A third major factor has been
the lack of effective mosquito control in areas where dengue is endemic. To reduce Dengue in
our country the National Dengue control programmes must follow all WHO recommended
Dengue control guidelines. The important focused points in WHO guidelines are the public
health education, community participation, detection of breeding sites, environmental
management by reactive insecticide fogging, geo referenced entomologic and clinical
surveillance systems. ( Humaira Zafar et al)
The four serotypes of DENV have been co-circulating in Sri Lanka for more than three decades
and their distribution has not changed drastically in the last 30 years. Epidemiologically, during
the 19th century DF was considered as a sporadic disease, causing epidemics at long intervals.
However, dramatic changes in the epidemic pattern have occurred and DF/DHF currently is
ranked as the most important mosquito-borne viral disease in the world. DF is now one of the
leading causes of hospitalization and death among children in the tropical regions of the world.
The rapid increase in DENV activity in India and Sri Lanka suggests its potential to cause even
more severe epidemics in the future. (P.D.N.N. Sirisena et al)
The principal vector of dengue, Aedes aegypti, is found worldwide between latitudes 35°N and
35°S . It is an efficient vector for several reasons: it is highly susceptible to dengue virus; it feeds
preferentially on human blood; it thrives in close proximity to humans; it is a daytime feeder; its
bite is almost imperceptible; and it is a restless mosquito as the slightest movement interrupts
feeding, thus several people may be bitten in a short period for one blood meal. Unlike most
mosquitoes, A aegypti takes more than one blood meal during a gonotropic cycle—that is, before
the eggs are laid. In many areas, dengue epidemics occur during the warm, humid, rainy seasons,
which favour abundant mosquitoes and shorten the extrinsic incubation period. Dengue
haemorrhagic fever is distinguished from dengue by the presence of increased vascular
permeability, not by the presence of haemorrhage. Patients with dengue may have severe
haemorrhage without meeting WHO criteria for dengue haemorrhagic fever. In these cases the
pathogenesis probably derives from thrombocytopenia or a consumptive coagulopathy, not from
the vascular leak syndrome seen in dengue haemorrhagic fever. Dengue haemorrhagic fever may
(grades III and IV) or may not (grades I and II) include clinical shock, referred to as dengue
shock syndrome . Dengue virus antigen has been found in a variety of tissues, predominately the
liver and reticuloendothelial system.Viral replication is thought to occur primarily in the
macrophages, although dendritic cells(Langerhans cells) in the skin may be an early target of
infection. (Robert V Gibbons et al)
All four serotypes have been associated with dengue haemorrhagic fever. Variations in virus
strains within and between the four serotypes may influence disease severity. Secondary
infections (particularly with serotype 2) are more likely to result in severe disease and dengue
haemorrhagic fever. This is explained by the theory of antibody dependent enhancement,
whereby cross reactive but non­neutralising antibodies from a previous infection bind to the new
infecting serotype and facilitate virus entry into cells resulting in higher peak viral titres. In
primary and secondary infections, higher viral titres are associated with more severe disease.
Higher titres may result in an amplified cascade of cytokines and complement activation causing
endothelial dysfunction, platelet destruction, and consumption of coagulation factors, which
result in plasma leakage and haemorrhagic manifestations. (Robert V Gibbons et al)
Dengue is becoming a serious public health problem in India. Although dengue infection
has been endemic in India since the nineteenth century, DHF has become endemic in
various parts of India since 1987, with the first major widespread epidemics of DHF and DSS
occurring in 1996, involving areas around Delhi and Lucknow, Uttar Pradesh, and spreading
to other regions in India. However, the epidemics of Delhi and Pune in western India in
2006 and in Kerala state in 2008 marked the changing epidemiology of dengue infection,
with all four serotypes of dengue viruses found in co-circulation, leading to an increase in
secondary dengue infection and, in some cases, co-infections with DENV-1 and DENV-3,
DENV-2 and DENV-3 and DENV-1, DENV-2 and DENV-3. In West Bengal state, nearly 61%
of dengue cases reported between 2005 and 2007 were secondary dengue infection cases.
(Dengue Bulletin Vol 35,Dec 2011)
Epidemiologic studies have identified young age, female sex, high body-mass index, virus strain,
and genetic variants of the human major-histocompatibility- complex class I–related sequence B
and phospholipase C epsilon 1 genes as risk factors for severe dengue. Secondary infection, in
the form of two sequential infections by different serotypes, is also an epidemiologic risk factor
for severe disease. ( Cameron P. Simmons et al)
The mechanisms that have been considered to cause DHF include antibody-dependent
enhancement (ADE), T cell response, and a shift from Th-1 to Th-2 response. The combined
effect of all of these is cytokine tsunami resulting in movement of body fluids in extravascular
space. It has been suggested that in dengue a Th1 response is linked to recovery from infection
while a Th2 type response leads to severe pathology and exacerbation of the disease. ( Nivedita
Gupta et al)
Severe complications related to DHF may occur very early in disease, it is important to explore
prognostic markers of severe disease during acute phase. The hyperendemicity with cocirculation of two or more serotypes during the same time period have been widely suspected as
one of the major cause of disease severity in South East Asia.Hepatic dysfunction is common in
patients with severe dengue manifestations. Liver dysfunction might be responsible for the
decreased synthesis of specific factors of the intrinsic pathway, an unbalance of which may
consequently cause hemorrhage.( Rajni Kumaria)
One of the largest outbreaks in north India occurred in Delhi and adjoining areas in the 1996
which was mainly due to dengue-2 virus. Thereafter, in 2003, another outbreak occurred in Delhi
and all four dengue virus serotypes were found to be cocirculating. However, dengue-3 was
reported to predominate in certain parts of North India in 20039. Dengue has been rampant in
parts of Tamil Nadu in the past two decades. The prevalence of dengue vector and silent
circulation of dengue viruses have been detected in rural and urban Tamil Nadu, which is
ever increasing.The vulnerability of children to dengue infection was re-established in our study,
as has been reported earlier. (P. Gunasekaran et al)
Upon analyzing the year-wise distribution of dengue cases in the study population, steady
increase in the number of dengue patients over the past few years was noted. The study revealed
that majority of the cases were in the age group of 15–44 years. (Ashwini Kumar et al)
Cyclic dengue epidemics in Kerala state have been occurring since 2001, even though the first
dengue report was brought on record from Kottayam district in 1997 with 14 cases and 4 deaths
(Unpublished observation). This was followed by a more sever dengue outbreak implicating 67
cases (nearly 5-fold increase) and a toll of 13 human lives (3-fold increase) in 1998, again in the
same district. (Apr-May 2006 ICMR Bulletin)
Pathogenesis and clinical manifestations
Duration of viremia ranged from 1 to 7 days . Viremia during primary infection was prolonged
compared with secondary infections.The patients initially develop an abrupt onset of high fever
(39–40°C) with headache, retro-orbital pain, malaise, nausea, vomiting, and myalgia. The acute
febrile stage lasts 2–7 days and may be followed by recovery but patients feel weakness. During
defervescence some patients develop haemorrhagic manifestation that may be mild petechial
haemorrhage, and bleeding at the nose, gastrointestinal tract and gums, which may be severe. .
(David W. Vaughn et al) Hepatomegaly is common with soft and tender liver.
Thrombocytopoenia and rising haematocrit due to plasma leakage are usually detectable before
the onset of the subsequent stage of shock (DSS) with an abrupt fall to normal or subnormal
levels of temperature, varying degrees of circulatory disturbances lasting for 24–48 h.( U C
Chaturvedi et al)
Dengue virus infection in humans causes a spectrum of illness ranging from inapparent or mild
febrile illness to severe and fatal hemorrhagic disease. Important risk factors influencing the
proportion of patients who have severe disease during epidemic transmission include the strain
and serotype of the infecting virus and the immune status, age, and genetic background of the
human host. Classic dengue fever is primarily a disease of older children and adults. It is
characterized by the sudden onset of fever and a variety of nonspecific signs and symptoms. The
rash usually begins on the trunk and spreads to the face and extremities. Hemorrhagic
manifestations in dengue fever patients are not uncommon and range from mild to severe. Skin
hemorrhages, including petechiae and purpura, are the most common, along with gum bleeding,
epistaxis, menorrhagia, and gastrointestinal (GI) hemorrhage.
The primary pathophysiologic abnormality seen in DHF and DSS is an acute increase in vascular
permeability that leads to leakage of plasma into the extravascular compartment, resulting in
hemoconcentration
and
decreased
blood
pressure.
Dengue
virus
infections
of
monocytes/macrophages is enhanced by ADE. This enhancement is facilitated by the fact that
the dengue virus-specific CD41 T lymphocytes produce IFN-g, which in turn up-regulates the
expression
of
FC-g
receptors.
The
increased
number
of
dengue
virus-infected
monocytes/macrophages results in increased T-cell activation, which results in the release of
increased levels of cytokines and chemical mediators. Kurane and Ennis hypothesized that the
rapid increase in the levels and the synergistic effects of mediators such as TNF, IL-2, IL-6, IFNg, PAF, C3a, C5a, and histamine induce increased vascular permeability, plasma leakage, shock,
and malfunction of the coagulation system, which may lead to hemorrhage.( Duane J. Gubler )
DENV is an enveloped positive-strand RNA virus belonging to the Flaviviridae family.Mature
virions contain three structural proteins, the capsid protein C, membrane protein M, and the
envelope protein E. Multiple copies of the C protein (11 kDa) encapsulate the RNA genome to
form the viral nucleocapsid . The nucleocapsid is surrounded by a host-cell-derived lipid bilayer,
in which 180 copies of M and E are anchored. The M protein is a small (approx. 8 kDa)
proteolytic fragment of its precursor form prM (approx. 21 kDa). The E protein is 53 kDa and
has three distinct structural domains .( Izabela A et al)
Plasma leakage and coagulopathy are the fundamental pathological changes responsible for
clinical manifestations, morbidity, and mortality in DHF. Both humoral and cell-mediated
immune mechanisms eventually result in the release of cytokines responsible for changes in the
selective microvascular permeability and the resultant plasma leakage. (Kolitha H. Sellahewa et
al)
Laboratory diagnosis
Viremia generally lasts from 4 to 5 days. The circulating viable DENV particles remain readily
detectable in the blood for up to 5 days after the onset of symptoms, and then rapidly disappear
following the appearance of DENVspecific antibody. DENV-specific immunoglobulin M (IgM)
antibody becomes positive on the sixth day following the onset of illness in most patients.
During primary DENV infection, the IgG antibody appears a few days following IgM antibody.
In secondary DENV infection, an anamnestic IgG response occurs, causing serotiter to rapidly
increase initially, almost immediately following the onset of disease, and then remain high in
most patients .Therefore, laboratory diagnosis must consider the timing of the clinical course of
dengue patients.
The most sensitive virus isolation method is in vivo amplification through mosquito inoculation,
the mosquito-derived cell cultures, such as C6/36 (Aedes albopictus), AP61 (Aedes
pseudoscutellaris) and TRA- 284 (Toxorhynchites amboinensis). The diluted serum sample is
inoculated on to the cell culture monolayer on screw cap tubes, dishes or flasks. Following
incubation at 28°C for 1 week, the inoculated cells are screened for DENV by use of IF stain
with 4 different serotype-specific monoclonal antibodies . The disadvantages of this method are
the time-consuming procedures required to obtain the culture results, and the need for cell
culture facilities and experienced laboratory professionals.
The presence of DENV antigen in acute sera and peripheral blood mononuclear cells (PBMC)
from DENV-infected patients has been determined using biotin-streptavidine enzyme-linked
immunosorbent assay (ELISA).
Molecular diagnosis typically provides more sensitive and rapid detection than traditional virus
isolation methods, because it amplifies nucleic acid even for inactivated virus. However, the
specimens and RNA must be carefully handled to avoid RNA degradation.To minimize
contamination and maximize costeffectiveness, Harris et al established a modified single tube
multiplex RT-PCR. Real-time RT-PCR assay represents another choice for quantifying DENV
RNA. PCR products were detected in real time on a Light Cycler.
Historically, hemagglutination inhibition (HI), complement fixation test (CF) and neutralization
tests were used extensively for measuring anti-DENV antibodies. Three main disadvantages of
the HI test have limited its wide application. First, tested serum samples must be pretreated with
acetone or kaolin, to remove nonspecific inhibitors of hemagglutination. Second, the accurate HI
test requires paired (acute and convalescent) serum samples. Third, difficulty in discrimination
between the DENV infections from other closely related flaviviruses.to minimize the
disadvantages of HI test, the plaque reduction neutralization test (PRNT) is mainly used to
determine the serotype of DENV with which the patients are infected. The disadvantages of
PRNT include the time-consuming titration of DENV and the careful handling of cell culture
techniques required in each step. Unlike traditionally complicated HI and PRNT tests, a simple
and rapid ELISA method has been adapted to detect antibodies against DENV. Several formats
of ELISA are designed for detecting DENV antibodies. Classical indirect ELISA and
immunoglobulin antibody capture ELISA are the 2 most common formats. Recently, anti-human
IgM capture ELISA (MACELISA) and anti-human IgG ELISA (GAC-ELISA) were developed
and widely used for detecting human anti-dengue IgM and IgG antibodies.( Chuan-Liang Kao et
al)
Conclusion
Natural concurrent infection with dengue viruses may occur in highly endemic areas where
different dengue serotypes have been transmitted for many years. Many cases of simultaneous
infection by more than one arbovirus species in mosquito or human hosts have been documented
elsewhere. Simultaneous infection by different strains of dengue viruses in human or mosquito
host cells affords the potential for virus recombination. In this regard, recent evidence provided
by phylogenetic and rigorous nucleotides sequence analyses of dengue virus genomes showed
that recombination is an important, yet largely ignored, mechanism responsible for generation of
dengue virus diversity. Therefore, the genetic exchange between dengue strains, although rarely
reported in positive-strand RNA viruses might be, in addition to mutation, important factor
involved in genetic variation of dengue virus8. Due to the complexity of dengue infection and
the difficulties in obtaining a safe and effective vaccine, recombination and its role in the genetic
diversity of dengue virus must be investigated further. Strains isolated from cases of natural
concurrent infection could be good models in such studies.( Cecília Luiza Simões dos SANTOS
et al)
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