Review Article

Indian J Med Res 123, May 2006, pp 615-628

Diagnostic methods for detection & isolation of dengue viruses

from vector mosquitoes

P. Philip Samuel & B.K. Tyagi

Centre for Research in Medical Entomology (ICMR), Madurai, India

Received July 9, 2004

Dengue is a deadly mosquito-borne infection warranting urgent attention for its containment
particularly in the tropical and subtropical countries. In the absence of a vaccine or any specific
drug for its treatment, an early diagnosis is considered indispensable to prevent any casualty.
Detection of viruses in human sera particularly in endemic areas is cumbersome, difficult and also
not desirable. Therefore, as an alternative approach, detection of the dengue virus antigen in
mosquitoes has provided a reliable tool to (i) comprehend the types of viruses circulating in nature;
and (ii) help in designing vector-specific control strategies. A mlange of diagnostic techniques are
currently available with some advantages or disadvantages. Traditionally, cell cultures and suckling
mice have been employed for virus isolations. While the virus isolation method in baby mice is
time consuming, slow and expensive, the mosquito cell cultures offer a good degree of specificity.
Mosquito inoculation techniques have been reported for detection and propagation of flaviviruses.
Though this technique is sensitive for routine virological confirmation of dengue fever, it requires
large number of infected mosquitoes, besides being time consuming. Insect bioassays (Toxo-IFA)
are generally cumbersome requiring special facilities and are not suitable for large-scale
epidemiological surveillance. ELISA has been shown to be a rapid and sensitive alternative to
insect bioassays for monitoring arboviruses in wild populations. Reverse transcriptase polymerase
chain reaction (RT-PCR) is a recent molecular diagnostic technology used for detecting virus
infections in mosquitoes, which gives rapid results but is expensive and prone to contamination.

This review describes the development of various techniques involved in detection and isolation of
dengue viruses in mosquitoes. Definite diagnosis of the impending dengue epidemic can be made
using ELISA for virological surveillance system on dengue virus antigen in the mosquito vectors.
Therefore, ELISA offers a potential tool and a convenient system for quickly screening large number
of samples up to the serotype level which can be employed effectively and efficiently for large scale
dengue surveillance programmes on wild caught mosquito vectors. ELISA positive samples can be
screened further by Toxo-IFA system for virus isolation. On the other hand, techniques like
mosquitoes cell culture, mosquito inoculation (Toxo-IFA) and RT-PCR techniques can be employed
for dengue virus amplification.

Key words Dengue - diagnostics - vector mosquitoes - virus

615
616 INDIAN J MED RES, MAY 2006
Dengue, a mosquito-borne viral infection is
regarded as a major public health problem globally.
The two main clinical manifestations, namely dengue
haemorrahagic fever (DHF) and dengue shock
syndrome (DSS), are responsible for exacting heavy
morbidity and mortality every year and continues to
be serious public health problem1,2 . The severity of
this disease can be judged from simple statistics that
over 40 per cent (2.5 billion) of the worlds
population in 100 tropical and subtropical countries,
continue to live under the threat of contracting
dengue infection 3, while close to 50 million new
infections and 24,000 deaths are reported annually
worldwide. Besides, every year the disease forces
nearly 500,000 people to hospitalization, of which
90 per cent are children. It is therefore, not surprising
that disability adjusted life years (DALY) for dengue
infection exacts a disease burden more or less equal
to that of malaria (465,000) 4. Mostly these deaths
are due to lack of early diagnosis of dengue virus
infection caused by four distinct serotypes, DEN-1,
DEN-2, DEN-3 and DEN-4. DHF case fatality rate
that can generally exceed 20 per cent in a non-
endemic population, may be reduced to less than
1 per cent with the aid of modern supportive therapy
based on early diagnosis of the type of viral
infection5. Dengue viruses are, however, amongst the
most difficult arboviruses to be isolated and
propagated in vitro. In most parts of South Asia,
including India, both Aedes aegypti and Ae.
albopictus are considered as the two main vectors
for dengue transmission. The geographical
distribution of the disease is characteristically
parallel to that of the principal vector species, Ae.
aegypti6.
Each year large numbers of dengue cases among
children occur in many places coinciding with
monsoon season and period of high vector prevalence.
Sudden onset of dengue epidemics can be greatly
mitigated using rapid and reliable diagnostic methods
for the control and management of this disease. A
definite diagnosis, which depends on isolation of the
virus, can be made only in the laboratory by detecting
viral antigen. Until recently detection of the virus
relied solely on viral isolations, however, current
procedures can detect dengue virus RNA and specific
virus antigens in the different tissues. Surveillance of
mosquitoes infected with dengue viruses can help
monitor the infection rates within vector mosquito
population harbouring specific serotype and provides
an early warning sign to predict an impending
epidemic7. Some workers have recently advocated use
of virus infection rates in vector species as well as
density of vectors in developing early warning tool8.
This fact underscores the importance of vector
mosquitoes in being employed to yield quick and
reproducible results without any ethical implications.
Various types of sensitive, specific and rapid
diagnostic tools are available, each having its own
advantages and disadvantages, and can be effectively
used to find out the infected mosquitoes under a given
set of laboratory conditions, albeit extensive cross-
reactions among the flaviviruses obfuscating
identification of the particular serotype. The best
method for diagnosis is considered the one that is
rapid, specific and inexpensive. Obviously there is no
one best method for all arboviruses, therefore, a
melange of approaches fulfills the requirement. The
present review brings together the available
information on diagnostic methods employed for this
purpose on vector mosquitoes and the relative
significance of each technique to yield dependable
results. This will help to get reliable estimate of the
dengue infection in the mosquito vectors that can be
included in the dengue surveillance programmes in
future.
Virus isolation
Methods selected for virus isolation depend upon
the laboratory facilities available. No single isolation
system is adequate for all arboviruses. Identification
of the infecting dengue virus serotype depends upon
isolation of virus in a sensitive host system followed
by serotype identification using reference
monoclonal antibodies. A successful isolation of
virus followed by serotyping usually takes more than
two weeks which procrastinates the inevitable vector
control under epidemic situations. Dengue viruses
do not grow well in vitro normally. However, at
present some more sensitive isolation systems
comprising certain insect species such as non blood-
sucking Toxorhynchites mosquitoes are available
wherein the inoculation and isolation of dengue
viruses are successfully accomplished.
 SAMUEL & TYAGI: DETECTION & ISOLATION OF DENGUE VIRUS IN MOSQUITOES
(i) Suckling mice: All the four dengue viruses have
been successfully isolated in BS-C-1 cells (African
green monkey kidney cells) or 1-3 days old baby mice
using a soup prepared from Ae. aegypti9,10. Baby mice
are very insensitive before inculcating an evidence
of infection 7. In spite of this, suckling mice are
important as it is generally not possible to detect the
virus in other animal host body (e.g., mosquitoes,
ticks) when in low quantity. Mice are inoculated
intracranially with classified suspensions of clinical
specimens or macerated arthropod pools or animal
tissues. Since the suckling mice are readily available
in all laboratories and have certain practical
advantages over others, the supernatant of the
mosquito soup after centrifugation is inoculated
intracerebrally into suckling mice for virus isolation.
Dengue serotypes 1 and 4 were isolated from Ae.
aegypti in 1961 from Vellore, in Tamil Nadu State,
by inoculating infant mice11. In Singapore during 1960-
1961, six strains of DEN-2 serotype were isolated, five
from pools of Ae. aegypti and one from a pool of Ae.
albopictus. The dengue virus infection rates based on
minimum virus isolation rates per 1000 were 18.6 for
Ae. aegypti, and 0.8 for Ae. albopictus. The majorities
of the virus isolates were adapted to infant mice with
difficulty and required many serial passages before
illness appeared 12. During 1962-1963 nearly 200
isolates of dengue viruses were recovered from human
and arthropod materials in Thailand by passage in
suckling mice and/or tissue culture13. All the five pools
of Ae. aegypti mosquitoes from Rajasthan were
positive in mice14. Using mouse inoculation test, many
investigators were unsuccessful in isolating virus from
human cases during epidemics in India15-17. However,
using the same system, virus isolations were made
from about 24 per cent18 and 30 per cent of patients19.
Prasad Rao et al20 reported only 3 per cent isolation
rate during 1978 outbreak20. One advantage of using
baby mice, is that other arboviruses that cause dengue-
like illness may be isolated with this system2. Virus
isolation using suckling mice is time consuming, slow
and expensive. Moreover, because of low sensitivity
of this method many viruses cannot be isolated with
baby mice. Those that are isolated frequently required
numerous passages to adapt the virus growth in mice.
This method is no longer recommended for isolation
of dengue viruses21.
617
(ii) Mosquito cell cultures: The choice depends on
the availability of a host-cell cultures or mice that
serve an indicator of virus infection, i.e., cytopathic
effects in cell cultures, sign of illness or death in
mice. This is the most common method for virus
isolation. Grace22 reported the establishment of the
first mosquito cell line in the world from Ae. aegypti
mosquitoes. Singh23 established two cell lines at the
National Institute of Virology (NIV) Pune, from the
larval tissues of Ae. aegypti and Ae. albopictus. The
first Culex cell line in the country was established
from the embryonic tissue of Cx. bitaeniorhynchus
mosquitoes24. Cell lines from other two species of
Culicine mosquitoes viz., Cx. infula, Cx. ambiguour,
which are members of Cx.bitaeniorhynchus complex
were also established subsequently25. These cell lines
also supported the multiplication of other arboviruses
of public health importance in India viz.,
chikungunya, dengue and sindbis viruses26. A new
cell line from the forest mosquito Ae. krombeini was
established in the early 1990s, which proved highly
sensitive to several arboviruses 27. It was extremely
sensitive to Japanese encephalitis (JE) and dengue
viruses and very low titre of these viruses could be
detected using indirect immunofluorescent technique
(IFA) while Cx. bitaeniorhynchus and C6/36 cell
lines could not detect these viruses when infected
with same dilution28. Recently, a new cell line from
the embryonic tissue of Cx. tritaeniorhynchus
mosquitoes was established. IFA technique is now
routinely being used in the detection of virus antigen
in infected cell lines and mosquitoes 29,30. Mosquito
cell cultures particularly C6/36 (Ae. albopictus), AP-
61 (Ae. pseudoscutellaris), Ae. krombeini, TR-248
(Toxorhynchites amboinensis), and other established
mammalian cell culture lines (LLC-MK2 cells), are
commonly methods for virus isolation27,31-35. These
are not only the most widely used but also provide a
relatively simple and economical method for dengue
virus isolation and assay36. The development of direct
plaque assay using LLC-MK2 cells provided faster
and more sensitive method, but still had the
disadvantage of requiring adoption of the virus to
the cell culture37. Several continuous mosquito cell
lines have been shown to be highly susceptible to
dengue virus infection. The C6/36 clone of Ae.
albopictus cells was chosen for virus isolation
because it demonstrated high sensitivity to dengue
618 INDIAN J MED RES, MAY 2006
virus infection and ease of handling31,36. These cell
lines are highly stable and have optimal growth at
lower temperatures than mammalian cells. The virus
titre can be determined in all groups of mosquitoes
using BHK-21 cells38. In Peninsular Malaysia, virus
isolation was carried out using cell culture (C6/36
clone) of Ae. albopictus and detection of dengue virus
by peroxidase staining. All positive isolations were
further confirmed by the reverse transcriptase-
polymerase chain reaction (RT-PCR)39.
For dengue virus isolation mosquito cell cultures
have proved to be more sensitive than mice or
mammalian cell culture systems. Some prefer to use
cytopathic effect to detect infection especially with
AP-61 cells. This has the disadvantage of not
producing cytopathic effects (rounding, refraction of
light, detachment from the substrate) and requires
secondary step for recognizing presence of virus in
the culture. Intra-thoracic inoculation of
Toxorhynchites mosquitoes (which do not take blood
meals) or Aedes mosquitoes has also been used. In
routine diagnosis, the C6/36 cell lines have become
most widely used. Following a period of 7-10 days
post-inoculation in cell lines or 14 days post-
inoculation in mosquitoes, virus identification is done
by immunofluorescence assay with serotype-specific
monoclonal antibodies 35,40,41 . Viral isolation rates
were obtained up to 36 per cent with C6/36 cell lines
or up to 80 per cent by direct mosquito
inoculation 41,42 . A rapid centrifugation assay for
dengue virus improved the isolation rate, even in
tissue samples43. IFA has become an important tool
in the detection of virus antigen with its simplicity
and specificity 44. Several virus isolations were made
in mosquito cell lines from field collected mosquitoes
at NIV, Pune 45-48. The Cx. bitaeniorhynchus cell line
for JE virus and Ae. albopictus cell line or C6/36
cells 31 for DEN viruses are mainly being used in the
isolation of these viruses from mosquitoes collected
during epidemic periods. Ravi et al 49 reported
isolation rates of 3.7 per cent using Ae. albopictus49
and 7.5 per cent using Ae. pseudoscutellaris cell
cultures50.
A new cell line from Cx. tritaeniorhynchus
supported the multiplication of JE and West Nile
viruses but not of any of the dengue serotypes51. Due
to the presence of certain cytoplasmic inclusions in
the cells 52, this cell line was not used for further
research especially for isolation of virus from field-
collected mosquitoes53. However, this system was
less sensitive than mosquito inoculation 54 . The
sensitivity of the mosquito cell lines may also vary
with the strain of the virus. In samples from an
epidemic in Mozambique, more than twice as many
DEN-3 viruses were isolated by mosquito inoculation
than by the use of mosquito cells 55 . None of the
standard laboratory animals nor mammalian cell
cultures are sufficiently susceptible to dengue virus
infection to use for routine dengue virus isolation56.
Although the use of this method continues in some
laboratories, it is not recommended41,54.
(iii) Mosquito inoculation: Mosquito inoculation
techniques are reported for detection and viral
amplification45,46,57-62. This technique provided for the
first time a sensitive method for isolation and assay
of dengue viruses. Tx. splendens, a non
haematophagous mosquito, was evaluated as a
bioassay host for the detection and propagation of
dengue viruses. All dengue virus serotypes and
strains attained titres in Tx. splendens comparable to
those observed for 2 strains of Ae. aegypti. Peak virus
titres occurred in Tx. splendens approximately 6 days
post-inoculation; however, specific fluorescence for
all viruses was not observed in 100 per cent of
mosquito heads until 12 days post-inoculation. A 100
per cent correlation was noted between specific
fluorescence in Tx. splendens heads and the recovery
of virus from corresponding thorax, abdomens. The
volume of inoculum tolerated by Tx. spendens was
approximately 5 times greater than that injected into
Ae. aegypti. The overall survival rate for Tx.
splendens following intra-thoracic inoculation with
dengue viruses was higher compared to Ae. aegypti.
These findings imply that Tx. splendens would be
more efficient than Ae. aegypti as a laboratory assay
host for detecting dengue viruses in blood of infected
patients and for use in experimental investigations63.
The mosquito inoculation technique was used for all
titrations 64. After incubation for 10 days at 32C
individual mosquitoes were examined for the
presence or absence of viral antigen in the salivary
glands and brain tissue by the direct
immunofluorescence antibody technique (IFA).
 SAMUEL & TYAGI: DETECTION & ISOLATION OF DENGUE VIRUS IN MOSQUITOES
Mourya65 demonstrated fluorescence 24 h after intra-
cerebral inoculation of Tx.splendens larvae. This is
a simple technique to determine dengue virus
infection in the mosquito 57 . Variations of the
mosquito inoculation technique include inoculation
of adult64,66 and larval Toxorhynchites mosquitoes67,68.
Isolation of viruses by intracerebral inoculation of
the fourth instars of Tx. splendens larvae is routinely
followed for dengue diagnosis46,60,62,69-72. The viruses
are detected by indirect immunofluorescence (IIFA)
using a type-specific dengue monoclonal
antibody65,73. Mosquito inoculation techniques have
been shown to be sensitive for isolation of
flaviviruses67. Inoculation of Tx. splendens larvae is
relatively simple and safe and has been employed
for isolation of dengue virus62 and JE virus from field-
caught mosquitoes74.
Both the inoculation techniques have similar
sensitivity to intrathoracic inoculation of adult
mosquitoes, but are more difficult and labour-
intensive. The main advantage is that viruses can be
isolated in a few days. Inoculation of samples directly
into mosquitoes specially adult or larval inoculation
in Tx. amboinensis and Tx. splendens is the best
isolation system in terms of sensitivity66,71. NIV, Pune
and Centre for Research in Medical Entomology
(CRME), Madurai have developed necessary
expertise in this discipline8,67,74-87. Detection of virus
antigen is a promising tool for surveillance. Distinct
advantage is that it can be performed under field
conditions88. With the availability of a fluorescence
microscope objective attachment to a standard
laboratory microscope for field use89,90, it should now
be possible to perform the virus antigen detection
test even in peripheral laboratories. Insect bioassays
are cumbersome and special facilities are required91.
It has the disadvantage of being labour-intensive and
requires rearing and maintenance of mosquitoes67, the
expertise for which is not available in most virology
laboratories. The mosquito inoculation technique has
the disadvantages of being labour-intensive and
requiring an insectary to produce large numbers of
mosquitoes for inoculation. Also, unless strict safety
precautions are maintained, the chance of laboratory
infections increase, although this risk can be
eliminated by using male Aedes mosquitoes or non-
biting Toxorhynchites species for inoculation 64 .
619
However, the possibility of laboratory acquired
infections through the bite of mosquitoes should be
borne in mind and, therefore, rigid safety precautions
must be followed in utilizing this technique. The use
of non-biting mosquitoes92 can eliminate the risk of
laboratory infection through infective bite, but the
slow breeding and protracted life history of this
mosquito make it rather difficult to get them in
sufficiently large numbers for experimental purposes.
Unfortunately, this method is not available in most
endemic countries.
Direct visualization (detection) of viral antigens
in field-collected mosquito vectors of dengue
(i) Detection of antigen by enzyme linked
immunosorbant assay (ELISA): ELISA is the most
widely used in routine practice93-95. Dengue viruses are
difficult to isolate and propagate, as they do not grow
well in laboratory animals or cultures of mammalian
cells. Although there are very sensitive methods to
isolate dengue viruses in mosquito cell lines 36 and
mosquitoes 64 , they are time consuming, labour-
intensive and expensive33,66. Isolation and recovery of
arboviruses from field-collected mosquitoes can be
most efficiently accomplished by pooling adult
specimens prior to testing in various in vivo and in
vitro assay systems96. Such pooling of larvae has also
become a routine practice for evaluating the role of
transovarial transmission in the ecology of
arboviruses81-83. It is sometimes important to detect
virus in individual mosquitoes, although this is more
labour-intensive than detection in pooled specimens.
Use of individual specimens greatly reduces the
chance of mistakes caused by introducing a
misidentified specimen into a large pool. More
importantly, evaluation of individual mosquitoes can
generate a precise estimate of vector infection rate.
This parameter, combined with survival rate and
human biting rate, can be used to make a meaningful
estimation of local transmission risk97.
Direct detection of virus can provide a rapid
diagnosis. In the epidemic situations, the early
detection will help to devise appropriate public health
measures. Therefore the more rapid, sensitive and
specific diagnostic methods would be better in
situations for which time is a critical element. In
620 INDIAN J MED RES, MAY 2006
order to develop dengue-specific ELISA, dengue
serotype-specific monoclonal antibody (MABs) that
captures only dengue DEN1-DEN4 was used. By this
method studies undertaken in the rural dengue
surveillance in Vellore district and dengue epidemic
in Chennai, south India, were investigated. In
Vellore, pools of mosquitoes of adult Ae. aegypti and
Ae. albopictus were tested by ELISA and found
positive for flavivirus antigen. By employing this
ELISA and/or Toxo-IFA system the natural vertical
transmission of dengue viruses in Ae. aegypti in
Vellore district was also investigated and DEN-2
virus was identified in Vellore 8,73 . ELISA can be
used as an inexpensive way to screen large
number of mosquito specimens with relatively little
effort 8,67,74,77-87,98-100. Detection of virus infection in
wild-caught Aedes species should form an essential
component of a surveillance system for dengue.
ELISA technique is shown to be a sensitive
alternative to insect bioassay for monitoring
arboviruses in wild mosquito population. This
technique will be useful in dengue virus surveillance
for monitoring the dengue virus activity in endemic
areas and also to develop an early warning to plan
control strategies79. Thus ELISA based methods using
specific MABs can also lead to definite
diagnosis33,96,98,101,102. The antigen capture ELISA for
virus detection is the most useful procedure currently
available and it is widely recommended for
virological surveillance. During routine surveillance
in our study, initially some 600 pools were tested by
Toxo-IFA and those found positives were confirmed
by ELISA. This procedure took nearly 1 yr. After
adapting the revised strategy, in about 6 months, over
4,000 pools were first screened by ELISA and more
than 70 per cent of about 300 pools were examined
by Toxo-IFA also67.
(ii) Detection of antigens by immunofluorescence:
Identification of dengue virus isolates by using
monoclonal antibodies in an indirect
immunofluorescence assay was developed40,103. The
method of choice for dengue virus identification is
IFA with serotype-specific monoclonal antibodies
produced in tissue culture or mouse ascitic fluids and
an anti-mouse immunoglobulin G-fluorescein
isothiocyanate conjugate. This test can be easily
performed with infected cell cultures, mosquito brain
or tissue squashes, mouse brain squashes, or even
on formalin-fixed tissues embedded in paraffin and
sectioned for histopathological testing. It is a simple
and reliable method which allows the detection of
multiple viruses in patients with concurrent
infections with more than one serotype.
In those laboratories that do not have
immunofluorescence capability, dengue viruses can
also be identified using monoclonal antibodies in an
antigen capture ELISA 33,54. These methods usually
take two or more weeks delaying after the laboratory
diagnosis for initiating the control operation.
(iii) Immune electron microscopy: Its advantages
include rapidity and lack of requirement for specific
reagents. High viral density in liquids is required.
Only small tissue areas can be examined by this
sectioning, however, techniques have been described
for enhancing chances for virus detection. The most
difficult task in diagnosing viruses by electron
microscopy is to determine whether unusual structure
are indeed viruses rather than spherical structures and
membrane debris in negative stains and normal
cellular organelles in thin sections. Electron
microscopy can be an important adjunct to other
methods for virus identification, but possibly cannot
be relied on its own. Recently structural identity of
Chandipura virus was confirmed by electron
microscopy from a large encephalitis outbreak in
children in many districts of Andhra Pradesh104.
Modern diagnostic technologies
Polymerase chain reaction (PCR) has been applied
to dengue diagnosis with sera, tissue from fatal cases,
mosquito pool, infected cell cultures, and mosquito
larvae, mosquito surveillance and genetic strain
characterization. Several PCR protocols for dengue
detection have been described that vary in the RNA
extraction methods, genomic location of primers,
specificity, sensitivity and the methods to detect PCR
products and to determine the serotype105-108. Reverse
transcriptase (RT)-PCR has provided one of the most
important steps in the molecular diagnosis of dengue
virus34. A rapid assay has been developed followed
by a second PCR with specific primers allowing
serotype identification (DNA products) of different
sizes according to the dengue serotype obtained105.
 SAMUEL & TYAGI: DETECTION & ISOLATION OF DENGUE VIRUS IN MOSQUITOES
(i) Polymerase chain reaction (PCR): In recent years
RT-PCR has been developed for a number of RNA
viruses, including dengue viruses109. Unlike most
other techniques which require screening of pools of
mosquitoes to detect viruses, RT-PCR carries out the
job with solitary specimen. The technique allows for
the multifold biological amplification of viral nucleic
acid and has been used to rapidly diagnose viral
diseases 34,110-112. The primary advantage of this
molecular tool lies in the speed at which specimens
can be screened for the presence of dengue viruses
and also by its highly sensitive and specific detection.
It is able to monitor the infection rate in mosquitoes,
both adults and larvae, with a high degree of
precision 1,113 . RT-PCR can also detect small
quantities of virus 3. This method has also been
employed for detecting and typing dengue virus RNA
in the field caught Aedes mosquitoes, besides
determining the infection rate in local Aedes
mosquitoes 114. Chung et al114 have developed a single
RT-PCR followed by a semi-nested PCR using an
upstream consensus primer and four type-specific
primers within the non-structural protein gene (NS3)
of dengue viruses to type dengue viruses in field
populations of female Aedes mosquitoes.
Alternatively, RT-PCR offers the potential for the
rapid, highly sensitive and specific detection of
dengue viruses up to the serotype level.
In the dengue-sensitive areas in Singapore, a rapid
and sensitive, semi-nested, RT-PCR assay using non-
structural protein 3 gene primers for the type-specific
detection of dengue viruses in artificially infected
and in field-collected adult Aedes mosquitoes were
employed115. In laboratory experiments, the assay was
sensitive enough to detect one virus infected
mosquito head in pools of up to 59 uninfected heads.
Use of RT-PCR in epidemiological investigations has
been intensified recently as geographic locations of
the virus-infected Aedes mosquitoes, detected as
early as six weeks before the start of the dengue
outbreaks, were traced to have a correlation with the
residence or workplaces of patients. Virologic
surveillance using RT-PCR for detecting dengue
virus-infected Aedes mosquitoes in the field may
serve as an additional early warning monitoring
system for predicting dengue outbreaks115.
621
Since a change in serotype is particularly
important to bring about a surge in DF/DHF cases,
RT-PCR is further advantageous in detecting the
specific serotype circulating in natural populations
of mosquitoes116. The PCR can be designed to be
broadly cross-reacting or extremely specific. The
extraction techniques used to isolate RNA destroy
the infectivity of the virus, therefore, the risk of
exposure to bio-hazardous materials is eliminated
once the RNA is extracted from the sample. Despite
its advantages, the PCR is not routinely employed in
arbovirus diagnostic laboratories because it is
expensive. Improvements in automated handling of
PCR as well as detection of product are currently
being developed but are not available to most
laboratories performing arbovirus diagnosis. PCR is
notoriously prone to contamination. Every batch of
assays of mosquitoes should accompany a no-DNA
(negative control) sample, and stringent precautions
must be taken to avoid carryover contamination 117.
PCR is used for quick detection and it requires a small
quantity of the sample.
Although RT-PCR has similar sensitivity to virus
isolation systems that use C6/36 cell cultures, poor
handling, storage and time of sample collection and
quantum of virus present in the samples greatly affect
the results. The presence of antibody usually do not
influence the outcome of PCR as they do in virus
isolation. A number of methods involving primers
from different locations in the genome and different
approaches to detect the RT-PCR products have been
developed over the past several years118,35,41. Since
RT-PCR is highly sensitive to amplicon
contamination, without proper controls false-positive
results may occur.
(ii) Hybridization probes for detection of viral nucleic
acid: Recent advances in the molecular technology
such as RT-PCR and hybridization, nucleic acid probes
are made possible for the detection of viral genomic
materials in mosquitoes. Hybridization techniques are
used for the detection of many important arboviruses.
Hybridization probe method detects viral nucleic acids
with cloned hybridization probes 119. Probes with
variable specificity ranging from dengue complex to
serotype-specific can be constructed depending on the
622 INDIAN J MED RES, MAY 2006
genome sequences used. A PCR-amplified serotype-
specific complimentary DNA (cDNA) was cloned and
used as a non-radioactive nucleic acid hybridization
probe in subsequent assays 120 . Hybridization
techniques are rapid and specific and relatively simple
especially good for diagnosis of viruses that are
difficult to cultivate. Using degenerate primers, RT-
PCR forms a sensitive and specific method for the
detection of dengue viruses in clinical specimens. The
use of radiolabelled serotype probes allows for an
additional level of specificity (96-100% specific),
unavailable in assays using RT/PCR alone (89-
100%)111. Despite several advantages, hybridization
techniques have certain drawbacks like lack of
sensitivity. Moreover, a labelled probe specific for
each virus to be detected is required.
Preliminary data suggest121 that this method is less
sensitive than RT-PCR, but like PCR the outcome of
the test is not influenced by the presence of inhibitory
substances. Difficulties encountered during working
with RNA and the technical expertise required to
obtain reproducible results make this method more
suitable as a research tool than as a routine diagnostic
test2. Moreover, in this technique as a pre-requisite,
viraemia levels should also correlate with mosquito
infection rates121.
Discussion and conclusion
Reliable estimation of natural mosquito infection
with arboviruses forms a key element in any
surveillance system and is essential for vector
incrimination and monitoring control measures 67. A
laboratory diagnosis of dengue infection can be
accomplished by detecting either the virus or anti-
dengue antibodies. Methods selected for virus
isolation depend much upon the laboratory facilities
available. In the case of virus isolation the baby mice
virus isolation method is very time-consuming, slow
and expensive. Moreover, because of the low
sensitivity of the method, many wild-type viruses
cannot be isolated 21 . Those that are isolated
frequently require numerous passages to adapt the
viruses to grow in mice 122. This method is no longer
recommended for isolation of dengue viruses.
Mosquito cell cultures are a recent addition to
dengue virus isolation methodologies. Three cell
lines of comparable sensitivity are most frequently
used. The C6/36 clone of Ae. albopictus cells is less
sensitive than the mosquito inoculation method. Use
of these cell lines has provided a rapid, sensitive and
economical method for dengue virus isolation. The
sensitivity of mosquito cell lines may vary with the
strain of the virus. Even though cell cultures are less
sensitive than mosquito inoculation, large number
of samples can be processed in a relatively short time.
Mammalian cell cultures have many of the same
disadvantages as baby mice for isolation of dengue
viruses, such as being expensive, slow, and intensive.
Mosquito inoculation is a method sensitive
enough for routine successful virologic confirmation
of dengue. Moreover, there are many endemic dengue
virus strains that can be recovered only by this
method. Four mosquito species have been used for
virus isolation, viz., Ae. aegypti, Ae. albopictus, Tx.
amboinensis, and Tx. splendens. Male and female
mosquitoes are equally susceptible. A recent
variation on this method involves intracerebral
inoculation of larval and intrathoracic inoculation of
adult Toxorhynchities mosquitoes. However, intra-
cerebral innoculation not provided any advantage
over intrathoracic inoculation method since being
rapid. The mosquito inoculation technique has the
disadvantages of being labour-intensive and requires
an insectary to produce large numbers of mosquitoes
for inoculation. The possibility of laboratory acquired
infections through the bite is possible and therefore
safety precautions must be followed in utilizing this
technique 30 . Unless rigid safety precautions are
maintained, the chance of laboratory infections
increases, although this risk can be eliminated by
using male Aedes mosquitoes or non-biting
Toxorhynchites species for inoculation 92 .
Conventional methods, i.e., culture of virus in
suckling mice and the use of microinoculation of
Toxorhynchites adults or larvae and subsequent
detection with fluorescent antibody staining, for
detecting virues in the vector are laborious and time-
consuming 39 . Virus detection in the mosquito,
regardless of the species, is generally performed by
the IIFA test on mosquito tissues, usually brain or
salivary glands40,57,71,123. Another recent advance is the
development of specific monoclonal antibodies for
identification of dengue viruses36,124. For large-scale
 SAMUEL & TYAGI: DETECTION & ISOLATION OF DENGUE VIRUS IN MOSQUITOES
surveillance on wild-caught mosquito vectors of
dengue to estimate true positives, ELISA technique
can be used as a convenient and rapid system for
screening large number of samples 125 . For the
diagnosis up to serotype level, if the quantity of the
sample is sufficient, serotype-specific antibodies can
be used for ELISA.
In comparison to foregoing description of various
techniques employed in detection/isolation of dengue
viruses, the RT-PCR method is rapid, highly
sensitive, simple, reliable and reproducible for the
specific detection of dengue viruses. It can be
effectively used to detect viral RNA in mosquitoes 1.
The improvements in RT-PCR technology are being
continuously improvised which make it even more
useful in its application for dengue virus detection.
When the sample quantity is very less or insufficient
all the positives can be propagated by mosquito
inoculation technique/tissue culture/RT-PCR for
further virus isolation studies.
Dengue virus do not grow well in any of the
laboratory animals or mammalian cell cultures
normally used in virology laboratories. Dengue
viruses were first isolated by inoculation of baby
mice. This system is very insensitive and several
blind passages are usually required to allow
adaptation of the virus. The development of mosquito
inoculation technique provided, for the first time a
sensitive and relatively rapid method for isolation
and assay of dengue viruses. The IFA test provided
a simple technique to determine dengue virus
infection in the mosquitoes, but it requires expertise
to maintain a mosquito colony, which is not available
in many laboratories. This can be done using
mosquito cell lines that are less labour-intensive.
Even though mosquito cell lines are highly
susceptible to dengue virus infection, the technique
is less sensitive than the mosquito inoculation
technique for routine isolation of dengue viruses.
Toxorhynchites inoculation technique followed by
IFA is very sensitive for the detection of infection in
wild-caught vector mosquitoes and gives an estimate
of true positives. But it is time causing and
cumbersome for testing large number of samples.
ELISA test on the other hand is a convenient and
rapid system and can be used for large scale screening
623
of vectors. Further ELISA positives should be again
screened by Toxo-IFA system. By this new strategy,
a large number of pools can be screened within a
short time. The cost and labour involved are greatly
reduced. Thus utilization of these two techniques will
give a better estimate of mosquito infection rates and
help to forecast the associated risk of dengue
epidemic. Other methods can be employed for
propagation of these viruses and can be used for virus
isolation later. According to the laboratory facility
available, certain technical improvements can be
made on the diagnostic procedures which can also
be standardized and selected for the routine
surveillance use. This system allows the dengue
viruses being transmitted in an area to be monitored
with a minimal amount of effort and provides the
early warning capability necessary to predict
epidemic dengue.
References
1. C h a n S Y , K a u t n e r I M , L a m S K . D e t e c t i o n a n d
serotyping of dengue viruses by PCR: a simple, rapid
method for the isolation of viral RNA from infected
mosquito larvae. Southeast Asian J Trop Med Public
Health 1994 ; 25 : 258-61.
2. Gubler DJ. Dengue and dengue hemorrhagic fever.
Clin Microbiol Rev 1998; 11 : 480-96.
3. Fonseca BAL, Fonseca SNS. Dengue virus infections.
Curr Opin Pediatr 2002; 14 : 67-71.
4. Renganathan E, Parks W, Lloyd L, Nathan MB, Hosein
E, Odugleh A, et al. Towards sustaining behavioral
impact in dengue prevention and control. Dengue Bull
2003; 27 : 8-12.
5. WHO information Dengue and Dengue hemorrhagic fever.
Revised November 1998 Fact sheet No 117 HYPERLINK
http://www.who.int/inf-fs/en/fact - http://www.who.int/inf-
fs/en/fact 117.html, accessed on 26.06.2004.
6. Jacobs M. Dengue: emergence as a global public health
problem and prospects for control. Trans R Soc Trop Med
Hyg 2000; 94 : 7-8.
7. Lorono-Pino MA, Cropp CB, Farfan JA, Vorndam
AV, Rodriguez-Angulo EM, Rosado-Paredes EP, et al.
Common occurrence of concurrent infections by multiple
dengue virus serotypes. Am J Trop Med Hyg 1999 ; 61 :
725-30.
624 INDIAN J MED RES, MAY 2006
8. Tewari SC, Thenmozhi V, Katholi CR, Manavalan R,
Munirathinam A, Gajanana A. Dengue vector prevalence
and virus infection in a rural area in South India. Trop Med
Int Health 2004; 4 : 499-507.
9. Singhraj P, Simasathien P, Sukhavachana P, Halstead SB,
Scanlon JE. Recovery of dengue and other viruses in
mice and tissue culture from Thai mosquitoes.
Bull World Health Organ 1966; 35 : 67-8.
10. Carey DE, Myers RM, Reuben R. Dengue types 1 and 4
viruses in wild caught mosquitoes of south India. Science
1964; 143 : 131-2.
11. Carey DE, Myers RM, Reuben R, Rodrigues FM. Studies
on dengue in Vellore South India. Bull World Health Organ
1966; 35 : 61.
12. Rudnick A. Dengue viruses isolated from mosquitoes in
Singapore, 1960-61. Bull World Health Organ 1966; 35 :
63.
13. Udomsakdi S, Nisalak A, Halsted SB. Identification of
dengue and chikungunya viruses. Bull World Health Organ
1966; 35 : 68.
14. Ghosh SN, Pavri KM, Singh KRP, Sheikh BH,
Dlima LV, Mahadev PV, et al. Investigations on the
outbreak of dengue fever in Ajmer city, Rajasthan state in
1969. Part I. Epidemiological, clinical and virological study
of the epidemic. Indian J Med Res 1974; 62 : 511-22.
15. Chakraborty MS, Chakraborty SK, Mukherjee KK,
Mitra AC, Mitra KK, Gupta B, et al. Recurrent Japanese
encephalitis epidemics in West Bengal. Indian J Med Res
1980; 72 : 1-6.
16. Mohan Rao CVR, Prasad SR, Rodrigues JJ, Sharma NGK,
Shaikh BH, Pavri KM, et al. The first laboratory proven
outbreak of Japanese encephalitis in Goa. Indian J Med Res
1983; 78 : 745-50.
17. Mathur A, Chaturvedi UC, Tandon HO, Agarwal AK,
Mathur GP, Nag D, et al. Japanese encephalitis epidemic in
Uttar Pradesh, India during 1978. Indian J Med Res 1982;
75 : 161-9.
18. Rashmi K, Asha M, Arvind K. Clinical features and
prognostic indicators of Japanese encephalitis in children
in Lucknow (India). Indian J Med Res 1990; 91: 321-7.
19. Mathur A, Rashmi K, Sandhya S. Rapid diagnosis of
Japanese encephalitis by immunofluorescent examination of
cerebrospinal fluid. Indian J Med Res 1990; 19 : 1-4.
20. Prasad Rao GLN, Rodrigues FM, Nambiapan M, Nagarajan
M, Ghalsasi GR, Rodrigues JJ, et al. Aetiology of the 1978
outbreak of encephalitis in Tirunelveli and other district of
Tamil Nadu. Indian J Med Res 1982; 76 : 36-46.
21. Gubler DJ, Kuno G, Sather GE, Valez M, Oliver A.
Mosquito cell cultures and specific monoclonal antibodies
in surveillance for dengue viruses. Am J Trop Med Hyg
1984; 33 : 158-65.
22. Grace TDC. Establishment of a line of mosquito (Aedes
aegypti, L) cells grown in vitro. Nature 1966; 211 : 366-7.
23. Singh KRP. Cell cultures derived from larvae of Aedes
albopictus (Skuse) and Aedes aegypti (L). Curr Sci
1967; 36 : 506-8.
24. Pant U, Dhanda V. Establishment of a cell line from Culex
bitaeniorhynchus. J Tissue Culture Methods 1980; 6 : 61-3.
25. Pant U, Dhanda V. Arthropod tissue culture and its uses in
arboviral research. ICMR Bull 1985; 16 : 33-8.
26. Pant U, Banerjee K, Athawale SA, Dhanda V. Susceptibility
of Culex bitaeniorhynchus cell line to some arboviruses.
Indian J Med Res 1982; 76 : 789-94.
27. Pant U, Sudeep AB, Dhanda V, Mourya DT, Banerjee K.
New embryonic cell line from Aedes krombeini (H)
(Diptera: Culicidae). In Vitro Cell Dev Biol (Animal)
1992; 28 : 567-8.
28. Sudeep AB, Pant U. Aedes krombeini (H) cell line- A new
approach to arboviral research. Paper presented at the Ivth
international symposium on vectors and vector borne
diseases. Proceedings of the National Academy of Vector
Borne Diseases, Bhubaneswar and Defence Research and
Development Establishment, Gwalior; 1999 p. 45.
29. Sudeep AB, Pant U, Dhanda V, Banerjee K. A new Aedes
krombeini cell line and its susceptibility to some arboviruses.
In: Maramorosch K, Mitsuhashi J, editors. Invertebrate cell
culture: Novel directions and biotechnology applications.
USA: Science Publishers Inc: 1997 p. 11-7.
30. Ilkal MA, Dhanda V, Rodrigues JJ, Mohan Rao CVR,
Mourya DT. Xenodiagnosis of laboratory acquired infection
by mosquito inoculation and immunofluorescence.
Indian J Med Res 1984; 79 : 587-90.
31. Igarashi A. Isolation of a Singhs Aedes albopictus cell
clone sensitive to dengue and chikungunya viruses.
J Gen Virol 1978; 40 : 531-44.
32. Race MW, Williams MC, Agostini CFM. Dengue in the
Caribbean: virus isolation in a mosquito ( Aedes
pseudoscutellaris) cell line. Trans R Soc Trop Med Hyg
1979; 73 : 18-22.
33. Kuno G, Gubler DJ, Santiago de Well, N. Antigen capture
ELISA for the identification of dengue viruses. J Virol
Methods 1985; 12 : 93-103.
 SAMUEL & TYAGI: DETECTION & ISOLATION OF DENGUE VIRUS IN MOSQUITOES
34. Maneekarn N, Morita K, Tanaka M, Ugarashi A,
Usawattanakul W, Srisanthana V, et al. Application of
polymerase chain reaction for identification of dengue virus
isolated from patient sera. Microbiol Immunol
1993; 37 : 41-7.
35. Guzman MG, Kouri G. Advances in dengue diagnosis.
Clin Diagn Lab Immunol 1996; 3 : 621-7.
36. Tesh RB. A method for the isolation and identification of
dengue viruses using mosquito cell cultures. Am J Trop
Med Hyg 1979; 28 : 1053-9.
37. Yull TM, Sukhavachana P, Nisalak A, Russell PK. Dengue-
virus recovery by direct and delayed plaques in LLC-MK2
cells. Am J Trop Med Hyg 1968; 17 : 441.
38. Morens MD, Halstead SB, Repik MP. Simplified plaque
reduction neutralization assay for dengue viruses by
semimicro methods in BHK-21 cells. Comparison of BHK
suspension tests with standard plaque reduction
neutralization. J Clin Microbiol 1985; 22 : 205-14.
39. Ahmad R, Ismail A, Saat Z, Lim LH. Detection of dengue
virus from field Aedes aegypti and Aedes albopictus adults
and larvae. Southeast Asian J Trop Med Public Health 1997;
28 : 138-42.
40. Henchal EA, Mc Cown JM, Seguin MC, Gentry MK, Brandt
WE. Rapid identification of dengue virus isolates by using
monoclonal antibodies in an indirect immunofluorescence
assay. Am J Trop Med Hyg 1983; 32 : 164-9.
41. Vorndam V, Kuno G. Laboratory diagnosis of dengue virus
infections. In: Gubler DJ, Kuno G, editors. Dengue and
dengue haemorrhagic fever. New York: CAB International;
1977 p. 313-33.
42. Kuno G, Gubler DJ, Velez M, Oliver A. Comparative
sensitivity of three mosquito cell lines of dengue viruses.
Bull World Health Organ 1985; 63 : 279-86.
43. Rodriguez R, Alvarez M, Guzman MG, Morier L, Kouri G.
Isolation of dengue 2 in C6-36/HT cells by rapid
centrifugation/shell vial assay. Comparison with
conventional virus isolation method. J Clin Microbiol 2000;
38 : 3508-10.
44. Gubler D, Trent DW. Emergence of epidemic dengue/
dengue hemorrhagic fever as a public health problem in the
Americas. Infect Agents Dis 1994; 2 : 383-93.
45. Dhanda V, Mourya DT, Mishra AC, Ilkal MA, Pant U, Jacob
PG, et al. Japanese encephalitis virus infection in mosquitoes
reared from field collected immatures and in wild caught
males. Am J Trop Med Hyg 1989; 41 : 732-6.
46. Mourya DT, Ilkal MA, Mishra AC, George JP, Pant U,
Ramanujam S, et al. Isolation of Japanese encephalitis virus
625
from mosquitoes collected in Karnataka state, India, from
1985 to 1987. Trans R Soc Trop Med Hyg 1989; 83 : 550-2.
47. Naik PS, Ilkal MA, Pant U, Kulkarni SM, Dhanda V.
Isolation of Japanese encephalitis virus from Culex
pseudovishnui colless, 1957 (Diptera: Culicidae) in Goa.
Indian J Med Res 1990; 91 : 331-3.
48. Pant U, Ilkal MA, Soman RS, Shetty PS, Kanojia PC, Kaul
HN. First isolation of Japanese encephalitis virus from the
mosquito Culex triateniorhynchus Giles 1901 (Diptera:
Culicidae) in Gorakhpur district, Uttar Pradesh. Indian J
Med Res 1994; 99 : 149-51.
49. Ravi V, Vanajakshi S, Gowda A, Chandramuki A.
Laboratory diagnosis of Japanese encephalitis using
monoclonal antibodies and correlation of findings with the
outcome. J Med Virol 1989; 29 : 221-3.
50. Burke DS, Lorsomrudee W, Leake CJ. Fatal outcome
in Japanese encephalitis. Am J Trop Med Hyg 1985;
34 : 1203-10.
51. Athawale SS, Sudeep AB, Barde PV, Jadi RS, Pant U,
Mishra AC, et al. A new cell line from the embryonic tissues
of Culex tritaeniorhynchus and its susceptibility to certain
flaviviruses. Acta Virol 2002; 46 : 237-40.
52. Sreenivasan MA, Pant U, Athawale S, Arankalle V.
Detection by electron microscopy of endogenous viruses in
Aedes krombeini (H) line. In Vitro Cell Dev Biol (Animal)
1994; 30 : 142-4.
53. Sudeep AB, Mourya DT, Mishra AC. Insect cell culture
in research: Indian scenario. Indian J Med Res 2005;
121 : 725-38.
54. Gubler DJ, Sather GE. Laboratory diagnosis of dengue
and dengue hemorrhagic fever. In: Homma A, Cunha JF,
editors. Proceedings of the International Symposium
on yellow fever and dengue. Riode Janeiro, Brazil,
May 15-19, 1988 p. 201-302.
55. Gubler DJ, Sather GE, Kuno G, Cabral JR. Dengue 3 virus
transmission in Africa. Am J Trop Med Hyg 1986;
35 : 1280-4.
56. Gubler DJ. Current research on dengue. In: Kerry F. Haris,
editor. Current topics in vector research. New York:
Springer-Verlag; 1987 p. 337-56.
57. Kuberski TT, Rosen L. A simple technique for the detection
of dengue antigen in mosquitoes by immunofluoresence.
Am J Trop Med Hyg 1977; 26 : 533-7.
58. Gubler DJ, Reed D, Rosen L, Hitchcock JC Jr.
Epidemiologic, clinical and virologic observations on
dengue in the Kingdom of Tonga. Am J Trop Med Hyg
1978; 27 : 581-9.
626 INDIAN J MED RES, MAY 2006
59. Gubler DJ, Suharyono W, Lubis I, Eram S, Saroso JS.
Epidemic dengue hemorrhagic fever in rural Indonesia I.
Virological and epidemiological studies. Am J Trop Med
Hyg 1979; 28 : 701-10.
60. Thet-Win. Detection of dengue virus by
immunofluorescence after intracerebral inoculation of
mosquitoes. Lancet 1982; i : 53-4.
61. Pang T, Lam SK, Chew CB, Poon GK, Ramalingam S.
Detection of dengue virus by immunofluorescence following
inoculation of mosquito larvae. Lancet 1983; 1 : 1271.
62. Lam SK, Chew CB, Poon GK, Ramalingam S, Seow SC,
Pang T. Isolation of dengue viruses by intracerebral
inoculation of mosquito larvae. J Virol Methods 1986;
14 : 133-40.
63. Rosen L, Shroyer DA. Comparative susceptibility of five
species Toxorhynchites mosquitoes to parenteral infection
with dengue and other flaviviruses. Am J Trop Med Hyg
1985; 34 : 805-9.
64. Rosen L, Gubler DJ. The use of mosquitoes to detect and
propagate dengue viruses. Am J Trop Med Hyg 1974; 23 :
1153-60.
65. Mourya DT. Rapid detection of Japanese encephalitis virus
by immunofluorescence after intra cerebral inoculation of
mosquito larvae. Trans R Soc Trop Med Hyg 1990; 84 : 580.
66. Gubler DJ, Rosen L. A simple technique for demonstrating
transmission of dengue virus by mosquitoes without the use
of vertebrate hosts. Am J Trop Med Hyg 1976; 25 : 146-50.
67. Gajanana A, Rajendran R, Thenmozhi V, Philip Samuel P,
Tsai TF, Reuben R. Comparative evaluation of bioassay and
ELISA for detection of Japanese encephalitis virus in field
collected mosquitoes. Southeast Asian J Trop Med
Public Health 1995; 26 : 91-7.
68. Padbidri VS, Mahadev PVM, Thakare JP, Pant U, Ilkal MA,
Geevarghese G, et al. Virological and entomological
investigations of an outbreak of dengue fever in Dhule
district, Maharashtra. Indian J Med Microbiol 1996;
14 : 5-32.
69. Thu HM, Aye KM, Thein S. The effect of temperature and
humidity on dengue virus propagation in Aedes aegypti
mosquitoes. Southeast Asian J Trop Med Public Health
1998; 29 : 280-4.
70. Mitchel CJ, Miller BR. Vertical transmission of dengue
viruses by strains of Aedes albopictus recently introduced
into Brazil. J Am Mosq Cont Assoc 1990; 6 : 251-3.
71. Rosen L, Roseboom LE, Gubler DJ, Lien JC, Chaniotis B.
Comparative susceptibility of mosquito species and strains
to oral and parenteral infection with dengue and Japanese
encephalitis viruses. Am J Trop Med Hyg 1985; 34 :
603-15.
72. Freier JE, Grimstad RP. Transmission of dengue virus by
orally infected Aedes triseriatus. Am J Trop Med Hyg 1983;
32 : 1429-34.
73. Thenmozhi V, Tewari SC, Manavalan R, Balasubramanian
A, Gajanana A. Natural vertical transmission of dengue
viruses in Aedes aegypti in southern India. Trans R Soc Trop
Med Hyg 2000; 94 : 507.
74. Gajanana A, Rajendran R, Philip Samuel P, Thenmozhi V,
Tsai TF, Kuroda JK, et al. Japanese encephalitis in South
Arcot District, Tamil Nadu, India: a three-year longitudinal
study of vector abundance and infection frequency. J Med
Entomol 1997; 34 : 651-9.
75. Mahadev PVM, Kollali VV, Rawal ML, Pujara PK, Shaikh
BH, Ilkal MA, et al. Dengue in Gujrat State, India during
1988 & 1989. Indian J Med Res 1993; 97 : 135-44.
76. Mahadev PVM, Prasad SR, Ilkal MA, Mavale MS, Bedekar
SS, Banerjee K. Activity of Dengue -2 virus and prevalence
of Aedes aegypti in the Chirimiri colliery area, Madhya
Pradesh, India. Southeast Asian J Trop Med Public Health
1997; 28 : 126-37.
77. Philip Samuel P, Hiriyan J, Thenmozhi V, Balasubramanian A.
A note on first isolation of Japanese encephalitis virus from
Culex infula Theobald (Diptera:Culicidae) . J Commun Dis
1998; 30 : 199-200.
78. Philip Samuel P, Hiriyan J, Thenmozhi V, Balasubramanian
A. A system for studying vector competence of mosquitoes
for Japanese encephalitis virus. Indian J Malariol 1998;
35 : 146-50.
79. Philip Samuel P. Japanese encephalitis in India with special
reference to the surveillance tools and control measures.
In: William SJ, Vincent S, editors. Proceedings of the
Symposium on recent trends in combating mosquitoes.
School of Entomology and Center for Natural Resources
Management Post Graduate and Research Department of
Zoology. October 3-4, 2000; p. 420-52.
80. Philip Samuel P, Hiriyan J, Gajanana A. Japanese
encephalitis virus infection in mosquitoes and its
epidemiological implications. ICMR Bull 2000; 30 : 37-43.
81. Thenmozhi V, Rajendran R, Philip Samual P, Hiriyan J,
Ayanar K, Balasubramanian A, et al. Natural vertical
transmission of JE virus in south Indian mosquitoes.
Trop Biomed 2001; 18 : 19-27.
82. Arunachalam N, Philip Samuel P, Hiriyan J, Thenmozhi V,
Balasubramanian A, Gajanana A, et al. Vertical transmission
of Japanese encephalitis virus in Mansonia species, in an
epidemic-prone area of southern India. Ann Trop Med
Parasitol 2002; 96 : 419-20.
 SAMUEL & TYAGI: DETECTION & ISOLATION OF DENGUE VIRUS IN MOSQUITOES
83. Arunachalam N, Philipsamuel P, Hiriyan J, Thenmozhi V,
Gajanana A. Japanese encephalitis in Kerala, South India:
Can Mansonia (Diptera: Culicidae) play a supplemental role
in transmission? J Med Entomol 2004; 41 : 456-61.
84. Arunachalam N, Murthy S, Kabilan L, Balasubramanian A,
Thenmozhi V, Narahari D, et al. Studies on dengue in rural
area of Kurnool district in Andhra Pradesh. J Am Mosq
Cont Assoc 2004; 20 : 87-90.
85. Das BP, Kabilan L, Sharma SN, Lal S, Regu K, Saxena VK.
Detection of dengue virus in wild caught Aedes albopictus
(Skuse) around Kozhikode Airport, Malappuram district,
Kerala, India. Dengue Bull 2004; 28 : 210-2.
86. Thenmozhi V, Kabilan L, Philip Samuel P, Dash AP.
Detection of dengue virus antigens in desiccated mosquitoes:
an improved surveillance system. Trop Med Int Health
2005; 10 : 187-9.
87. Dhanda V, Thenmozhi V, Kumar NP, Hiriyan J,
Arunachalam N, Balasubramanian A, et al. Virus isolation
from wild-caught mosquitoes during a Japanese encephalitis
outbreak in Kerala in 1996. Indian J Med Res 1997;
106 : 4-6.
88. Burke DS, Chatiyanonda K, Anandrik S. Improved
surveillance of Japanese encephalitis by detection of virus
specific IgM in desiccated blood specimens. Bull World
Health Organ 1985; 63 : 1037-42.
89. Polsuwan C, Lumlertdaccha B, Tepsumethanon W, Wilde
H. Using the UV paralens adapter on a standard laboratory
microscope for fluorescent rabies antibody detection.
Trans R Soc Trop Med Hyg 1992; 86 : 107.
90. Makler MT. Fluorescent microscope objective. Trans R Soc
Trop Med Hyg 1992; 86 : 108.
91. Scott TW, Olson JG. Detection of eastern equine
encephalomyelitis viral antigen in avian blood by enzyme
immunoassay: a laboratory study. Am J Trop Med Hyg
1986; 35 : 611-8.
92. Watt DM, Harrison BA, Nisalak A, Scott R, Mc N, Burke
DS. Evaluation of Toxorhynchites splendens (Diptera:
Culicidae) as a bioassay host for dengue virus. J Med
Entomol 1982; 19 : 54-9.
93. Kuno G, Gomez I, Gubler DJ. An ELISA procedure for the
diagnosis of dengue infections. J Virol Methods 1991; 33 :
101-3.
94. Miagostovich MP, Nogueira RMR, Santos FB, Schatzmayr
HG, Araujo ESM, Vorndam V. Evaluation of an IgG
enzyme-linked immunosorbent assay for dengue diagnosis.
J Clin Virol 1999; 14 : 183-9.
627
95. Nawa M, Takasaki T, Yamada KI, Akatsuka T, Kurane I.
Development of dengue IgM capture enzyme-linked
immunosorbent assay with higher sensitivity using
monoclonal detection antibody. J Virol Methods 2001;
92 : 65-72.
96. Broom AK, Hall RA, Johansen CA, Oliveria N, Howard
MA, Lindsay MD, et al. Identification of Australian
arboviruses in inoculated cell cultures using monoclonal
antibodies I ELISA. Pathology 1998; 30 : 286-8.
97. Sithiprasna R, Strickman D, Innis BL, Linthicum KJ. ELISA
for detecting dengue and Japanese encephalitis viral antigen
in mosquitoes. Ann Trop Med Parasitol 1994; 88 : 397-404.
98. Tsai TF, Bolin RA, Montoya M, Bailey RE, Francy DB,
Jozen M, et al. Detection of St.Louis encephalitis virus
antigen in mosquitoes by capture enzyme immunoassay.
J Clin Microbiol 1987; 25 : 370-6.
99. Scott TW, Olson JG, Le2wis TE, Carpenter JW, Lorenz LH,
Lembeck LA, et al. A prospective field evaluation of an
enzyme immunoassay. Detection of eastern equine
encephalomyelitis virus antigen in pools of Culiseta
melanura. J Am Mosq Cont Assoc 1987; 3 : 412-7.
100.Murthy US, Singh TG, Arunachalam A, Philip Samuel P.
Epidemiology of Japanese encephalitis in Andhra Pradesh,
India- a brief overview. Trop Biomed 2000; 17 : 97-102.
101.Hunderkar SL, Thakare JP, Gokhle MD, Barde PV,
Argade SV, Mourya DT. Development of monoclonal
antibody based antigen capture ELISA to detect
chikungunya virus antigen in mosquitoes. Indian J
Med Res 2002; 115 : 144-8.
102.Knox TB, Kay BH, Hall RA, Ryan PA. Enhanced vector
competence of Aedes aegypti (Diptera: Culicidae) from the
Torres strait compared with mainland Australia for Dengue
2 and 4 viruses. J Med Entomol 2003; 40 : 950-6.
103. Henchal EA, Gentry MK, McCown JM, Brand WE. Dengue
virus specific and flavivirus group determinants identified
with monoclonal antibodies by indirect
immunofluorescence. Am J Trop Med Hyg 1982;
31 : 830-6.
104. Rao BL, Basu A, Wairagkar NS, Gore MM, Arankalle VA,
Thakare JP, et al. A large outbreak of acute encephalitis
with high fatality rate in children in Andhara Pradesh,
India, in 2003, associated with Chandipura virus.
www.thelancet.com September 4, 2004; 364 : 869-74.
105. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam
AV. Rapid detection and typing of dengue viruses from
clinical samples using reverse transcriptase-polymerase
chain reaction. J Clin Microbiol 1992; 30 : 545-51.
628 INDIAN J MED RES, MAY 2006
106. Morita K, Meomoto T, Honda S, Onish K, Murata M,
Tanaka M, et al. Rapid detection of virus genome from
imported dengue fever and dengue hemorrhagic fever
patients by direct polymerase chain reaction. J Med Virol
1994; 44 : 54-8.
107. Harris E, Roberts G, Smith L, Selle S, Kramer LD,
Valle S, et al. Typing of dengue virus in clinical specimens
and mosquitoes by single-tube mutiplex reverse
transcriptase PCR. J Clin Microbiol 1998; 36 : 2634-9.
108. Rosario D, Alvarez M, Diaz J, Contreras R, Rodrigues R,
Vazques S, et al. Rapid detection and typing of dengue
viruses from clinical samples using reverse transcriptase
polymerase chain reaction. Pan Am J Public Health
1998; 4 : 1-5.
109. Sudiro TM, Ishiko H, Green S, Vaughn DW, Nisalak A,
Kalayanarooj S, et al. Rapid diagnosis of dengue viremia
by reverse transcriptase- polymerase chain reaction using
3-noncoding region universal primers. Am J Trop Med Hyg
1997; 56 : 424-9.
110. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R,
Horn GT, et al. Primer directed enzymatic amplification
of DNA with a thermostable DNA polymerase. Science
1988; 239 : 487-91.
111. Henchal EA, Polo SL, Yaemsiri VVC, Hoke CH.
Sensitivity and specificity of a universal primer set for the
rapid diagnosis of dengue virus infections by polymerase
chain reaction and nucleic acid hybridization. Am J Trop
Med Hyg 1991; 45 : 418-28.
112. Morita K, Tanka M, Igarashi A. Rapid identification of
dengue virus serotype by polymerase chain reaction. J Clin
Microbiol 1991; 29 : 2107-10.
113. Rohani A, Ismail A, Saat Z, Lee HL. Detection of dengue
virus from field Aedes aegypti and Aedes albopictus adults
and larvae. Southeast Asian J Trop Med Public Health
1997; 28 : 138-42.
114. Pinheir VC, Tadei WP, Barros PM, Vascocelos PF, Cruz
AC. Detection of dengue virus serotype 3 by reverse
transcription-polymerase chain reaction in Aedes aegyptis
Diptera-Culicidae captured in Manaus, Amazonas.
Mem Inst Oswaldo Cruz 2005; 100 : 833-9.
115. Chow VT, Chan YC, Yong R, Lee KM, Lim LK, Chung
YK, et al. Monitoring of dengue viruses in field-caught
Reprint requests: Dr P. Philip Samuel, Centre for Research in Medical Entomology (ICMR)
4, Sarojini Street, Chinna Chokkikulam, Madurai 625002, India
e-mail: crmeicmr@icmr.org.in
Aedes aegypti and Aedes albopictus mosquitoes by type-
specific polymerase chain reaction and cycle sequencing.
Am J Trop Med Hyg 1998; 58 : 578-86.
116. Armstrong PM, Rico-Hesse R. Efficacy of dengue serotype
2 virus strains to infect and disseminate in Aedes aegypti.
Am J Trop Med Hyg 2003; 68 : 539-44.
117. Kow CY, Koon LL, Yin PF. Detection of dengue viruses
in field caught male Aedes aegypti and Aedes albopictus
(Diptera: Culicidae) in Singapore by type-specific PCR.
J Med Entomol 2001; 38 : 475-9.
118. Deubel V. The contribution of molecular techniques to the
diagnosis of dengue infection. In: Gubler DJ, Kuno G,
editors. Dengue and dengue hemorrhagic fever. London,
United Kingdom: CAB International; 1997 p. 335-6.
119. Henchal EA, Narupiti S, Feighny R, Padmanabhan R,
Vakharia V. Detection of dengue virus RNA using nucleic
acid hybridization. J Virol Methods 1987; 15 : 187-200.
120. Deubel V, Laille M, Hugnot JP, Chungue E, Guesdon JL,
Drouet MT, et al. Identification of dengue sequences by
genomic amplification: rapid diagnosis of dengue virus
serotypes in peripheral blood. J Virol Methods 1990; 30 :
41-54.
121. Gubler DJ, Suharyono W, Tan R, Abidin M, Sie A.
Viraemia in patients with naturally acquired dengue
infections. Bull World Health Organ 1981; 59 : 623-30.
122. Gubler DJ. Dengue. In: Monath TP, editor. Epidemiology
of arthropod-borne viral diseases. Inc, Boca Raton, Fla:
CRC Press; 1988 p. 223-60.
123. Waterman SH, Monath TP. Fluorescent antibody techniques
applied to the identification of dengue virus in infected
tissues. Acta Virol 1982; 26 : 367-81.
124. Gentry MK, Henchal EA, McCown JM, Brandt WE,
Dalrymple JM. Identification of distinct determinants on
dengue 2 virus using monoclonal antibodies. Am J Trop
Med Hyg 1982; 31 : 548-55.
125. Peiris JSM, Amerasinghe FP, Amerasinghe PH,
Ratnayake CB, Karunaratne SHP, Tsai TF. Japanese
encephalitis in Sri Lanka- a study of an epidemic: vector
incrimination, porcine infection an human disease.
Am J Trop Med Hyg 1992; 86 : 307-13.