Diagnostic Methods For Detection & Isolation of Dengue Viruses From Vector Mosquitoes

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 mélange 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

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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 world’s 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 nonendemic 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 crossreactions 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 bloodsucking 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 19601961, 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 denguelike 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.

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(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), AP61 (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

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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 transcriptasepolymerase 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 postinoculation 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 fieldcollected 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 32ºC 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 intracerebral 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 fieldcaught mosquitoes74. Both the inoculation techniques have similar sensitivity to intrathoracic inoculation of adult mosquitoes, but are more difficult and labourintensive. 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 nonbiting Toxorhynchites species for inoculation 64 .

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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, labourintensive 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

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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 nonstructural 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.

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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

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genome sequences used. A PCR-amplified serotypespecific 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, RTPCR 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 (89100%)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 antidengue 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, intracerebral 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 timeconsuming 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

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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/inffs/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.

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Reprint requests: Dr P. Philip Samuel, Centre for Research in Medical Entomology (ICMR) 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002, India e-mail: [email protected]