Detection of transovarial dengue virus from field-caught Aedes aegypti and Ae. albopictus larvae using C6/36 cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques A. Rohania , I. Zamreea, H.L. Leea, I. Mustafakamalb, M.J. Norjaizab and D. Kamilanc a
Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research, Kuala Lumpur b
Vector Borne Disease Centre Programme, Kuala Terengganu, Terengganu c
Vector Borne Disease Centre Programme, Kuantan, Pahang
Abstract Larvae of Aedes aegypti and Aedes albopictus were collected from a wide variety of artificial containers. Most samples were collected from used tyres and water-holding containers located in residential urban or rural areas. The identified mosquito larvae were pooled according to the species, date and locality and stored at –70 °C. A total of 378 pools of Ae. aegypti and 553 pools of Ae. albopictus were collected. Virus isolation was carried out using cell culture (C6/36 clone) of Ae. albopictus and virus detection by reverse-transcriptase polymerase chain reaction (RT-PCR). Transovarial transmission of dengue virus was demonstrated in both Ae. aegypti and Ae. albopictus in nature. Infected larvae were recovered from 16 localities (10 in Terengganu; 5 in Kuala Lumpur and 1 in Pahang). The study showed that both the cell culture and RT-PCR techniques can be used to detect dengue virus from mosquito larvae. Keywords: Dengue; Aedes aegypti; Aedes albopictus; Transovarial transmission; Malaysia.
Introduction Dengue is an acute arboviral human disease caused by four serotypes of dengue viruses, which are closely related but antigenically distinct. Dengue viruses are transmitted to humans through the bite of infected mosquitoes. For many years, members of the subgenus Stegomyia, especially Aedes aegypti (Linn.) and Aedes albopictus (Skuse.) have been recognized as the primary vectors of
dengue.[1,2,3] Ae. aegypti breeds in clean water collected in and around human settlements. Ae. albopictus can be found not only around and near human habitation, like Ae. aegypti, but also in forests and plantations. The emergence of dengue has been linked to economic and ecological changes that caused dramatic expansion of the urbanized vector of dengue viruses. Industrialization and rapid human population growth led to
E-mail: rohania@imr.gov.my 47
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Detection of transovarial dengue virus from field-caught larvae of Ae. aegypti and Ae. albopictus
uncontrolled urbanization in Malaysia. In the absence of proper water supply (for example in the squatter (slum) areas), residents have to store water for domestic use, creating the ideal ecological niche for Ae. aegypti and Ae. albopictus. These mosquitoes prefer to feed on humans and to lay eggs in artificial containers. The increase in air travel also contributed to the expansion of dengue, providing the means for viremic people to move very rapidly from one place to another.[4] The prevention and control of dengue outbreaks depends upon the surveillance of cases and mosquito vectors. Vector surveillance allows timely implementation of emergency mosquito control measures to limit an impending outbreak from spreading. In Malaysia, a comprehensive mosquito control programme administered by the VectorBorne Disease Control Programme, Ministry of Health, incorporates source reduction, public health education, community participation and law enforcement against mosquito breeding. The Aedes control strategy has focused mainly on surveillance for the elimination of Aedes larval breeding habitats and emergency control of adults using insecticidal fogging during outbreaks. Although this strategy has successfully reduced the Aedes mosquito population to a relatively low level, as indicated by the overall House index (HI), it has not prevented the emergence of progressively larger outbreaks in recent years.[5] Despite extensive research on vaccine development, there are at present no known methods of controlling dengue except by limiting the mosquito vectors. Virological surveillance, which involves the monitoring of dengue virus infection in humans, has been used as an early warning system to predict outbreaks.[6,7] Such surveillance is based on isolation of dengue virus from human serum by cell culture or mosquito inoculation and 48
type-specific identification by immunoflourescence or multiplex RT-PCR. This approach is less effective since the virus is already circulating in the human population. A more effective approach is to detect the virus in mosquitoes before it is introduced into the human population. This way, preventive vector control measures can be undertaken immediately to offset an outbreak. Surveillance of mosquitoes infected with dengue viruses provides an early warning sign for risk of transmission in an area and the specific predominant circulating serotype in the vector population.[8] Control programmes can be prioritized and focused more effectively in specific localities. The objective of this study was to detect dengue virus from field mosquitoes using C6/36 cell culture and reverse transcriptasepolymerase chain reaction (RT-PCR) techniques.
Materials and methods Larval collections Third and fourth instar Aedes aegypti and Aedes albopictus larvae were collected from the field in major towns in Malaysia, i.e. Terengganu, Pahang and Kuala Lumpur. The larvae were collected during dengue outbreaks for each state in 2005. Mosquito larvae collected in the survey were identified using standard taxonomic keys. Identified mosquito larvae were segregated according to species, site and date. Mosquito larvae were stored in pools of 10 larvae per pool in cryogenic vials at –70 °C for future virus isolation studies. The mosquito pools were selected randomly and dengue virus detection was carried out by RT-PCR and C6/ 36 cell culture technique. A total of 931 pools of Aedes mosquitoes were separated equally and assayed by C6/36 cell culture and RT-PCR.
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Detection of transovarial dengue virus from field-caught larvae of Ae. aegypti and Ae. albopictus
Detection of dengue virus using reverse transcriptase-polymerase chain reaction (RT-PCR) Extraction of RNA The larval pools were homogenized in a sterile homogenizer and RNA was extracted using High Pure Nucleic Acid Kit (Roche, Germany). The procedures of RNA extraction were as follows: 200 μ l sample were added subsequently into 1.5 ml eppendorf tubes containing 50 μl proteinase K. This was briefly vortexed and incubated at 27 °C for 10 minutes. Subsequently, 100 μl of isopropanol was added and vortexed briefly. The solution was then pipetted into the high pure filter tubes, placed into collection tubes and were then centrifuged at 14 000 rpm for 1 minute. The flow-through was discarded and filter tubes were placed into new collection tubes. Five hundred microlitres of inhibitor-remover wash buffer were added into the upper reservoir and centrifuged at 14 000 rpm for 1 minute. The flow-through was discarded and again the filter tubes were placed into new collection tubes. A volume of 450 μl wash buffer was added to the upper reservoir and centrifuged at 14 000 rpm for 1 minute. The flow-through was discarded and again the filter tubes were placed into new collection tubes. The wash step was repeated and continued with a short spin for 15 seconds. The collection tube was discarded and the filter tubes were inserted into clean 1.5 ml eppendorf tubes. RNA was eluted by adding 35 μl elution buffer into the middle spot of the filter tubes and centrifuged at 14 000 rpm for 2 minutes. The extracted RNA was kept at –70 °C until used. Detection of viral RNA using consensus primers RNA isolated from each pool was subjected to the RT-PCR assay using Lanciotti’s dengue Dengue Bulletin – Volume 31, 2007
consensus primers (TCAATATGCTGAAACGCG CAGAAACCG and TTGCACCAACAGTCAATGT CTTCAGGTTC).[9] The primers correspond to genome positions 131 to 161 and 616 to 644 of dengue virus respectively. Master mix was prepared using Titan One Tube RT-PCR Kit. Each reaction required 9.75 μl of double distilled water, 2 μl of dNTP mix, 1.25 μl of DTT, 0.5 μl RNAse inhibitor, 5.0 μl of RT-PCR buffer, 0.5 μl of enzyme mix, 0.5 μl of dengue universal sense primer and 0.5 μl of dengue universal anti-sense primer to reach 20 μl total volume of master mix. RNA products were prepared by heating the tubes at 65 °C for 5 minutes by block heater. Five microlitres of each RNA product were added to the master mix and then centrifuged at 8000 rpm. The RT step was carried out at 51 °C for one 30 minutes to produce cDNA which was then amplified by the following PCR steps: 92 °C for 3 minutes as initial denaturation, 92 °C for 30 seconds as denaturation steps, 51 °C for 45 seconds as annealing step and 72 °C for 1 minutes as extension step. The cycle was repeated 41 times before final extension at 72 °C for 5 minutes. For every RT-PCR run, a positive control (a confirmed dengue isolate) and negative control (C6/36 cell culture without dengue virus) were included. The PCR products were analysed by performing electrophoresis in a 2.0% Nusieve PCR gel (FC Bio, USA) stained with ethidium bromide and run at about 100 volts. The gel was viewed under ultraviolet illuminator (Ultra Lum Ins., California, USA) and the image of the resulting band was captured with a Polaroid camera.
Dengue virus detection using Ae. albopictus clone C6/36 cells[10] The method employed was modified from Maneekarn.[11] The C6/36 cells were grown in 49
Detection of transovarial dengue virus from field-caught larvae of Ae. aegypti and Ae. albopictus
minimum essential medium (MEM) growth media supplemented with 10% fetal calf serum (FCS) for 2 days until cell monolayers were formed in culture tubes. Pooled mosquitoes were homogenized on ice in 1.5 ml MEM medium with 5% fetal calf serum. 100 ul each of the homogenate were passed through 0.2 um filters and inoculated into respective culture tubes. The culture tubes were then vortexed and left to incubate for 2 hours at ambient temperature for adsorption. Maintenance medium containing 2% FCS was added and the culture tubes were incubated at 28 °C for 7 days. Smear preparation The culture tubes were vortexed and centrifuged after 7 days’ incubation period. Cells from the sediments were transferred onto the Teflon-coated, 12 well slides where each slide would have a maximum of 7 test smears, 4 positive control and a negative control. The smears were left to dry at 28 °C for 4 hours in a class II biohazard cabinet with the air blower on. The smears were then fixed with cold acetone for 20 minutes. The cultures were stored at –20 °C for confirmation by a second or third passage in cell culture if only the initial results were positive. Peroxidase-antiperoxidase (PAP) staining PAP staining procedures were performed as previously described by Igarashi.[12] The cells that were fixed with cold acetone reacted with dengue anti-serum at 1:1000 at room temperature for 40 minutes. The antibody is universally reactive to all 4 dengue serotypes. The cells were rinsed in PBS and reacted with rabbit anti-mouse IgG at 1:1000 for 40 minutes. The cells were rinsed in PBS again and then reacted with goat-anti rabbit IgG at 1:1000 for 40 minutes. The cells were then rinsed in PBS and exposed to peroxidase-rabbit anti50
peroxidase complex at 1:1000 for 40 minutes followed by washing and peroxidase reaction, using 0.2 mg/ml of 3,3 diaminobenzidine (DAB) and 0.2% H2O2 as the substrate. The cells were observed under a normal light compound microscope. The positive samples were reconfirmed by a second passage in cell culture. Minimum Infection Rate (MIR) The MIR was used to compare virus infection rates in Ae. aegypti and Ae. albopictus larvae at the sampling area. The MIR was calculated as (number of positive pools by species ÷ total number of that species tested) X 1000.[13] Virus Infection Rate (VIR) The VIR was calculated as (number of mosquitoes by species infected with dengue virus ÷ total number of that species tested) X 100.[14]
Results and discussion In urban areas of Malaysia, artificial containers are the major larval habitats of Aedes mosquitoes in and near human habitation. The type and location, presence of shade and water conditions usually associated with water receptacles are known to affect breeding of Aedes mosquitoes.[15] The dominant mosquito larvae collected in the present survey were Ae. albopictus, whereas only very low number of containers were positive for Ae. aegypti larvae. Ae. aegypti tend to be more selective in nature compared to Ae. albopictus. Ae. aegypti larvae preferred heavily shaded areas. This preferential choice of shaded breeding area was due to the stability of water temperature in the artificial containers and therefore more conducive to breeding.[15] Dengue Bulletin – Volume 31, 2007
Detection of transovarial dengue virus from field-caught larvae of Ae. aegypti and Ae. albopictus
A total of more than 500 containers from 422 collection sites were examined and yielded a total of 9310 Aedes larvae comprising 5530 of Ae. albopictus and 3780 of Ae. aegypti. Ae. albopictus was more abundant than Aedes aegypti. The preferred sites for Ae. albopictus included artificial receptacles: tyres – 23.9%; plastic containers – 23.5%; tins – 16.5%; and earthen jars – 7.8% (Table 1). All these containers were found not only around houses and shops in urban areas but also in rubber plantations, coconut plantations and in the vicinity of houses in suburban areas. Overall,
179 artificial containers were positive for Ae. aegypti breeding and the preferred sites included artificial receptacles: tyres – 26.3%; plastic containers – 21.2%; tins – 16.2%; and paint cans – 9.5% (Table 1). A total of 378 Ae. aegypti pools were selected randomly for virus detection. Of these, 33 pools were positive for dengue virus by cell culture and 19 pools were positive by RT-PCR. A total of 553 Ae. albopictus pools were assayed and 17 pools were positive by cell culture technique and 6 pools were positive by RT-PCR.
Table 1: Comparison of breeding of mosquito larvae in different types of containers
*10 larvae per pool Dengue Bulletin – Volume 31, 2007 51
Detection of transovarial dengue virus from field-caught larvae of Ae. aegypti and Ae. albopictus
The positive pools were collected from tyres, plastic containers, abandoned washing machine, glass and wheel barrow. We detected dengue virus in larvae by RT-PCR and cell culture technique because it was easier to collect larvae from field compared with adult mosquitoes. Infection rates of Ae. aegypti and Ae. albopictus in the survey are expressed as MIR and compared. Our study showed that MIR for Ae. aegypti was higher compared to Ae. albopictus despite higher number of Ae. albopictus samples. More positive pools came from tyres and plastic containers. Table 2 and 3 summarizes the results of the virus isolation. The figure shows agarose gel electrophoresis of RT-PCR of dengue virus. The correct size of the DNA product (480 bp) was obtained for each of the positive pools after amplification with universal dengue primers. The serotyping showed that the viruses belonged to DENV-1 and DENV-3.
Our findings suggest the occurrence of transovarial transmission of dengue virus by Ae. aegypti and Ae. albopictus in nature. Infected larvae were recovered from 16 localities (10 in Terengganu; 5 in Kuala Lumpur and 1 in Pahang). The virus infection rates (VIR) were higher in Ae. aegypti (13.7%) compared to Ae. albopictus (4.2%). Field isolations of dengue virus from Ae. albopictus larvae were reported in China [16] and Brazil. [17] Chung [14] and Rohani[3] reported that field-caught male mosquitoes of both Ae. aegypti and Ae. albopictus are capable of being infected with dengue virus in the natural environment. If the mechanism of infection in these infected maternal parents and the sex ratio for infection in the offspring was equal, it is likely that a similar proportion of females in the field were also infected via the same mode of transovarial transmission, without the need for the presence
Table 2: Detection of dengue virus by RT-PCR
MIR – Minimum infection Rate per 1000 mosquito larvae * 10 larvae per pool
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Table 3: Detection of dengue virus by C6/36 cell culture
MIR – Minimum infection rate per 1000 mosquito larvae *10 larvae per pool
of infected hosts.[17]. The infected males also could pass the virus to the females during mating, and the latter in turn could pass it on to their offspring transovarially.[18] In Malaysia, Ae. aegypti is the main vector involved in dengue virus transmission. Ae. albopictus plays an important but secondary Dengue Bulletin – Volume 31, 2007
role.[19] However, in an urban and suburban environment, they can share a breeding site.[20] Ae. aegypti is a very efficient vector for dengue viruses, highly receptive to oral infection, well adapted to urban environment (e.g. laying eggs only in artificial containers), and feeding exclusively on humans. On the other hand, Ae. albopictus, which is not highly orally receptive 53
Detection of transovarial dengue virus from field-caught larvae of Ae. aegypti and Ae. albopictus
Figure: Detection of dengue virus in pools of Aedes mosquitoes electrophoresis of RT-PCR products on 3% agarose gels. Lane M – marker; lanes 1 - positive control of laboratory infected Ae. albopictus; lane 2 - negative control of uninfected Ae. albopictus; Lane 3, 4 - RT-PCR products from field Ae. albopictus pools positive for dengue virus; Lane 5 - negative control of uninfected Ae. aegypti; Lane 6,7 - RT-PCR products from field Ae. aegypti pools positive for dengue virus; Lane 8 - positive control of laboratory infected Ae. aegypti; Lane 9 - negative control from cell culture and lane 10 - positive control from dengue virus culture.
to dengue virus, is present mainly in rural areas and does not feed exclusively on humans. Ae. albopictus could bridge a putative sylvatic and an urban cycle of dengue since it colonizes both rural and suburban breeding sites.[21,22] This study showed that both cell culture and RT-PCR techniques can be used to detect dengue virus from mosquito larvae. The results showed that the rate of dengue virus detection was significantly higher by cell culture technique (28.0%) than RT-PCR technique (9.5%). However, no conclusive observation can be made because these field-collected larvae were separated randomly for the study. Our study observed heavy cytopathic effects in dengue-infected C6/36 cell culture as 54
observed by Singh and Paul.[23] Gubler[24] reported that some viruses grow slowly in C6/ 36 cells. The mosquito inoculation technique and cell culture in C6/36 cell line are widely practised for the isolation of dengue virus from human serum. The availability of a number of tissue culture systems for the propagation of dengue virus has greatly facilitated studies on this agent. However, the low efficiency of infected cells and the slow development of virus within them remain major obstacles. Dengue-infected cells need 7–10 days to grow and, therefore, virus detection methods using mosquito cell line are time-consuming and relatively labour-intensive. Another Dengue Bulletin – Volume 31, 2007
Detection of transovarial dengue virus from field-caught larvae of Ae. aegypti and Ae. albopictus
disadvantage to this method of flavivirus detection in field-caught mosquitoes has been the requirement to keep captured mosquitoes alive or frozen fresh to detect viral infection. RT-PCR for the detection of flaviviruses in human clinical samples has been used for nearly a decade now.[25] The accuracy and speed of the RT-PCR assay makes it an appealing test for the diagnosis of dengue and epidemiologic surveillance. The present study indicates that PCR could detect dengue virus in mosquitoes using larvae collected from the field. Similar findings were also reported by Chao et al. (2007). [26] These techniques constitute practical molecular diagnostic and epidemiological tools for the virological surveillance of dengue virus-infected Aedes mosquitoes to serve as an early warning system for dengue outbreaks. In typical infections many types of mosquito tissues are eventually infected and the virus can persist in these tissues throughout the life of the vector. It is generally assumed that once a mosquito becomes infected with dengue it retains that infection for life.[27] Therefore, the collection of wild-caught mosquitoes would appear to be a reasonable approach to virus detection compared with attempts at virus recovery in human
populations, which typically have very short periods (4-5 days) of detectable viremia. Active surveillance for dengue virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. A need exists for better techniques to conduct timely, accurate and meaningful vector surveys and virus detection for estimating transmission risk among susceptible population in endemic areas. PCR could be considered for processing large numbers of samples in operational programmes, mapping areas with different levels of endemicity and stratification of areas, provided the technique is further refined to have higher specificity and sensitivity. A better understanding of dengue virus-vector relationships in the field and their association with human disease can help establish reliable vector population target levels for initiating measures to control and reduce virus transmission.
Acknowledgements The authors wish to thank the Director, Institute for Medical Research, Kuala Lumpur, for permission to publish. Thanks are also due to the staff of the Medical Entomology Unit/IDRC, IMR, and the Vector-Borne Disease Centre Programme, Terengganu and Pahang.
References [1] Boromisa RD, Rai KS, Grimstad PR. Variation in the vector competence of geographic strains of Aedes albopictus for dengue 1 virus. J Am Mosq Control Assoc. 1987 Sep; 3(3): 378-86. [2] Gubler DJ. Dengue and dengue hemorrhagic fever in the Americas. P R Health Sci J. 1987 Aug;6(2):107-11. [3] 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 Mar; 28(1): 138-42. [4] Gubler DJ. The global pandemic of dengue/ dengue haemorrhagic fever: current status and prospects for the future. Ann Acad Med Singapore. 1998 Mar; 27(2): 227-34. [5] VBDCP . Annual report vector borne disease control programme , Ministry of Health, Malaysia. 2004: 177.
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[6] Lam SK. Two decades of dengue in Malaysia. Trop Med. 1993; 35(4): 195-200. [7] Seah CL, Chow VT, Tan HC, Can YC. Rapid, single-step RT-PCR typing of dengue viruses using five NS3 gene primers. J Virol Methods. 1995 Feb; 51(2-3): 193-200. [8] Loroño-Pino MA, Cropp CB, Farfán JA, Vorndam AV, Rodríguez-Angulo EM, RosadoParedes EP , Flores-Flores LF, Beaty BJ, Gubler DJ. Common occurrence of concurrent infections by multiple dengue virus serotypes. Am J Trop Med Hyg. 1999 Nov; 61(5): 725-30. [9] Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992 Mar; 30(3): 545-51. [10] Igarashi A. Isolation of a Singh’s Aedes albopictus cell clone sensitive to dengue and chikungunya viruses. J Gen Virol. 1978 Sep; 40(3): 531-44. [11] Maneekarn N, Morita K, Tanaka M, Igarashi A, Usawattanakul W, Sirisanthana V, Innis BL, Sittisombut N, Nisalak A, Nimmanitya S. Applications of polymerase chain reaction for identification of dengue viruses isolated from patient sera. Microbiol Immunol. 1993; 37(1): 41-7. [12] Igarashi A, Fujita N, Okura Y. Isolation of dengue viruses from patients with dengue hemorrhagic fever (DHF) and those with fever of unknown origin (FUO) in Jakarta, Indonesia in the year of 1981 and 1982. Annals of International Council for Medical Research 1992;2:7-17. [13] Chow VT, Chan YC, Yong R, Lee KM, Lim LK, Chung YK, Lam-Phua SG, Tan BT. Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a typespecific polymerase chain reaction and cycle sequencing. Am J Trop Med Hyg. 1998 May; 58(5): 578-86.
[14] 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 Jul; 38(4): 475-9. [15] Rohani A, Abdullah AG, Ong YF, Saadiyah I, Zamree I, Lee H L. Survey of mosquito larvae distribution in container habitats collected from urban and rural areas in major towns of Malaysia. Trop Biomed. 2001; 18(1): 41-49. [16] Zuo L, Shu LP . Isolation, identification, and phylogenetic analysis of a dengue virus strain from Aedes albopictus collected in Mawei town in Guizhou Province, China. Chin Med J (Engl). 2004 Dec; 117(12): 1847-9. [17] Mitchell CJ, Miller BR. Vertical transmission of dengue viruses by strains of Aedes albopictus recently introduced into Brazil. J Am Mosq Control Assoc. 1990 Jun; 6(2): 251-3. [18] Tu WC, Chen CC, Hou RF. Ultrastructural studies on the reproductive system of male Aedes aegypti (Diptera: Culicidae) infected with dengue 2 virus. J Med Entomol. 1998 Jan; 35(1): 71-6. [19] Lee HL. A nation wide resurvey of the factors affecting the breeding of Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera:Culicidae) in urban town of Peninsular Malaysia 19881999. Trop Biomed. 1991;8:185-189. [20] Lee HL. Environmental friendly approaches to mosquito control. Symposium on “Towards A mosquito-safe environment” 29-30 June 1999. Legend Hotel, Kuala Lumpur, Malaysia. [21] Rudnick A. Ecology of dengue virus. Asian J Infectious Disease. 1978; 2: 156-160. [22] Vazeille M, Rosen L, Mousson L, Failloux AB. Low oral receptivity for dengue type 2 viruses of Aedes albopictus from Southeast Asia compared with that of Aedes aegypti. Am J Trop Med Hyg. 2003 Feb; 68(2): 203-8. [23] Singh KR, Pavri KM. Experimental studies with chikungunya virus in Aedes aegypti and Aedes albopictus. Acta Virol. 1967 Nov; 11(6): 51726.
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[24] Gubler DJ, Kuno G, Sather GE, Velez M, Oliver A. Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. Am J Trop Med Hyg. 1984 Jan; 33(1): 158-65. [25] Eldadah ZA, Asher DM, Godec MS, Pomeroy KL, Goldfarb LG, Feinstone SM, Levitan H, Gibbs CJ Jr, Gajdusek DC. Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. J Med Virol. 1991 Apr; 33(4): 260-7.
[26] Chao DY, Davis BS, Chang GJ. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes. J Clin Microbio. 2007 Feb; 45(2): 584-9. [27] Rodhain F and Rosen L. Mosquito vectors and dengue virus relationships. In: DJ Gubler and G Kuno, editors. Dengue and dengue hemorrhagic fever. Wallingford (UK): CAB; 1997. p. 45-60.
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