Symposium on Dengue - 2015
Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/10378
Papers presented at the Symposium on Current Research Activities on Dengue, organized by the Molecular Medicine Unit, on 13th of November 2015
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Item Engineering miR-shRNA based molecule to interfere replication of dengue virus in transgenic Aedes aegypti mosquitoes: Bioinformatics approach(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Kajan, M.; Abeyewickreme, W.BACKGROUND: The genus Flavivirus of the family Flaviviridae includes several vector-borne viruses to which the four serotypes of dengue viruses (DENV-1,-2,-3 and 4) belong to and DENV viruses have a messenger like positive polarity, single-stranded RNA genome approximately 11kb in length which encodes three structural proteins (C-prM-E) and seven Non-Structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). RNA interference (RNAi) and its properties as a tool has heralded a new era in functional genomics and short double stranded RNAs mediated by RNAi has become a powerful tool for post transcriptional gene silencing. Therefore, this study took the advantage the latter biological phenomenon and designed a multiple miR-shRNA (multi-mir-shRNA) molecule using bioinformatic approach to be effective to block the replication of all dengue serotypes of Sri Lanka. METHODS: Genome sequences of DENV strains belonged to serotypes 1 and 3 isolated from Sri Lanka deposited in GenBank were analyzed for potential sequences for the best siRNA target sites and identified two such sites from DENV 1 and DENV 3 from non structural protein coding sequence of NS5 and structural protein coding sequence of prM consensus regions, respectively. Two more siRNA targets reported from previous study chosen from upstream and downstream of non coding region effective for silencing all DENV serotypes were also used in designing mir-shRNA sequences. The stem region of miR1175 pre-miRNA sequence (miRBase ref: MI0013470) of Aedes aegypi was then replaced with each selected siRNA targets to generate the DENV effective miR-shRNAs and generated miR-shRNAs connected together by placing restriction endonuclease sites between each other to obtain multi-mir-shRNA containing four loop and stem structures. The transgenic gene cassette containing Ae. aegypti carboxypeptidase A promoter, multi-miR-shRNAs and poly adenylation signal of Simian Virus 40 Major Capsid VP1 was then constructed. Expression of this effecter molecule can be achieved by the induction of Ae. aegypti carboxypeptidase A (AeCPA) promoter following blood meal which then ensures activation of RNAi at the time of virus enter into the midgut of mosquito. RESULTS: Folding patterns of the transcript of the designed multi-mir-shRNA cluster were analyzed using online bioinfomatic tool, mfold and the secondary structure of this transcript shown to have optimum endogenous miRNA cleavge/processing with the lowest -ΔG indicating the ability of this design to exert RNAi in mosquito Ae. aegypti. CONCLUSION: Designing multi-miR-shRNA in bioinformatic means an effective way to construct the effector molecule that could exert the maximum RNAi against DENV. However, the effect of design will have to be demonstrated first by transforming to Ae. aegypti mosquitoes and then by evaluating the inhibition of DENV replication in mosquito.Item Maternal transformation of Wolbachia isolated from infected mosquito hosts to Aedes aegypti using micro-injection based procedure: an approach towards integrated dengue vector control(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Ruanareerate, T.; Kittayapong, P.; Abeyewickreme, W.BACKGROUND: The maternal transformation of Wolbachia (Wb) to Aedes mosquitoes is an approach that has been widely used in Aedes mosquito population control strategies in the integrated dengue vector control efforts. This study details usage of such approach in the maternal transformation of Aedes aegypti using the Wb isolated from infected mosquito hosts using micro-injection based procedure for the first time in Sri Lanka. METHODS: Twenty five ovaries of each adult female Aedes albopictus and Culex quinquifaciatus mosquitoes (14-18 days old) were dissected and Wb was isolated and suspended in 20 μl of 1X Phosphate Buffered Saline (PBS). The isolated samples were kept on ice to maximize the bacteria viability. The exudate was then used in the trans-infection of Ae. aegypti mosquitoes. Infection of Wb into the mosquito Ae. aegypti was carried out by maternal/adult microinjection. To create stable trans-infected lines, the regions near the 3rd leg emerged from the thorax of Ae. aegypti were microinjected to incorporate Wb into the developing germ line and then to facilitate the transmission of Wb to offspring. The volume used for injection based on the swelling of the abdomen, which was monitored throughout the experiment while maintaining the injection pressure, time and number of injecting times. Virgin female Aedes mosquitoes survived after microinjection was fed with 1 ml of 10% sugar solution containing 10,000 units Penicillin/Streptomycin per 100 ml. Survival rate was monitored at 1, 3 and 24 hour/s following microinjection procedure. Survived female mosquitoes resulted from injected line (generation 0 [G0]) was placed in vials with three uninfected male mosquitoes to establish iso-female lines. After egg laying 3 times, G0 females were sacrificed and total DNA was extracted, and PCR was carried out (wsp primers 81F and 691R) using DNA extracted from Ae. aegypti to detect the presence of Wb DNA and its density in the latter mosquito using dilution series of extracted DNA. To select for a stable infection, only offspring from females that came positive for Wb by PCR screening was used as parental stocks. For the samples positive by wsp PCR, strain identification was carried out by performing PCR with 4 different primer sets; wPip, wAlbA, wAlbB and wsp specific for Wb. Females that tested negative for Wb were discarded along with their progeny. The resulting triple infected line will be monitored periodically by PCR to confirm infection status. RESULTS: Wolbachia-triple infected line of Ae. aegypti mosquito was created by using isolated wAlbA+wAlbB+wPip Wolbachia strains. Infection status of newly developed line was monitored by PCR up to G2 and will be continued to G8. Experimental work will be started from G5 to evaluate the use of newly develop Ae. aegypti mosquito line for Wb-based approach under laboratory condition. CONCLUSION: Direct maternal micro-injection technique was used to create Wb infected mosquito line of Ae aegypti, which could be applied in Wb-based dengue vector control activities in Sri Lanka once the line is established.Item Transgenic mosquitoes to control vector-borne diseases(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Sampath, L.D.S.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.The mosquito-borne diseases have achieved the world wide attention due to the high number of cases and deaths reported to WHO annually. Such diseases include malaria and dengue fever. Control of these diseases has been difficult due to drug resistance in parasites (e.g. malaria), insecticide resistance in mosquitoes, lack of effective vaccines and failure of the vector control strategies to minimize human-mosquito contact. As a novel approach for the rising situation, mosquito population replacement strategies and, genetic based mosquito population suppression and elimination strategies such as Release of Insects carrying a Dominant Lethal (RIDL) gene have been introduced. Both strategies used for the development of transgenic mosquitoes (TMs). The development of TMs that have impaired competency to transmit pathogens or population suppression and elimination ability as result of alteration of genetics of mosquitoes is a promising approach that could potentially reduce threats exerted by mosquitoes to humans. The work carried out so far on TMs has shown successful results in the laboratory and open field release experiments indicating TMs to be the leading strategy in the future to control mosquito borne diseases.