Browsing by Author "Ramyasoma, H.P.B.K.D."

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Assessment of developmental and reproductive fitness of dengue-resistant transgenic Aedes aegypti and Improvement of fitness using antibiotics
(Hindawi Pub. Co., 2021) Ramyasoma, H.P.B.K.D.; Gunawardene, Y.I.N.S.; Hapugoda, M.; Dassanayake, R.S.
BACKGROUND: Genetic modification offers opportunities to introduce artificially created molecular defence mechanisms to vector mosquitoes to counter diseases causing pathogens such as the dengue virus, malaria parasite, and Zika virus. RNA interference is such a molecular defence mechanism that could be used for this purpose to block the transmission of pathogens among human and animal populations. In our previous study, we engineered a dengue-resistant transgenic Ae. aegypti using RNAi to turn off the expression of dengue virus serotype genomes to reduce virus transmission, requiring assessment of the fitness of this mosquito with respect to its wild counterpart in the laboratory and semifield conditions. METHOD: Developmental and reproductive fitness parameters of TM and WM have assessed under the Arthropod Containment Level 2 conditions, and the antibiotic treatment assays were conducted using co-trimoxazole, amoxicillin, and doxycycline to assess the developmental and reproductive fitness parameters. RESULTS: A significant reduction of developmental and reproductive fitness parameters was observed in transgenic mosquito compared to wild mosquitoes. However, it was seen in laboratory-scale studies that the fitness of this mosquito has improved significantly in the presence of antibiotics such as co-trimoxazole, amoxicillin, and doxycycline in their feed. CONCLUSION: Our data indicate that the transgenic mosquito produced had a reduction of the fitness parameters and it may lead to a subsequent reduction of transgenic vector density over the generations in field applications. However, antibiotics of co-trimoxazole, amoxicillin, and doxycycline have shown the improvement of fitness parameters indicating the usefulness in field release of transgenic mosquitoes.
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.
Multiple dengue virus serotypes resistant transgenic Aedes aegypti fitness evaluated under laboratory conditions.
(Landes Bioscience, 2020) Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Hapugoda, M.; Capurro, M.L.; Silva Gunawardene, Y.I.N.
ABSTRACT:Dengue viruses (DENV) are the wildest transmitted arbovirus members of the family Flaviviridae, genus Flavivirus. Dengue viruses are composed of four serotypes, DENV1, 2, 3, and 4, and these viruses can cause dengue fever and dengue haemorrhagic fever or dengue shock syndrome, when infecting humans. RNA interference (RNAi) is a self-defence mechanism, which can be used to prevent invasions of RNA viruses to the host. Genetically engineering a host with an RNAi molecule that targets a single virus serotype may develop escape mutants, and can cause unusual dominance over other serotypes. Therefore, the simultaneous targeting of multiple serotypes is necessary to block DENV transmission. Here, we report the development of transgenic Aedes aegypti based on a bioinformatically designed multiple miRshRNA (microRNA-based shRNA) DNA sequence under the control of a blood-meal induced promoter, Carboxypeptidase A, to induce RNAi for DENV in Aedes aegypti, and demonstrate the expression of a synthetic multiple shRNA polycistronic cluster having RNA interference sequences to target DENV genomes. The transgenic mosquitoes have lower rates of infection, dissemination, and transmission for DENV2 and DENV4 compared to wild mosquitoes, with a significant reduction of dengue copy number and antigen levels in the midgut. These levels of DENV were low enough to make transgenic mosquitoes stop the DENV transmission from infected host to a susceptible host and refractory to DENV2 and DENV4 infection. Such multiple resistance in Ae. aegypti has not been documented previously. Laboratory fitness measurement of transgenic Ae. aegypti showed results comparable to other reported transgenic mosquitoes. KEYWORDS: RNA interference; aedes aegypti; dengue disease; multiple miRshRNA; piggyBac