Postgraduate student
Mr. H. P. B. K. D. Ramyasoma, Research Assistant/M.Phil (extending to Ph.D.) Student, Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka.

Supervisors
Prof. Dassanayake R. S.(Supervisor)
Prof.(Mrs) Gunawardene Y. I. N. S.(Co-Supervisor)
Dr. Chandrasekeran N. V.(Co-Supervisor)
Prof. Abeyewicreme W. (Co-Supervisor)

1. To construct the recombinant plasmid containing the miR-shRNA cluster that expresses siRNA to knockdown dengue viral replication in Aedes aegypti mosquitoes.
2. To construct the recombinant plasmid containing the viral gene that expresses an inverted-repeat (IR) RNA to knockdown dengue virus serotype 2 replication in Aedes aegypti mosquitoes.
3. To perform Germ-Line Transformation of Ae. aegypti mosquito with recombinant plasmid construct for the establishment of transgenic mosquito families using optimized protocols.

Project Summary 

Sri Lanka has been affected by epidemics of Dengue virus (DENV) for over 2 decades. In recent years, DENV has become the number one vector borne disease in Sri Lanka. DENV can cause severe flu-like illness called dengue fever (DF), and sometimes lethal complication called dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV categorized under 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 posttranscriptional gene silencing. The current study will focus on the possibility of using an alternative disease transmission control strategy of dengue resistant mosquitos, which is developed through RNA interference method as an additional useful tool in integrated dengue vector control in Sri Lanka. RNA interference has several well established approaches to knockdown gene expression and multiple miR-shRNA is a one of approaches used to block the expression of multiple genes/transcripts. The approach of multiple miR-shRNA has great potential for applications on DENV which evades serotype escape mutants and covers the all serotypes of DENV. Cluster of miR-shRNA effective for DENV was designed using bioinfomatic tools and Cluster of miR-shRNA including transgenic gene cassette to be expressed in Ae. aegypti was then constructed using Ae. aegypti carboxypeptidase A promoter (AeCPA), mir-shRNA cluster and downstream poly adenylation signal. AeCPA promoter ensures activation of RNAi at the time of virus entry into the mosquito midgut. Also a gene cassette of another RNAi approach from a previous study, an effecter sequence having inverted repeated dsRNA of prM protein encoding region of type 2 strain of DENV will be synthesized to express inverted repeated dsRNA under control of AeCPA promoter. Gene cassettes containing cluster of miR-shRNA or dsRNA will be transfer to Ae. aegypti by using piggybac transposable element present in donor vector plasmid and with use of helper plasmid, which express of trasposase enzyme for transposition. After transferring gene cassettes to Ae. aegypti mosquitoes, transgenic mosquitoes will be identified and analysed for the capability of resistance to transmission of DENV to establish DENV resistant transgenic mosquitoes.

DENV resistant transgenic mosquitoes and non-transgenic mosquitos will both compete in the environment for viramic blood and resistivity will vertically be transmitted to offspring. Scientists had developed the transgenic mosquitos with dengue resistance and had successful interruption of dengue transmission in some parts of the world. However, in Sri Lanka this strategy still not in the pipeline for dengue virus transmission control. Therefore, the proposed study will explore the possibility of using transgenic mosquitoes embedded virus transmission control as an additional tool in integrated vector management.