Generation of a Zika Virus Reverse Genetics System for Drosophila melanogaster

Zika Virus (ZIKV) is a positive-strand RNA [(+)RNA] arbovirus of the flavivirus genus that has recently caused a global health crisis associated with an increase of primary microcephaly, a congenital anomaly correlated with brain size and development. In this work, we are attempting to create a plasmid-based reverse genetics system for ZIKV that is functional in insect cell lines, specifically Drosophila melanogaster. Several plasmid-based ZIKV reverse genetics systems have been established for mammalian systems, though they do not work in insect cells due to incompatible promoters. Establishing Drosophila as a model insect system for ZIKV would allow us to take advantage of Drosophila’s fully sequenced and well-annotated genome and the many tools available for genetic manipulation. Initial attempts to construct a plasmid-based reverse genetics system indicated toxicity of ZIKV sequences in E. coli, consistent with the findings of others. Using the 1947 ZIKV cDNA plasmid developed by Schwarz et al. we replaced the cytomegalovirus (CMV) promoter with the baculovirus immediate early 1 (IE1) promoter. The IE1 promoter works well in Drosophila S2 cells and has been used for other (+)RNA virus insect cell replicon systems. Surprisingly, the presence of the IE1 promoter resulted in toxicity in E. coli despite the presence of introns and using a low-copy number plasmid backbone. Next, we replaced the CMV promoter with the Drosophila actin 5c promoter. Initial results indicate that this plasmid is stable in E. coli. We are currently testing this replicon system for ZIKV replication in Drosophila S2 cells