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

Isolation of 35 Mycobacteriophages and Genomic Analysis of the Novel Mycobacteriophage, Glass

Thirty-five new mycobacteriophages were isolated from soil samples collected on or nearby Hope College in Holland, Michigan. All were capable of infecting Mycobacterium smegmatis and produced a variety of plaque morphologies based on size, shape, and clarity, consistent with the isolation of an assortment of different phages. Both lytic and temperate phages appear represented in this collection. Purified phage stocks were used to prepare genomic DNA samples for restriction digest analysis. A comparison of those 35 digest results revealed few similarities among the group, further supporting our interpretation that most of the new phage isolates were distinct. One mycobacteriophage, Glass, was chosen for complete genome sequencing using the Illumina MiSeq platform and comparative genomic analysis. The predominant plaque produced by Glass at 32°C was turbid and 0.5-1.0mm in diameter, while plaque produced at 42°C was clear and 1.0-1.5mm in diameter. Genome sequence data for Glass revealed a relationship to a group of 12 mycobacteriophages in Cluster B2. The genome of Glass is 67.5 Kb, 69.0% GC, and contains 91 genes in agreement with the genome characteristics of closely related phage. A detailed analysis of the complete genome sequence and comparison with sequenced members of this small and unique group of mycobacteriophages is the subject of the second semester of this yearlong course and is presented