The purpose of this project was to develop several of the components required for an in vitro cell culture-based assay for detecting replication-competent dengue virus. This plan involved the stable transfection of a 293T cell-line with a recombinant plasmid encoding protease-cleavable dengue non-structural proteins NS4A/B-NS5 fused to a tetracycline transactivator (tTA). This same cell-line was also stably transfected with a reporter plasmid encoding green fluorescent protein (GFP) under the control of a tetracycline responsive element (TRE). Infection of this engineered cell line with replication-competent dengue virus results in the cleavage of the NS4A/B-NS5-tTA fusion protein by active NS3 protease, there by releasing free tTA, which activates expression of the GFP reporter, allowing for easy detection. To date, the NS4A/B-NS5-tTA plasmid has been partially constructed, while the TRE/GFP reporter plasmid has been successfully transfected into 293T cells.

• This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

Creator

• Maisey, Heather Clair
• Henry, Leah Marie

Publisher

• Worcester Polytechnic Institute

Identifier

• 02D214M

Advisor

• Adams, David S.

Year

• 2002

Sponsor

• University of Massachusetts Medical School

Date created

• 2002-01-01

Resource type

• Major Qualifying Project

Major

• Biology & Biotechnology

Rights statement

• In Copyright