Thesis Progress Report (Semester 1/2017)
Title: Drug Repositioning Against Dengue Virus Infection
Dengue virus (DENV) infection is a mosquito-borne infection common in tropical and sub-tropical areas of the world. It is transmitted by mosquitoes of Aedes family and has become a major global health concern in recent years. The clinical manifestation of DENV infection ranges from mild febrile illness to severe dengue syndrome that includes dengue hemorrhagic fever and dengue shock syndrome. Apparently, epidermal dendritic cells and keratinocytes are the first cell to be infected. Once the virus enters the body, its starts to multiply and spread into various organs through the blood stream and lymphatic system. There are clinical evidences of liver involvement during severe diseases manifestations. However, the disease pathogenesis is still poorly understood. Though this disease has been studies since decades, no any anti-dengue drug for human use has been identified yet. Therefore, the development drug against dengue infection has become utmost important. Drug repositioning can be an important tool for discovering potent anti-dengue agents. Here in this study we used drugs from US FDA DrugCollection Library asapotent anti-dengue candidate drugs.
Research Question: Does drug candidate from US FDA approved drug library exhibit anti-dengue activity?
Objective: To repurpose US FDA approved drugs against dengue virus infection in vitro
Materials and Methods: Human hepatoma cell line (HepG2), human adenocarcinomic alveolar basal epithelial cell line (A549) and endothelial hybrid cell line EA.hy 926 will be used in the experiments. Dengue virus (DENV) will be used at MOI of 1. Eight candidate Drugs from US FDA Drug Library will be used for the experiments.
The workplan is as follows-
The research will be divided into two main parts-
a) Determination on CC50 (dose at which 50% cells are viable) and,
b) Determine the effect of candidate drugs on DENV replication
I have finished the first part of my thesis research and now I am doing the second part. After determining the CC50 dose of the drugs, I proceeded to examine the effects of the candidate drugs in virus replication. HepG2 cells were seeded in 96 wells plate, 2×104 cells per well and incubated at 37°C for 24 h in presence of 5% CO2. Next, the cells were infected with DENV-2 at MOI 1 and after 2 h unbound virus were removed and drugs were added an incubated at 37°C for 24 h in presence of 5% CO2. After 24 h, cell viability, intracellular DENV E antigen and virus production from infected cells were determined. Among, the eight drugs, four drugs could decrease DENV production without affecting the cell viability.
For in detail study, the drug that could decrease virus production to the maximum was chosen. The selected drug impaired virus production of all four serotypes of DENV. The effect of the selected drug on different stages of virus replication namely, viral adsorption, internalization, RNA replication and protein synthesis was done by flow cytometry, real time PCR and western blot respectively. Our candidate drug decreased viral RNA replication and protein synthesis.In addition to HepG2 cells, we also observed inhibition of DENV production in A549 and EAhy.926 cell line upon treatment with the drug. At present, we are working on experiments to clarify the molecular mechanism of the drug.