Luciferase Based Plasmid Reporter System for the Detection and Quantification of

Human Respiratory Syncytial Virus

Group 14: Oral Report 4, 3/13/08Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

Human Respiratory Syncytial Virus is the most common cause of bronchiolitis and pneumonia in children under 1 year of age (CDC)

About 800000 children die per year worldwide due to RSV infection (~91 per hour)

There are currently two methods for the clinical confirmation of RSV infection

Viral isolation from culture (gold standard, requires several days) Direct antigen test (tests range from 20-75 mins)

There are currently no vaccines or drugs available to prevent or treat RSV

Background on RSV in the Clinic

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Background on RSV in the Lab Ongoing RSV research:

Understand mechanisms of RSV pathogenesis in order to develop drugs

Test vaccine candidates Mouse models are commonly used The current method to quantify RSV titer in mice is

the plaque assay

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Current Method: Viral Plaque Assay

CultureCells

WaitFor Cells to

Grow

3 days

InoculateCells with

Virus

Overlay Cellswith Methyl-

Cellulose

AllowPlaquesTo Form

5 days

Stain Cells withHematoxylinand Eosin

Wait for Cellsto becomeInfected

1 hour

CountPlaques

CalculateViralTiter

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

The Problem Viral plaque assay is

Labor intensive Costly Time consuming Partially subjective

Need high throughput, inexpensive system to quantify infectious RSV

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Our Solution Novel plasmid based reporter system

Luciferase plasmid Cell line

Luminesce upon infection with RSV

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

ComparisonPlaque Assay Luciferase System

Detection Method Staining/Counting Luminescence

Objectivity Partial Yes

Time (work/total) 10 hours/7 days 2.5hrs/2 days

Materials Cost $8 $1

Throughput 30 samples/experiment 240 samples/experiment

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Comparison: Evaluation Chart

    Plaque Assay Luciferase System

Criteria Weight (1-5) Value Product Value Product

Quick 5 2 10 4 20

Low Cost 3 2 6 4 12

Objective 3 4 12 5 15

Efficient 4 3 12 5 20

Total 40 67

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

MethodsRSV Genome

RSV Genome (truncated)NS1

L3’ 5’

NS1 Start L Stop

pcDNA

(Synthesized)

NS1NS2 SH M2

3’ 5’N M G LFP

Methods

Luciferase Gene (luc)

luc

NS1 Start L Stop

selectionpRSVlucM5

pRSVlucM5

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Development CostsItem Cost

pcDNA3.1 vector $361

pGEM-luc $83

Trailer minigenome plasmid $274

Leader oligonucleotides 2x at $78 and 2x at $98

Cloning discs 2x at $29

Misc. chemicals and disposable lab equip. $750*

TOTAL $1878*

* Approximate value

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Alternate Solutions PCR - polymerase chain reaction

Proven to work for the detection and quantification of viruses

Limitations: Measures amount of nucleic acid (cannot differentiate

between live virus and dead virus) Low throughput Costly

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Project Status Completed:

Design of all plasmid constituents in silco Ligation of the plasmid constituents Screening selection Demonstration that the plasmid works as designed Stable transfection of cells with plasmid Submitted Information Disclosure Form to Office of

Tech Transfer

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Luminescence Data

RLU vs. Amount of DNA Transfected

0

500

1000

1500

2000

2500

3000

0 0.2 0.4 0.6 0.8 1

Amt of DNA Transfected (ug)

RL

U

(-) control

2,1

2,2

3,1

3,2

Thursday March 13th, 2008Oral Report 4VUSE Senior Design

Project Status In Progress:

Test stable transfection Future Work:

Optimize the system Final write-up

Thursday March 13th, 2008Oral Report 4VUSE Senior Design