Nadgir, Sagar
(2012)
Development of an Infectivity Assay for Human Herpesvirus-8.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
An accurate method for determining HHV-8 infectivity is lacking. The most common method currently used is the measure of encapsidated (i.e. DNAse-resistant) DNA genomes using quantitative real-time PCR. This method, while highly sensitive, does not distinguish between infectious and non-infectious virus particles. Immunofluorescence imaging of infected cells can provide some idea of infectivity but this method is subjective and accurate measurements are difficult to obtain. We have developed a cell culture assay using the HHV-8 cellular receptor DC-SIGN and a β-galactosidase gene under the control of the replication trans-activator (RTA) responsive promoter, T1.1. Infection of these cells with HHV-8 results in RTA production (from the infecting genome), which in turn drives the T1.1-β-galactosidase reporter gene. The T1H6 cell line containing the β-galactosidase gene under the control of the HHV-8 T1.1 promoter, was transfected in our lab with a plasmid expressing DC-SIGN under the control of the CMV IE promoter producing the cell line T1H6-DC-SIGN. Expression of DC-SIGN in T1H6-DC-SIGN cells was confirmed by IFA. β-galactosidase levels were determined using a chemiluminescent β-galactosidase detection kit (Clontech). Levels of β-galactosidase were compared between HHV-8-infected T1H6 and T1H6-DC-SIGN cells. TCID50 values were determined using the Reed-Muench calculation. DNA copy numbers were determined using quantitative PCR specific for HHV-8 DNA. Levels of β-galactosidase were significantly increased in infected T1H6-DC-SIGN cells compared to T1H6 cells supporting the role of DC-SIGN as a viral receptor. A ratio of TCID50 values to DNA genome copy numbers demonstrated specific infectivity ranging from 10-4 to 10-6. Validation of TCID50 values was obtained by infection of immature dendritic cells using 1 and 2 TCID50s. Using this assay, we compared replication kinetics in de novo HHV-8 infected activated B cells in two donors. In terms of public health, this is a more sensitive and specific assay for data that is needed to study HHV-8 infection. A potential clinical application using this assay involves determination of neutralizing antibody titers.
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Details
Item Type: |
University of Pittsburgh ETD
|
Status: |
Unpublished |
Creators/Authors: |
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ETD Committee: |
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Date: |
21 September 2012 |
Date Type: |
Publication |
Defense Date: |
12 July 2012 |
Approval Date: |
21 September 2012 |
Submission Date: |
8 August 2012 |
Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
Number of Pages: |
72 |
Institution: |
University of Pittsburgh |
Schools and Programs: |
School of Public Health > Infectious Diseases and Microbiology |
Degree: |
MS - Master of Science |
Thesis Type: |
Master's Thesis |
Refereed: |
Yes |
Uncontrolled Keywords: |
Human Herpesvirus-8, HHV-8, KSHV, Kaposi's Sarcoma-Associated Herpesvirus, TCID |
Date Deposited: |
21 Sep 2012 15:40 |
Last Modified: |
15 Nov 2016 14:01 |
URI: |
http://d-scholarship.pitt.edu/id/eprint/13490 |
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