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Development of an Infectivity Assay for Human Herpesvirus-8

Nadgir, Sagar (2012) Development of an Infectivity Assay for Human Herpesvirus-8. Master's Thesis, University of Pittsburgh. (Unpublished)

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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:
CreatorsEmailPitt UsernameORCID
Nadgir, Sagarsagar.nadgir@gmail.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorJenkins, Frankfjenkins@pitt.eduFJENKINS
Committee MemberWang, Tianyitywang@pitt.eduTYWANG
Committee MemberRinaldo, Charlesrinaldo@pitt.eduRINALDO
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: Graduate 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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