%A Sagar Nadgir
%T Development of an Infectivity Assay for Human Herpesvirus-8
%X 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 ?-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-?-galactosidase reporter gene. The T1H6 cell line containing the ?-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. ?-galactosidase levels were determined using a chemiluminescent ?-galactosidase detection kit (Clontech).  Levels of ?-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 ?-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.
%D 2012
%K Human Herpesvirus-8, HHV-8, KSHV, Kaposi's Sarcoma-Associated Herpesvirus, TCID
%I University of Pittsburgh
%L pittir13490