Detection and Discrimination of Latent and Replicative Herpesvirus Infection at the Single Cell Level In Vivo

Abstract

Herpesviruses have two distinct phases to their life cycle. Characteristically, they persist as a latent infection for the lifetime of the infected host. This usually involves a very small number of infected cells in a particular tissue where the virus is present at very low copy number and there is limited or no viral gene expression. The other phase of the life cycle involves replication of the virus to produce infectious virus. This typically involves expression of a large number of genes and high copy numbers of the viral genome, but again can be highly restricted to a small number of cells in a specific location. One characteristic that distinguishes latent and lytic infection is the form of the viral genome. In latently infected cells, the genome is circular, whereas during lytic replication the genome is linear. We have taken a gel technique where linear and circular herpesvirus DNA migrate with known and different mobilities, the Gardella gel (1), and combined it with a highly sensitive DNA polymerase chain reaction (PCR) system (2,3, and Chapter 7 in this volume), that can be used to detect a single copy of the viral genome in 10⁶ uninfected cells. The result is a technique that can be used to determine if a single infected cell is present in a tissue sample and distinguish if the cell is latently or lytically infected. This technique has several advantages over other methods that typically involve some form of reverse transcriptase (RT)-PCR, for distinguishing latent from lytic infection.