In situ polymerase chain reaction: localization of HSV-2 DNA sequences in infections of the nervous system

Abstract

To detect and localize a herpes simplex virus type 2 (HSV-2) thymidine kinase gene sequence in paraffin sections of brains and trigeminal ganglia of infected mice, an in situ polymerase chain reaction (ISPCR) protocol was developed. Using a single pair of primers, a 110 base pair DNA target sequence, and incorporation of a digoxigenin-labelled nucleotide during amplification, this procedure permitted rapid, specific, reproducible detection of infected cells. During acute brain infection, cells labelled by ISPCR were in the same infected foci that, in adjacent sections, contained viral antigen. This, together with controls, gave evidence of method specificity. In mice surviving acute infection, latently infected cells were labelled by ISPCR. In brains, focal areas contained labelled cell nuclei, and in trigeminal ganglia, neuronal nuclei were likewise labelled. Latent infection was confirmed by several methods, including identification of an HSV-specific sequence in DNA extracts of brains and ganglia, virus isolation from explanted ganglia, and HSV-2 latency-associated transcript (LAT) RNA localization in ganglionic neurons by in situ hybridization. Evidence in brains of ISPCR-labelled cells in regions where HSV-2 LAT-positive cells were not detected, and in ganglia of more ISPCR-labelled neurons than were LAT-positive, indicated that ISPCR is more sensitive in detecting latently infected cells than previous methods.