PCR-based analysis of herpes simplex virus type 1 latency in the rat trigeminal ganglion established with a ribonucleotide reductase-deficient mutant

Abstract

Competitive quantitative PCR and reverse transcriptase-PCR were used to quantitate DNA and RNA from an attenuated ribonucleotide reductase-deleted herpes simplex virus type 1 (HSV-1) mutant in the rat trigeminal ganglion after peripheral inoculation following corneal scarification. Amplification of ganglionic DNA with oligonucleotide primers specific for the HSV-1 glycoprotein B (gB) gene and for the latency-associated transcript (LAT) gene indicated that there were approximately 2 x 10(5) genome equivalents per ganglion at 2 days, 7 days, and 8 weeks after inoculation. Amplification of ganglionic RNA with primers specific for HSV-1 LAT indicated that the amount of LAT RNA was also stable over 8 weeks, with 10(7) LAT molecules per ganglion at 2 days and at 7 days postinoculation and 1.4 x 10(7) LAT molecules per ganglion at 8 weeks. In situ hybridization with a digoxigenin-labeled riboprobe specific for LAT detected an average of one to two LAT-positive cells in each positive 6-microns section of trigeminal ganglion. In situ PCR detection of HSV-1 genomes in similar sections, using digoxigenin-labeled nucleotides with primers specific for HSV-1 gB, identified as many as 120 genome-positive cells per section. These results indicate that there are approximately 50 LAT molecules per latent HSV-1 genome in the trigeminal ganglion, compared with 15 LAT molecules per latent HSV-1 genome in the central nervous system (R. Ramakrishnan, D. J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994), but that cells with detectable LATs by in situ hybridization represent only a small proportion of those ganglionic neurons containing HSV-1 genomes. The presence of latent HSV-1 genomes in a large number of neurons suggests that HSV-1 may be more efficient in establishing the latent state than would be anticipated from previous reports.