Analysis of individual human trigeminal ganglia for latent herpes simplex virus type 1 and varicella-zoster virus nucleic acids using real-time PCR

Abstract

Herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) establish latent infections in the peripheral nervous system following primary infection. During latency both virus genomes exhibit limited transcription, with the HSV-1 LATs and at least four VZV transcripts consistently detected in latently infected human ganglia. In this study we used real-time PCR quantitation to determine the viral DNA copy number in individual trigeminal ganglia (TG) from 17 subjects. The number of HSV-1 genomes was not significantly different between the left and right TG from the same individual and varied per subject from 42.9 to 677.9 copies per 100 ng of DNA. The number of VZV genomes was also not significantly different between left and right TG from the same individual and varied per subject from 37.0 to 3,560.5 copies per 100 ng of DNA. HSV-1 LAT transcripts were consistently detected in ganglia containing latent HSV-1 and varied in relative expression by >500-fold. Of the three VZV transcripts analyzed, only transcripts mapping to gene 63 were consistently detected in latently infected ganglia and varied in relative expression by >2,000-fold. Thus, it appears that, similar to LAT transcription in HSV-1 latently infected ganglia, VZV gene 63 transcription is a hallmark of VZV latency.

FIG. 1

HSV-1 and VZV DNA burden in human TG. Total DNA was extracted from left and right human TG and the virus DNA copy number per 100 ng was determined by real-time PCR. Statistical analysis allowed condensation of the results from individual ganglia to yield virus DNA copy numbers in 100 ng per subject. Subject numbers refer to the individuals described in Table 1; data quantitation is shown in Tables 3 and 4. VZV DNA (○) was found in the TG from all 17 subjects, while HSV-1 DNA (●) was found in 12 of the 17 subjects.

FIG. 2

Sensitivity of real-time PCR. Dilutions from 10⁶ to 10⁻¹ copies of polyadenylated VZV gene 21, 29, and 63 transcripts and nonpolyadenylated HSV-1 LAT transcript per 10 μl of nuclease-free water were added to 500 ng of total RNA extracted from control African green monkey dorsal root ganglia. Each dilution was reverse transcribed, and the cDNA was subjected to real-time PCR using gene 29 reagents. (A) Standard curve generated using VZV genomic DNA as described in Materials and Methods. ●, standards; ○, unknowns (cDNA samples shown in panel B). (B) Calculated gene 21, gene 29, gene 63, and LAT copy numbers in cDNA generated from synthetic RNA, relative to the standard curve shown in panel A (gene 29) and the standard curves for gene 21, gene 63, and LAT (not shown).