Prevalence and abundance of latently transcribed varicella-zoster virus genes in human ganglia

Abstract

In human ganglia latently infected with varicella-zoster virus (VZV), sequence analysis has revealed that five viral genes (VZV genes 21, 29, 62, 63, and 66) are transcribed. However, their comparative prevalence and abundance are unknown. Here, using real-time PCR, we analyzed 28 trigeminal ganglia from 14 humans for RNA corresponding to the five virus genes known to be transcribed in latently infected human ganglia. The most prevalent transcript found was VZV gene 63 (78%), followed by gene 66 (43%), gene 62 (36%), and gene 29 (21%). No gene 21 transcripts were detected in any of the 28 ganglia. VZV gene 63 RNA was also the most abundant (3,710 +/- 6,895 copies per 1 microg of mRNA) transcript detected in latently infected human ganglia, followed by VZV gene 29 (491 +/- 594), VZV gene 66 (117 +/- 85), and VZV gene 62 (64 +/- 38). Thus, the repeated detection and high abundance of VZV gene 63 transcripts in latently infected ganglia suggests that VZV gene 63 may be more important for the maintenance of virus latency than the less abundantly transcribed and randomly detected VZV genes 21, 29, 62, and 66.

FIG. 1.

Efficiency of VZV DNA quantitation. VZV DNA diluted at 1 to 10,000 copies was mixed with 100 ng of herring sperm DNA and amplified in duplicate using TaqMan primers specific for VZV genes 21, 29, 62, 63, and 66. Each primer set amplified 1 to 10,000 copies of VZV DNA with similar efficiency (R² = 0.95). The data represent average C_T values obtained with various VZV DNA copy numbers for all genes.

FIG. 2.

Quantitative PCR analysis of VZV transcripts in VZV-infected MeWo cells. Duplicate dilutions of total RNA from VZV-infected cells were reverse transcribed, and cDNAs corresponding to VZV genes 21, 29, 62, 63, and 66 were quantitated. The regression line slopes were similar in all cases (0.97 ± 0.03).

FIG. 3.

Abundance of VZV transcripts in VZV-infected MeWo cells. Total RNA extracted from VZV-infected cells was diluted and reverse transcribed, and the number of cDNAs corresponding to VZV genes 21, 29, 62, 63, and 66 was quantitated and normalized to 1 μg of input RNA.

FIG. 4.

Integrity of RNA extracted from human ganglia. Total RNA extracted from the left (L) and right (R) TG from subjects 1 to 8, the brains from subjects 15 and 16, and from uninfected (U) and VZV-infected (V) MeWo cells was resolved by microfiltration. Distinct 28S and 18S rRNA bands are seen throughout except in samples from subjects 5 and 15. M, molecular weight markers.

FIG. 5.

Real-time PCR analysis of human ganglia and brain for cell transcripts. mRNA (•) and cDNA (○) from the left and right TG of subjects 1 to 14 and the brains from subjects 15 and 16 were amplified with GAPDH-specific primers DNA (A) or neurofilament heavy 200-kDa protein-specific DNA (B), and the C_T values were determined.

FIG. 6.

Prevalence (A) and abundance (B) of VZV transcripts in latently infected human TG. The left and right TG of 14 individuals were examined for the presence of transcripts corresponding to VZV genes 21, 29, 62, 63, and 66. For the prevalence data, VZV gene 63 transcripts were the most frequently detected, followed by gene 66, gene 62, and gene 29; no gene 21 transcripts were detected in any of the subjects. The average abundance (panel B) of transcripts in each positive TG normalized to 1 μg of input mRNA was highest for open reading frame (ORF) 63, followed by gene 29, gene 66, and gene 62.

FIG. 7.

Relative abundance of VZV gene 21, 29, 62, 63, and 66 transcripts in individual trigeminal ganglion. The 18 TG that were found to contain VZV gene 29, 62, 63, or 66 transcripts were selected for further analysis. For each TG the number of transcripts mapping to each VZV gene was compared to that of the most abundant virus gene. Thus, for 16 of the 17 individual ganglia in which VZV gene 63 transcripts were the most abundant, the number of VZV gene 29, 62, and 66 transcripts is <10% the number of VZV gene 63 transcripts. ORF, open reading frame.