Prevalence of varicella-zoster virus DNA in dissociated human trigeminal ganglion neurons and nonneuronal cells

Abstract

Previous analyses using in situ hybridization alone or together with PCR have yielded conflicting results regarding the cell type in which latent varicella-zoster virus (VZV) resides. We separated human trigeminal ganglia (TG) into neuronal and nonneuronal fractions, followed by primary and nested PCR to quantitate VZV DNA at the single cell level. Both TG from each of eight cadavers were dissociated and separated into neuronal and nonneuronal cell suspensions by differential filtration. Analysis of the neuron fraction (5,000 neurons per sample) revealed VZV DNA in 9 of 16 samples, with copy numbers ranging from 1 to 12, whereas only 2 of 16 nonneuronal cell samples were positive for VZV DNA, with 1 copy each. Further analysis of 10 samples of 100 neurons and the corresponding nonneuronal cell fractions from each TG of a single subject revealed VZV DNA in 3 of 10 samples of the left TG (range, 2 to 5 copies) and in 1 of 10 samples of the right TG (2 copies) but in none of the 20 nonneuronal cell fractions. These data indicate that latent VZV DNA is present primarily, if not exclusively, in neurons, at a frequency of two to five copies per latently infected neuron.

FIG. 1

Cell-type separation of dissociated human TG. (A) Large debris from a trigeminal ganglion captured by 70-μm mesh. (B) Flowthrough from 70-μm mesh contains neuronal and nonneuronal cells, including small debris. Arrows depict three large neurofilament-positive cells stained as described in Materials and Methods. (C) Neuron (arrow) retained by 20-μm mesh. (D) Three neurofilament-positive cells retained by 20-μm mesh (short arrows), together with two remaining nonneuronal cells (long arrows) and a cell fragment (arrowhead). (E) Small neurofilament-positive fragments (arrowhead) but no neurofilament-positive cells in the nonneuronal cell population after 20-μm mesh filtration. (F) Absence of neurofilament staining in control dissociated liver cells. (G) Positive actin staining in 20-μm filtrate (nonneuronal cells in E). (H) Luxol-fast blue staining of filamentous debris often seen in suspensions, revealing myelinated axon fragments. (I) Acridine orange staining of mixed-cell populations: diffuse orange cytoplasm represents retained cell RNA.

FIG. 2

Quantitation of VZV DNA. (A) PCR of dilutions from 10⁻¹ to 10⁴ copies of VZV gene 21 for various nested PCR cycles. (B) Graph of quantitation from part A, showing a linear relation between product and initial copy number for 10 cycles of nested PCR. Number of nested cycles: ●, 5; ○, 10; ▾, 15; ▿, 20; ■, 25; □, 30; ⧫, 35; ◊, 40.

FIG. 3

Cell-type distribution of VZV DNA in dissociated TG from eight subjects. Left (L) and right (R) TG from eight subjects were dissociated and processed into mixed-cell (A), neuronal (B), and nonneuronal (C) populations based on a 5,000-neuron count. The top blot in each panel shows the PCR amplification for β-actin in all samples. The middle blot in each panel shows VZV gene 21 DNA after 40 cycles of primary PCR, followed by 10 cycles of nested PCR. Positive signals were seen in six mixed-cell samples, nine neuronal-cell samples, and two nonneuronal-cell samples. Quantitative analysis revealed 1 to 12 copies of VZV DNA in five mixed-cell samples, seven neuronal-cell samples, and two nonneuronal-cell samples. VZV DNA corresponding to less than 0.5 copies was present in one mixed-cell sample and two neuronal-cell samples. The bottom blot in each panel shows VZV gene 21 DNA after 40 cycles of primary PCR, followed by 40 cycles of nested PCR. Intense signals were seen in seven mixed-cell samples, nine neuronal-cell samples, and four nonneuronal-cell samples. ND, no added DNA; SD, dissociated non-latently-infected human TG cells spiked with 1,000 copies of VZV gene 21 plasmid, digested with DNase attached to beads before proteinase K treatment, which did not amplify VZV gene 21 DNA (extracellular DNA contamination control); VD, DNA extracted from VZV-infected BSC-1 cells. Quantitation of 1 to 10⁴ copies of gene 21 was performed as described in Fig. 2.

FIG. 4

VZV DNA copy number in dissociated TG. Ten aliquots each from left (upper two blots in panels A and B) and right (lower two blots in panels A and B) TG from subject 8 were dissociated and processed into neuronal (A) and nonneuronal (B) cells based on 100-neuron counts. The top blot in each panel shows two to five copies of VZV DNA in three neuronal-cell samples and no VZV DNA in any nonneuronal-cell samples, respectively, after 40 cycles of primary PCR for VZV gene 21 followed by 10 cycles of nested PCR. The second blot in each panel shows a strong VZV DNA signal in five neuronal-cell samples and no VZV DNA in any of ten nonneuronal-cell samples, respectively, after PCR for 40 cycles of primary PCR for VZV gene 21 followed by 40 cycles of nested PCR. The third blot in each panel shows two copies of VZV DNA in a single neuronal-cell sample and no VZV DNA in any of 10 nonneuronal-cell samples, respectively, after 40 cycles of primary PCR for VZV gene 21, followed by 10 cycles of nested PCR. The bottom blot in each panel shows VZV DNA in one neuronal-cell sample and no VZV DNA in any of ten nonneuronal-cell samples, respectively, after 40 cycles of primary PCR for VZV gene 21 followed by 40 cycles of nested PCR. ND, SD, VD, and quantitation were as described in Fig. 2 and 3.