Latent varicella-zoster virus is located predominantly in neurons in human trigeminal ganglia

Abstract

Varicella-zoster virus (VZV) is a human herpesvirus that causes varicella (chicken pox) as a primary infection and, after a variable period of latency in trigeminal and dorsal root ganglia, reactivates to cause herpes zoster (shingles). Both of these conditions may be followed by a variety of neurological complications, especially in immunocompromised individuals such as those with human immunodeficiency virus (HIV) infection. There have been a number of conflicting reports regarding the cellular location of latent VZV within human ganglia. To address this controversy we examined fixed wax-embedded trigeminal ganglia from 30 individuals obtained at autopsy, including 11 with HIV infection, 2 neonates, and 17 immunocompetent individuals, for the presence of latent VZV. Polymerase chain reaction (PCR), in situ hybridization, and PCR in situ amplification techniques with oligonucleotide probes and primer sequences to VZV genes 18, 21, 29, and 63 were used. VZV DNA in ganglia was detected in 15 individuals by using PCR alone, and in 12 individuals (6 normal non-HIV and 6 positive HIV individuals, but not neonatal ganglia) by using PCR in situ amplification. When in situ hybridization alone was used, 5 HIV-positive individuals and only 1 non-HIV individual showed VZV nucleic acid signals in ganglia. In all of the VZV-positive ganglia examined, VZV nucleic acid was detected in neuronal nuclei. Only occasional nonneuronal cells contained VZV DNA. We conclude from these studies that the neuron is the predominant site of latent VZV in human trigeminal ganglia.

Figure 1

In situ hybridization of human trigeminal ganglia with DIG-labeled probes. (a) Sample 9 (normal) hybridized with VZV gene 29 probe. (b) Control, ganglion hybridized with non-VZV plasmid pGEM 3 DNA probe. (c and d) Sample 2 (HIV-positive) hybridized with VZV gene 63 probe and with VZV gene 29 probe respectively. Positive signals in neurons can be seen in a, c, and d, but not in b. (×450.)

Figure 2

PCR in situ (direct and indirect) of normal human trigeminal ganglia with VZV gene primers. (a) Sample 16 amplified with primers to VZV gene 18 (direct PCR in situ). (b and c) Sample 12 and sample 19 amplified with primers to VZV gene 29 (direct PCR in situ). (d) Sample 12 amplified with primers to VZV gene 29 and the section subsequently hybridized to the DIG-labeled internal 70-mer oligonucleotide probe (indirect PCR in situ). Positive signals in neurons can be seen in all panels. (×450.)

Figure 3

Southern blot analysis of amplicons from in situ PCR sections. Lane 1, BRL 123-bp DNA ladder; lane 2, DNA isolated from 8 5-μm sections of ganglia that had undergone direct PCR in situ with primers to VZV gene 21; lane 3, BRL 123-bp DNA ladder; lane 4, DNA isolated from 8 5-μm sections of ganglia that had undergone direct PCR in situ with primers to VZV gene 29. DNA was electrophoresed through 2% agarose gels, blotted onto GeneScreenPlus (DuPont), and hybridized with either the 285-bp gene 21 (lane 2) or the 185-bp gene 29 (lane 4) random primed ³²P-labeled VZV PCR product probe.