title: Laser-capture microdissection: refining estimates of the quantity and distribution of latent herpes simplex virus 1 and varicella-zoster virus DNA in human trigeminal Ganglia at the single-cell level.
abstract: There remains uncertainty and some controversy about the percentages and types of cells in human sensory nerve ganglia that harbor latent herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) DNA. We developed and validated laser-capture microdissection and real-time PCR (LCM/PCR) assays for the presence and copy numbers of HSV-1 gG and VZV gene 62 sequences in single cells recovered from sections of human trigeminal ganglia (TG) obtained at autopsy. Among 970 individual sensory neurons from five subjects, 2.0 to 10.5% were positive for HSV-1 DNA, with a median of 11.3 copies/positive cell, compared with 0.2 to 1.5% of neurons found to be positive by in situ hybridization (ISH) for HSV-1 latency-associated transcripts (LAT), the classical surrogate marker for HSV latency. This indicates a more pervasive latent HSV-1 infection of human TG neurons than originally thought. Combined ISH/LCM/PCR assays revealed that the majority of the latently infected neurons do not accumulate LAT to detectable levels. We detected VZV DNA in 1.0 to 6.9% of individual neurons from 10 subjects. Of the total 1,722 neurons tested, 4.1% were VZV DNA positive, with a median of 6.9 viral genomes/positive cell. After removal by LCM of all visible neurons on a slide, all surrounding nonneuronal cells were harvested and assayed: 21 copies of HSV-1 DNA were detected in approximately 5,200 nonneuronal cells, while nine VZV genomes were detected in approximately 14,200 nonneuronal cells. These data indicate that both HSV-1 and VZV DNAs persist in human TG primarily, if not exclusively, in a moderate percentage of neuronal cells.
authors.affiliation: Medical Virology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA. kwang@niaid.nih.gov
authors.forename: Kening
authors.initials: K
authors.lastname: Wang
authors.forename: Tsz Y
authors.initials: TY
authors.lastname: Lau
authors.forename: Melissa
authors.initials: M
authors.lastname: Morales
authors.forename: Erik K
authors.initials: EK
authors.lastname: Mont
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Straus
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D015967:Gene Expression Regulation, Viral; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000302:isolation & purification; D014645:Herpesvirus 3, Human / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007834:Lasers; D042282:Microdissection / Q000379:methods; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012668:Trigeminal Ganglion / Q000821:virology*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral