scholarly journals 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

2005 ◽  
Vol 79 (22) ◽  
pp. 14079-14087 ◽  
Author(s):  
Kening Wang ◽  
Tsz Y. Lau ◽  
Melissa Morales ◽  
Erik K. Mont ◽  
Stephen E. Straus
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Laser Capture Microdissection ◽  
Trigeminal Ganglia ◽  
Herpes Simplex Virus 1 ◽  
Varicella Zoster ◽  
Pcr Assays ◽  
Simplex Virus ◽  
Laser Capture ◽  
Hsv 1

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 ∼5,200 nonneuronal cells, while nine VZV genomes were detected in ∼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.

scholarly journals Varicella-Zoster Virus (VZV) Glycoprotein E Is a Serological Antigen for Detection of Intrathecal Antibodies to VZV in Central Nervous System Infections, without Cross-Reaction to Herpes Simplex Virus 1

2011 ◽  
Vol 18 (8) ◽  
pp. 1336-1342 ◽  
Author(s):  
Anna Grahn ◽  
Marie Studahl ◽  
Staffan Nilsson ◽  
Elisabeth Thomsson ◽  
Malin Bäckström ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Igg Antibodies ◽  
Herpes Simplex Virus 1 ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) cause serious central nervous system (CNS) diseases that are diagnosed with PCR using samples of cerebrospinal fluid (CSF) and, during later stages of such infections, with assays of intrathecal IgG antibody production. However, serological diagnoses have been hampered by cross-reactions between HSV-1 and VZV IgG antibodies and are commonly reported in patients with herpes simplex encephalitis (HSE). In this study we have evaluated VZV glycoprotein E (gE) as a new antigen for serological diagnosis of VZV-induced CNS infections. Paired samples of CSF and serum from 29 patients with clinical diagnosis of VZV CNS infection (n= 15) or HSE (n= 14), all confirmed by PCR, were analyzed. VZV gE and whole VZV were compared as antigens in enzyme-linked immunosorbent assays (ELISAs) for serological assays in which the CSF/serum sample pairs were diluted to identical IgG concentrations. With the gE antigen, none of the HSE patients showed intrathecal IgG antibodies against VZV, compared to those shown by 11/14 patients using whole-VZV antigen (P< 0.001). In the patients with VZV infections, significantly higher CSF/serum optical density (OD) ratios were found in the VZV patients using the VZV gE antigen compared to those found using the whole-VZV antigen (P= 0.001). These results show that gE is a sensitive antigen for serological diagnosis of VZV infections in the CNS and that this antigen was devoid of cross-reactivity to HSV-1 IgG in patients with HSE. We therefore propose that VZV gE can be used for serological discrimination of CNS infections caused by VZV and HSV-1.

scholarly journals Herpes Simplex Virus 1 Latency and the Kinetics of Reactivation Are Regulated by a Complex Network of Interactions between the Herpesvirus Entry Mediator, Its Ligands (gD, BTLA, LIGHT, and CD160), and the Latency-Associated Transcript

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Shaohui Wang ◽  
Alexander V. Ljubimov ◽  
Ling Jin ◽  
Klaus Pfeffer ◽  
Mitchell Kronenberg ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Latently Infected ◽  
Simplex Virus ◽  
Kinetics Of ◽  
Herpesvirus Entry Mediator ◽  
Hsv 1

ABSTRACTRecently, we reported that the herpesvirus entry mediator (HVEM; also called TNFRSF14 or CD270) is upregulated by the latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1) and that the absence of HVEM affects latency reactivation but not primary infection in ocularly infected mice. gD has been shown to bind to HVEM. LIGHT (TNFSF14), CD160, and BTLA (B- and T-lymphocyte attenuator) also interact with HVEM and can interfere with HSV gD binding. It was not known if LIGHT, CD160, or BTLA affected the level of latency reactivation in the trigeminal ganglia (TG) of latently infected mice. To address this issue, we ocularly infected LIGHT−/−, CD160−/−, and BTLA−/−mice with LAT(+) and LAT(−) viruses, using similarly infected wild-type (WT) and HVEM−/−mice as controls. The amount of latency, as determined by the levels of gB DNA in the TG of the LIGHT−/−, CD160−/−, and BTLA−/−mice infected with either LAT(+) or LAT(−) viruses, was lower than that in WT mice infected with LAT(+) virus and was similar in WT mice infected with LAT(−) virus. The levels of LAT RNA in HVEM−/−, LIGHT−/−, CD160−/−, and BTLA−/−mice infected with LAT(+) virus were similar and were lower than the levels of LAT RNA in WT mice. However, LIGHT−/−, CD160−/−, and BTLA−/−mice, independent of the presence of LAT, had levels of reactivation similar to those of WT mice infected with LAT(+) virus. Faster reactivation correlated with the upregulation of HVEM transcript. The LIGHT−/−, CD160−/−, and BTLA−/−mice had higher levels of HVEM expression, and this, along with the absence of BTLA, LIGHT, or CD160, may contribute to faster reactivation, while the absence of each molecule, independent of LAT, may have contributed to lower latency. This study suggests that, in the absence of competition with gD for binding to HVEM, LAT RNA is important for WT levels of latency but not for WT levels of reactivation.IMPORTANCEThe effects of BTLA, LIGHT, and CD160 on latency reactivation are not known. We show here that in BTLA, LIGHT, or CD160 null mice, latency is reduced; however, HVEM expression is upregulated compared to that of WT mice, and this upregulation is associated with higher reactivation that is independent of LAT but dependent on gD expression. Thus, one of the mechanisms by which BTLA, LIGHT, and CD160 null mice enhance reactivation appears to be the increased expression of HVEM in the presence of gD. Thus, our results suggest that blockade of HVEM-LIGHT-BTLA-CD160 contributes to reduced HSV-1 latency and reactivation.

scholarly journals Latent Herpes Simplex Virus 1 Infection Does Not Induce Apoptosis in Human Trigeminal Ganglia

2015 ◽  
Vol 89 (10) ◽  
pp. 5747-5750 ◽  
Author(s):  
Susanne Himmelein ◽  
Anja Lindemann ◽  
Inga Sinicina ◽  
Michael Strupp ◽  
Thomas Brandt ◽  
...  
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Caspase 3 ◽  
Trigeminal Ganglia ◽  
Herpes Simplex Virus 1 ◽  
Reverse Transcription Quantitative Pcr ◽  
Latently Infected ◽  
Simplex Virus ◽  
Hsv 1

Herpes simplex virus 1 (HSV-1) can establish lifelong latency in human trigeminal ganglia. Latently infected ganglia contain CD8+T cells, which secrete granzyme B and are thus capable of inducing neuronal apoptosis. Using immunohistochemistry and single-cell reverse transcription-quantitative PCR (RT-qPCR), higher frequency and transcript levels of caspase-3 were found in HSV-1-negative compared to HSV-1-positive ganglia and neurons, respectively. No terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay-positive neurons were detected. The infiltrating T cells do not induce apoptosis in latently infected neurons.

scholarly journals Quantitation of Latent Varicella-Zoster Virus and Herpes Simplex Virus Genomes in Human Trigeminal Ganglia

1999 ◽  
Vol 73 (12) ◽  
pp. 10514-10518 ◽  
Author(s):  
Stephanie R. Pevenstein ◽  
Richard K. Williams ◽  
Daniel McChesney ◽  
Erik K. Mont ◽  
John E. Smialek ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Ganglion Cells ◽  
Trigeminal Ganglia ◽  
Varicella Zoster ◽  
Glycoprotein G ◽  
Simplex Virus ◽  
Virus Genomes ◽  
Hsv 1

ABSTRACT Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia. Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902 ± 1,082 (standard error of the mean) or 109 genomes/105 cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258 ± 38 genomes/105 ganglion cells. These levels of latent viral genome loads have implications for virus distribution in and reactivation from human sensory ganglia.

scholarly journals Frequency and Abundance of Alphaherpesvirus DNA in Human Thoracic Sympathetic Ganglia

2014 ◽  
Vol 88 (14) ◽  
pp. 8189-8192 ◽  
Author(s):  
Maria A. Nagel ◽  
April Rempel ◽  
Jonathon Huntington ◽  
Forrest Kim ◽  
Alexander Choe ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Sympathetic Ganglia ◽  
Herpes Simplex Virus 1 ◽  
Varicella Zoster ◽  
Target Organs ◽  
Simplex Virus ◽  
Visceral Disease ◽  
Hsv 1

Alphaherpesvirus reactivation from thoracic sympathetic ganglia (TSG) and transaxonal spread to target organs cause human visceral disease. Yet alphaherpesvirus latency in TSG has not been well characterized. In this study, quantitative PCR detected varicella-zoster virus (VZV), herpes simplex virus 1 (HSV-1), and HSV-2 DNA in 117 fresh TSG obtained postmortem from 15 subjects. VZV DNA was found in 76 (65%) ganglia from all subjects, HSV-1 DNA was found in 5 (4%) ganglia from 3 subjects, and no HSV-2 was found.

scholarly journals Mutations Inactivating Herpes Simplex Virus 1 MicroRNA miR-H2 Do Not Detectably Increase ICP0 Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Dongli Pan ◽  
Jean M. Pesola ◽  
Gang Li ◽  
Seamus McCarron ◽  
Donald M. Coen
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Trigeminal Ganglia ◽  
Herpes Simplex Virus 1 ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Latently Infected ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) latency entails the repression of productive (“lytic”) gene expression. An attractive hypothesis to explain some of this repression involves inhibition of the expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to ICP0 mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 in HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2 but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to the level of ICP0 mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for the expression of ICP0 and other lytic genes in acutely and latently infected mouse trigeminal ganglia. The latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting ICP0 mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency. IMPORTANCE Herpes simplex virus 1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since miR-H2's discovery as a viral microRNA bearing complete sequence complementarity to the mRNA for the important viral gene activator ICP0, inhibition of ICP0 expression by miR-H2 has been a major hypothesis to help explain the repression of lytic gene expression during latency. However, this hypothesis remained untested in latently infected animals. Using a miR-H2-deficient mutant virus, we found no evidence that miR-H2 represses the expression of ICP0 or other lytic genes in cells or mice infected with HSV-1. Although miR-H2 can repress ICP0 expression in transfection assays, such repression is weak. The results suggest that other mechanisms for miR-H2 activity and for the repression of lytic gene expression during latency deserve investigation.

scholarly journals Differences in the Likelihood of Acyclovir Resistance-Associated Mutations in the Thymidine Kinase Genes of Herpes Simplex Virus 1 and Varicella-Zoster Virus

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Hikaru Fujii ◽  
Shizuko Harada ◽  
Tomoki Yoshikawa ◽  
Souichi Yamada ◽  
Natsumi Omura ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Thymidine Kinase ◽  
Low Frequency ◽  
Herpes Simplex Virus 1 ◽  
Varicella Zoster ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTAcyclovir (ACV) resistance-associated mutations in two recombinant herpes simplex virus 1 (HSV-1) clones were compared. Recombinant HSV-1 lacking its thymidine kinase (TK) and expressing varicella-zoster virus (VZV) TK ectopically had no mutations in the VZV TK gene. In contrast, recombinant HSV-1 expressing HSV-1 TK ectopically harbored mutations in the HSV-1 TK gene. These results suggest that the relatively low frequency of ACV-resistant VZV is a consequence of the characteristics of the TK gene.

scholarly journals CD8+T Cells Play a Bystander Role in Mice Latently Infected with Herpes Simplex Virus 1

2016 ◽  
Vol 90 (10) ◽  
pp. 5059-5067 ◽  
Author(s):  
Kevin R. Mott ◽  
David Gate ◽  
Harry H. Matundan ◽  
Yasamin N. Ghiasi ◽  
Terrence Town ◽  
...  
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Link Type ◽  
Latently Infected ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTBased on an explant reactivation model, it has been proposed that CD8+T cells maintain latency in trigeminal ganglia (TG) of mice latently infected with herpes simplex virus 1 (HSV-1) [T. Liu, K. M. Khanna, X. Chen, D. J. Fink, and R. L. Hendricks, J Exp Med 191:1459–1466, 2000, doi:10.1084/jem.191.9.1459; K. M. Khanna, R. H. Bonneau, P. R. Kinchington, and R. L. Hendricks, Immunity 18:593-603, 2003, doi:10.1016/S1074-7613(03)00112-2]. In those studies, BALB/c mice were ocularly infected with an avirulent HSV-1 strain (RE) after corneal scarification. However, in our studies, we typically infect mice with a virulent HSV-1 strain (McKrae) that does not require corneal scarification. Using a combination of knockout mice, adoptive transfers, and depletion studies, we recently found that CD8α+dendritic cells (DCs) contribute to HSV-1 latency and reactivation in TG of ocularly infected mice (K. R. Mott, S. J. Allen, M. Zandian, B. Konda, B. G. Sharifi, C. Jones, S. L. Wechsler, T. Town, and H. Ghiasi, PLoS One 9:e93444, 2014, doi:10.1371/journal.pone.0093444). This suggested that CD8+T cells might not be the major regulators of HSV-1 latency in the mouse TG. To investigate this iconoclastic possibility, we used a blocking CD8 antibody and CD8+T cells in reactivated TG explants from mice latently infected with (i) the avirulent HSV-1 strain RE following corneal scarification or (ii) the virulent HSV-1 strain McKrae without corneal scarification. Independently of the strain or approach, our results show that CD8α+DCs, not CD8+T cells, drive latency and reactivation. In addition, adoptive transfer of CD8+T cells from wild-type (wt) mice to CD8α−/−mice did not restore latency to the level for wt mice or wt virus. In the presence of latency-associated transcript (LAT(+); wt virus), CD8+T cells seem to play a bystander role in the TG. These bystander T cells highly express PD-1, most likely due to the presence of CD8α+DCs. Collectively, these results support the notion that CD8+T cells do not play a major role in maintaining HSV-1 latency and reactivation.SIGNIFICANCEThis study addresses a fundamentally important and widely debated issue in the field of HSV latency—reactivation. In this article, we directly compare the effects of anti-CD8 antibody, CD8+T cells, LAT, and CD8α+DCs in blocking explant reactivation in TG of mice latently infected with avirulent or virulent HSV-1. Our data suggest that CD8+T cells are not responsible for an increase or maintenance of latency in ocularly infected mice. However, they seem to play a bystander role that correlates with the presence of LAT, higher subclinical reactivation levels, and higher PD-1 expression levels.

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