A5-Positive Primary Sensory Neurons Are Nonpermissive for Productive Infection with Herpes Simplex Virus 1 In
Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. There are a few quantitative studies of the viral load within the trigeminal ganglion, but none that investigate other cranial nerve ganglia. The sensitivity, specificity and reproducibility of this PCR protocol were determined on uninfected and HSV-infected mouse tissues and on HSV DNA from infected tissue culture cells. The primary goal of this report is to generate a recombinant HSV-1 constitutively expressing Egr-1 and to investigate the regulation of viral replication in different cell types or in animals with Egr-1 overexpression. HHV-1 causes gingivostomatitis, keratoconjunctivitis, encephalitis, herpes labialis, herpetic whitlow among others. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV. Thirty four cell line isolates and sixteen clinical samples taken from a group of adult patients with neurological signs were tested for the presence of HSV-1/2 DNA in the LightCycler® instrument.
HHV co-infection was also frequent (10.2%) in the patients with FUO. DNA from YP2 (lanes 1 and 2) and CW1 (lanes 3 and 4) served as the templates for PCR amplification using primer pairs specific for HSV-TK (lanes 1 and 3) and gD (lanes 2 and 4), producing the expected 1,131- and 262-bp amplicons, respectively. Although HSV infection was not associated with sperm motility and morphological defects, it was correlated with lower sperm count in the seminal fluid. (c) Southern hybridization of YP2. HSV-specific CD4 and CD8 T cells infiltrate herpetic lesions (16, 17, 19). This may not be the complete list of references from this article. (iii) The requirement for ICP22 and UL13 protein kinase for the stabilization of cdc2 was investigated in two series of experiments.
). A second transgenic mouse, containing the first 2.5 kb of the HSV-2 LAT (5′ exon and 2.2-kb intron) under control of its native promoter, was constructed (34). Comparing developed and developing nations, infection is consistently the most common cause of FUO, but the types of infection vary –. In mouse sensory ganglia, the A5 and KH10 markers identify functionally distinct nociceptive neuronal populations. A5+ neurons are nerve growth factor (NGF) responsive, are immunoreactive for the calcitonin gene-related peptide (CGRP), and project Aδ and C fibers to laminae I and II (outer) of the dorsal horn (14). KH10+ neurons are colabeled with Bandeiraea simplicifolia isolectin B4 (BSL-IB4) and are small-diameter, RET-positive neurons that express the ATP-gated ion channel P2X3 and receptors for glial-cell-derived neurotrophic factor (GDNF) and neurturin (4, 14, 28, 33, 41, 54). They project C fibers to lamina II (inner) of the dorsal horn (14).
Animal models of infection and latency have been valuable in the study of HSV pathogenesis but have limitations for studying mechanisms that regulate the establishment and maintenance of viral latency. These limitations include the relatively small proportion of ganglionic neurons in which latency is established, the asynchronicity of events, the very small number of neurons that can be induced to reactivate, and the difficulty of manipulating the molecular state of infected neurons. A temperature-sensitive mutant of HSV-2 has a conditional defect in virion stability that maps to the VP16 gene (Moss, 1989). Detection and direct typing of herpes simplex virus by polymerase chain reaction. Furthermore, in vitro models with embryonic or neonatal sensory neurons do not reflect the mature heterogeneous populations of neurons in the adult sensory ganglia. The primer pairs used in these studies identified HSV types 1 and 2 and did not cross react with other herpesviruses including cytomegalovirus (CMV), varicella-zoster virus (VZV), human herpesvirus-6, and EBV. None of the men or their spouses had reported any clinically confirmed genital herpetic infection in their medical history.
Using this in vitro model, we determined that A5+ trigeminal ganglion neurons are relatively nonpermissive for productive HSV-1 infection compared to other populations of trigeminal ganglion neurons. In this model we also determined that preferential permissiveness for productive infection is regulated at, or before, the level of immediate early (IE) viral gene expression. Neuronal cultures.Six-week-old female Swiss Webster mice (Simonsen Labs, Gilroy, CA) were euthanized by CO2, followed by transcardial perfusion with cold, calcium- and magnesium-free (CMF) phosphate-buffered saline (PBS). Trigeminal ganglia (TG) were removed, incubated at 37°C for 20 min in papain (25 mg) (Worthington, Lakewood, NJ) reconstituted with 5 ml Neurobasal A medium (Invitrogen) and for 20 min in Hanks balanced salt solution (HBSS) containing dispase (4.67 mg/ml) and collagenase (4 mg/ml) (Sigma) on a rotator, and mechanically dissociated by triturating with a 1,000-μl pipette. The resultant cell suspension was layered on a 5-step OptiPrep (Sigma) gradient. OptiPrep was first diluted with 0.8% sodium chloride (50.5:49.5) to make a working solution and was then further diluted with Neurobasal A medium to make gradient steps as follows: 150 μl of OptiPrep working solution and 850 μl of Neurobasal A, 250 and 750 μl, 300 and 700 μl, 350 and 650 μl, and 400 and 600 μl, respectively. The PCR thermal cycling incubations used for screen 1 were as follows: reverse transcription and initial amplification were performed in a single reaction by incubation at 37°C for 15 min and 94°C for 40 s preceding 33 cycles of incubation at 94, 60, and 72°C for 20 s each; further amplification with the nested primers was by 33 cycles of incubation at 94, 55, and 72°C for 20 s each.
The lower end of the centrifuged gradient (∼3.5 ml), minus the pellet, was then transferred to a new tube and was washed twice with Neurobasal A medium supplemented with 2% B27 supplement (Invitrogen) and 1% penicillin-streptomycin (PS). Neurons were counted and plated on poly-d-lysine- and laminin-coated 8-well chamber slides (BD Biosciences) at a density of 3,000 per well. These temporally separate presentations were defined as separate episodes. The medium was then replaced with fresh medium without fluorodeoxyuridine and aphidicolin (growth factors were from R&D Systems, and other supplements were from Sigma). Neonatal trigeminal ganglia were cultured using identical methods and conditions. Our focus is on the identification of the cells and molecular determinants that mediate initial entry into tissue. Viruses.The wild-type HSV-1 strains KOS, RE, and 17syn+ and the wild-type HSV-2 strain 333, as well as all mutant virus strains, were propagated in rabbit skin cells (52).
HSV1-VP26-GFP and HSV2-VP26-GFP were generated in Vero cells by cotransfection and homologous recombination of plasmid pK26GFP (kindly provided by Prashant Desai, Johns Hopkins University) with purified viral DNA from either HSV-1 strain 17+ or HSV-2 strain 333 by using previously described methods (6). The PCR was carried out using Failsafe PCR enzyme Mix (Epicentre: Cat#: FS99250) and Buffer Mix E (FSP995E). KOS/58, an HSV-1-based virus expressing lacZ under the control of the neurofilament light (NFL) promoter at the gC locus, and KOS/62, an HSV-1-based virus expressing lacZ inserted between SacII and the second HpaI sites downstream of the LAT promoter, have been described previously (29). SC16 was a gift from Dr A. RE-pgC-EGFP and RE-pICP0-EGFP have been described previously (11). Statistical analysis The eligibility and classification of the clinical FUO syndromes were determined from the original record of each item in the medical history and an examination of the database. To develop the RE-pICP27-EGFP virus, the upstream portion of ICP27 from 37 bp upstream of the ICP27 ATG to a position approximately 1,000 bp further upstream was amplified by PCR using primers 5′-gcagatctGTCGGATATGGCCTCTGGTGTGGCGCA and 5′-gagtaagcttCCTACACGAAAATTACCCGCCT (lowercase sequences encode restriction sites for cloning).
All statistical analyses were performed using the statistical software SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Viruses were derived and purified by selection for EGFP-positive plaques. Antigen was detected by sequential incubation with a 1:100 dilution of MAb P43, specific for the UL49 gene product, VP22 (11), affinity-purified goat anti-mouse immunoglobulin M-peroxidase conjugate (Sigma, St. The gB promoter amplification product was placed into the gC-EGFP plasmid to drive EGFP and then was linearized and used to derive and purify fluorescent virus. The sequence of plasmid PGEX4T-1/cdc2-dn was verified. This fragment was used to construct transgenic mice in a C57BL/6 background (NCI-Frederick, Frederick, Md.) at the National Institute of Allergy and Infectious Diseases (NIAID) Transgenic Mice Facility in accordance with protocols approved by the Frederick Cancer Research and Development Center and NIAID Animal Care and Use Committees. Hybridization was performed with random hexamer-primed (random labeling kit; Roche) [32P]dCTP-labeled pATD19 probe (see below) to detect expression of the 2.0-kb LAT intron.
After a 1-h adsorption period, virus was removed and replaced with complete neuronal medium (without fluorodeoxyuridine and aphidicolin). For infections lasting longer than 15 h, pooled human IgG was added to the medium to inhibit viral spread through the medium after the first productive cycle. IgG was removed for assays of infectious virus and viral reactivation. For immunofluorescent (IF) assays, paraformaldehyde (PFA) was added directly to the medium of the cultured cells after the time periods designated in the figure legends, to a final concentration of 2%, for 5 to 10 min. The fixative was then removed; the cultures were immunostained for the antigens designated in the figure legends; and neurons were counted using a Nikon Microphot fluorescent microscope. For infectious virus assays, 150 μl of the medium was transferred to a cryotube; cells were scraped and suspended in the remaining 150 μl of medium in each well before being transferred to a second cryotube; and the samples were frozen at −80°C. Samples were freeze-thawed to release infectious virus, and titers of the medium and homogenate were determined on Vero cells using a standard plaque assay.
All experiments were carried out in parallel with uninfected cultures, which served as negative controls. Whitley RJ, Roizman В. Neurons were initially differentiated from satellite glial cells (SGCs) by anti-NeuN antibody staining (Santa Cruz), and subsequently by morphology. GFP expression by VP26-GFP-expressing viruses was not immunohistochemically amplified. Since neuronal cells are slightly autofluorescent in the GFP spectral range, neurons in infected cultures were compared to neurons in uninfected cultures for determination of the presence of GFP expression. EGFP expression by immediate early (IE), early (E), and late (L) gene reporter viruses was amplified by incubation with a rabbit anti-GFP antibody (Santa Cruz) followed by FITC-labeled anti-rabbit IgG (Santa Cruz). 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining or rabbit IgG against β-galactosidase (β-gal; Abcam) as the primary antibody with FITC-labeled anti-rabbit IgG (Abcam) as the secondary antibody was used to visualize β-galactosidase.
Immunolabeled neuronal cultures were evaluated by fluorescence microscopy. Cultured adult murine trigeminal neurons mimic in vivo neuron characteristics.In previous studies, we used combined fluorescent in situ hybridization (FISH) for the LAT in conjunction with immunofluorescence (IF) for neuronal markers to demonstrate that HSV-1 preferentially establishes latent infection of murine sensory ganglia in A5-positive neurons while HSV-2 preferentially establishes latent infection in KH10-positive neurons after ocular or footpad infection of mice (21, 28). One mechanism that could account for these findings is differential permissiveness of different types of sensory neurons for productive infection with HSV-1 and HSV-2. To test this hypothesis directly, we developed an in vitro system for studying direct viral infection of dissociated adult murine trigeminal ganglion neurons, thus minimizing the confounding roles of the immune system and the variability of the efficiency of viral delivery to the axons in vivo. In a comparison of the two protocols, this assay was between approximately 10- and 100-fold more sensitive for the detection of identical isolates of HSV-1, VZV, and poliovirus type 2. 1) (21, 28, 52). In the adult neuron cultures, the A5 marker colocalized with immunoreactivity for the calcitonin gene-related peptide (CGRP) but not for staining with the lectin BSL-IB4.
A similar statistically significant pattern was observed when the analysis was limited to those CSF samples that originated from patients thought likely to have CNS viral infections. These results demonstrate that our in vitro cultures maintain neuronal heterogeneity and some of the well-established in vivo features of adult murine trigeminal neurons that we have reported previously (28). A5+ and KH10+ neuron distribution in uninfected neuron cultures. Staining of F-actin was performed with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma) for 15 min at room temperature. (B) Representative fluorescent microscopy images of cultured adult trigeminal neurons positive for the A5 (top) and KH10 (bottom) markers, revealed by use of monoclonal antibodies and a rhodamine-labeled secondary antibody.