Copyright © 2014 Yun Ji Hong et al. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. A single specific amplicon of 268 bp was obtained from 71 VZV clinical isolates and several laboratory strains. We previously generated human iPS cells from skin fibroblasts by introducing four reprogramming genes with non-integrating adenovirus. These cells supported productive infection by both laboratory and clinical VZV isolates, including the live varicella vaccine. Primers for human endogenous retrovirus-3 (HERV-3), an internal control, were adopted. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively.
While VZV infections are usually mild, they sometimes result in severe disease, particularly in immunocompromised patients (5, 6, 11, 22). After differentiation, approximately 80% of the total cell population expressed the neuron-specific protein, βIII-tubulin. VZV reactivation is characterized by the transition from latent to productive neuronal infection in which the subsequent anterograde transport of the virus back to the skin results in herpes zoster (18). Development of suitable animal models for the study of VZV infection has been hampered by the strict human-specific tropism of the virus (2, 38). In the context of neuronal infection, SCIDhu mice grafted with human fetal dorsal root ganglia (DRG) and infected cotton rats have both been reported as models to study VZV persistence or latency (1, 9,–,13, 29, 40,–,44, 52,–,56). Newer PCR methods have eliminated the need to propagate virus for VZV detection (18,19, 21, 30). The work is made available under the Creative Commons CC0 public domain dedication.
Neuroblastoma cell lines have previously been shown to provide highly valuable models in the analysis of the neuropathogenesis and neurotropism of a range of viruses due to the ability of the cells to mimic the morphological and biochemical characteristics of primary neurons (45). Our current study utilized the SH-SY5Y cell line, a derivative of primary neuroblastoma cells obtained by successive cloning (4), as these cells have previously been used successfully for viral tropism studies using human cytomegalovirus (36), assessment of neurovirulent poliovirus strains (33), and as a model of herpes simplex virus latency and reactivation (50). However, few studies have endeavored to enhance these characteristics by using the cells in a fully differentiated form, a step thought to be crucial in the development of the neuronal phenotype (16). In this study we examined various clinical samples and confirmed the ability of this PCR-RFLP assay to differentiate vaccine strain from isolates obtained from patients. The remaining authors have declared that no competing interests exist. In particular, a model of this nature may overcome many of the limitations of models requiring fresh primary human neural tissue, including donor heterogeneity and availability, limited and/or variable cell yield, complex and costly experimental procedures, and ethical considerations. SH-SY5Y cells grown in culture flasks were first induced to differentiate with culture medium (Dulbecco’s modified Eagle’s medium F-12 [DMEM F-12] containing 5% fetal calf serum and 50 IU/ml penicillin and streptomycin; Gibco, CA) supplemented with 10 μM all-trans retinoic acid (ATRA) (Sigma, Australia) for 10 days.
The cells were seeded onto coverslips coated with Matrigel (BD Biosciences, Australia) within 24-well culture plates at a density of 1 × 105 cells/well and allowed to adhere overnight. VZV DNA samples obtained from cells infected with the Oka vaccine strain and three laboratory VZV strains (Webster, vzv11, and ROD) were also examined. In the United States, the societal costs for treating patients with herpes zoster and its complications are estimated to exceed $1 billion annually . 1 A to C). While untreated cells possessed large, flat cell bodies, differentiated cells formed vast, branching neuritic networks with small, rounded cell bodies. Quantitation of the proportion of cells exhibiting neurites following each treatment period (Fig. Sequences were compared with the VZV ORF 62 sequences of the VZV Dumas strain (GenBank accession number X04370), which were used to design the PCR primers.
dorsal root ganglia) for xenografts and technical challenges limits its application. 1D). A rabbit antisynaptophysin antibody (Invitrogen, Australia) followed by an Alexa Fluor 488-conjugated anti-rabbit IgG antibody (Invitrogen, Australia) were used in immunofluorescence assays (IFAs) to assess the expression of neuron-specific synaptophysin protein (Fig. 1E and F), a neuronal differentiation marker (6, 46). PCR assays were completed in a volume of 100 μl of a solution that contained 500 ng of template DNA; 50 mM KCl; 10 mM Tris hydrochloride, pH 8.3; 5 mM MgCl2; a 200 μM concentration (each) of dATP, dCTP, dGTP, and dTTP; a 250 μM concentration of each primer; and 2.5 U ofTaq DNA polymerase (PCR Core kit [Boehringer Mannheim Biochemicals, Indianapolis, Ind.] or GeneAmp PCR reagent kit with AmpliTaq or AmpliTaq Gold DNA polymerase [Perkin-Elmer Cetus, Norwalk, Conn.]). Pathogenesis studies with guinea pig enteric neurons are based on a non-permissive host, which is likely to provide results substantially different from pathogenesis in humans. In contrast, undifferentiated cells exhibited minimal staining (Fig.
1E). Evaluation of SH-SY5Y cell differentiation using all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF). (18). iPS cells are derived from somatic cells that are reprogrammed by the introduction of key stem cell genes to become pluripotent cells that closely resemble ES cells. Values are means plus standard errors of the means (error bars) from three independent experiments. Significant difference was determined using a one-tailed, paired Student’s t test (*, P < 0.05; **, P < 0.01). RA, retinoic acid.
An initial PCR hot-start step of 96°C for 15 min was followed by 30 cycles of amplification (1 min at 94°C, 1 min at 72°C) and a final extension step at 72°C for 3 min (Mastercycler gradient, Eppendorf Scientific Inc.). In the present report, we describe the conditions required for the reproducible differentiation of human iPS cells to sensory neurons. Highly permissive human foreskin fibroblasts (HFFs) infected with VZV strain rOka were first labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) and then inoculated onto differentiated SH-SY5Y cells at a density of 1 × 105 cells/well in parallel with CFSE-labeled mock-infected HFFs. Labeling the inoculating HFFs with CFSE provided an additional means to readily distinguish these cells from SH-SY5Y cells. The cells were harvested at various time points postinfection (p.i.) for analysis by IFA. The 50- and 100-bp DNA ladders (GIBCO BRL, Gaithersburg, Md.) were used as DNA size markers. Mitotically inactivated mouse embryonic fibroblasts (MEFs; Millipore, Temecula, CA) were seeded on gelatin-coated tissue culture plates and grown in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum, and 1% penicillin-streptomycin-L glutamine (P/S/G).
Cells were incubated with antibodies against IE62 (Meridian Life Science Inc.), an IE protein, pORF29 (kindly provided by P. Kinchington, University of Pittsburgh), an E protein, and gE (Millipore, Australia), a L protein. Bound antibodies were detected using species-specific Alexa Fluor 594-conjugated antibodies. Amplicons within that size range usually provide optimal sensitivity for an assay, particularly for DNA amplification from clinical samples that may contain limited quantities of template. The supernatant containing iPS cells was carefully collected and centrifuged at 1000 rpm for 5 min. 2 A) and pORF29 (Fig. 2B) detected in the nucleus and gE (Fig.
2C) expressed on the cell surface and throughout the cytoplasm (15, 32, 35, 39). Twenty-one primer combinations were tested altogether, representing each of the three upper primers with each of seven lower primers. The media was replaced every 2–3 days for two weeks. 2D). No specific staining was observed in VZV-infected SH-SY5Y cells incubated with isotype control antibodies (data not shown). Comparable results were obtained in three independent experiments. On this basis, eight of the experimental primers were excluded from further analysis.
Blocking was performed for one hour in blocking buffer (5% normal serum, 0.3% Triton-X 100 in PBS). Differentiated SH-SY5Y cells were inoculated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled VZV rOka-infected human foreskin fibroblasts (HFFs) (green) and fixed 36 h p.i. IFA staining was performed using antibodies against VZV IE62 (A), pORF29 (B), or gE (red) (C), followed by a DAPI counterstain (blue). Insets show enlarged images of VZV antigen-positive cells. Furthermore, the reaction products resulting from PCR using the primer pair PKVL6U-PKVL1L during SmaI RFLP analysis could be easily differentiated by gel electrophoresis. The cells were fixed and permeabilized in FIX and PERM (Invitrogen) and the following primary antibodies were added for 30–45 min at RT: βIII-tubulin Alexa 647 (BD Pharmingen), goat peripherin (Santa Cruz), and rabbit Brn3a (Epitomics, Burlingame, CA). In addition, the proportion of SH-SY5Y cells infected with VZV strain rOka was compared with infection of these cells using a clinical isolate, VZV S.
Furthermore, in parallel with this analysis of SH-SY5Y infection, uninfected HFFs were seeded 24 h prior to infection at a density of 1 × 105 cells/well and inoculated in the same manner as differentiated SH-SY5Y cells to allow a direct comparison of infection efficiency between cell types. In both infection settings, the VZV-infected HFFs used as the inoculum were labeled with CFSE to enabled newly infected cells to be readily distinguished from the inoculating HFFs. Amplification products were produced with gradient annealing temperature cycling (ranging from 55 to 75°C) with the following primer pairs: PHKR1-PHKR2 (A), PHKR1-PKVL4L (B), PKVL7U-PKVL2L (C), and PKVL6U-PKVL1L (D). Immunocytochemistry was observed with a Nikon TS100 fluorescence microscope (Nikon Instruments, Melville, NY) and images were captured with a Nikon DS-Qi1 cooled camera head. VZV antigen-positive SH-SY5Y cells were readily detected following infection with both VZV rOka and VZV S, whereas no specific staining was observed in isotype control or mock-infected cultures (Fig. 3 A). The combined results of three independent experiments showed that the proportions of IE62- and gEgI-positive SH-SY5Y cells were comparable in cells infected with VZV rOka and VZV S at each time point, with no significant difference in the efficiency of infection of these two viruses (Fig.
Shown are results for the SmaI RFLP assay for VZV ORF 62 amplicons obtained with wild-type viruses (lanes 1 to 3 and 5 to 9 correspond to samples 44 to 46 and 48 to 52 in Table 1) and Oka vaccine strain (lane 4). Recording micropipettes (5–10 MΩ), pulled from thick-walled borosilicate glass (1.5 mm outer diameter, 0.85 mm inner diameter, WPI Sarasota, FL), were filled with (in mM): 122.5 Cs-gluconate, 17.5 CsCl, 10 HEPES (CsOH), 0.2 Na-EGTA, 2 Mg-ATP, 0.3 Na-GTP, and 8 NaCl at pH 7.3. 3B). Cytopathic effect (CPE) was detected at later times postinfection, as indicated by rounding of cells and detachment from the culture flask surface (data not shown). Taken together, these experiments demonstrated that differentiated SH-SY5Y cells were permissive to productive infection by both VZV rOka and VZV S. Adjustment of the reaction mixture pH to below 8.0 substantially decreased the sensitivity of detection (data not shown). RNA was extracted from undifferentiated and differentiated iPS cells using the RNeasy Micro Kit (Qiagen, Valencia, CA).
In parallel, cultures of HFFs were infected using the same method. (A) Flow cytometry scatter plots of cells 72 h after infection with rOka or VZV S following staining with antibodies against VZV IE62 or gEgI. Mock-infected cultures and VZV-infected cultures stained with isotype control antibody were included as controls. All VZV-positive samples generated a single specific amplicon 268 bp in size (Fig. The relative Brn3a expression in undifferentiated and differentiated iPS cells was analyzed using the ΔΔCt method . Values are means plus standard errors of the means (error bars) from three independent experiments. It has previously been reported that pure neuronal cell cultures inoculated with cell-free VZV undergo persistent infection, in contrast to cell-associated inoculation, which results in productive VZV infection (5).
We therefore sought to investigate infection of differentiated SH-SY5Y cells with cell-free VZV (at a multiplicity of infection [MOI] of 0.014) using the commercially available varicella vaccine (Varivax; Merck) which comprises a cell-free preparation of VZV strain vOka. No amplicons were detected in PCR assays using these specimens. The assay was performed in triplicate, and the data represent the mean number of foci ± SEM. 4 A), pOR29 (Fig. 4B), and gEgI (Fig. 4C) in these cultures indicated that the cells were undergoing productive VZV replication. We determined that these primers detected VZV DNA in every specimen from a panel of scab and vesicle fluid clinical samples (12 specimens), even when as little as 1/50 of the DNA preparation was used for the PCR.
1B) and expressed the neural progenitor markers Pax6 (Figure 1C) and nestin (Figure 1D). In order to confirm that these infected SH-SY5Y cells were capable of transmitting infectious VZV to another permissive cell type and thus complete the cycle of productive infection, infectious center assays were performed. Single wells containing vOka-infected SH-SY5Y cultures were trypsinized and inoculated onto uninfected HFF monolayers. Immunofluorescent staining performed 7 days p.i. Additionally, nine DNAs were typed as Oka vaccine strain (BglI+PstI−), among which only two specimens, our Oka vaccine virus control specimen and one U.S. (B) Brightfield images of iPS cells after 4 and 10 days of exposure to small molecule inhibitors. Our demonstration that inoculation of SH-SY5Y neuronal cells with cell-free VZV resulted in productive infection rather than a latent infection contrasts with a study using guinea pig enteric neurons (5).
This may be due to differences in the nature of the inoculum used, the different properties of cell line cultures compared with primary tissues, and the species from which the cells or tissues were derived. Nonetheless, it may be possible in the future to modify the SH-SY5Y cell productive infection model (e.g., by inclusion of viral DNA synthesis inhibitors) to derive a model which would abort viral replication infection and enable viral genome persistence. These data are shown in Table 2. We observed a dramatic change in the morphology of neural progenitor cells during this period, which included the formation of colonies and the extension of multiple, long neurites (Figure 2A). (D and E) No antigen staining was observed in cultures stained with corresponding isotype control antibodies. (F) To confirm the spread of VZV from Varivax-inoculated differentiated SH-SY5Y cultures, the cells were trypsinized and inoculated onto HFF monolayers. After 7 days, HFF monolayers were stained with a VZV gEgI (red) antibody and DAPI counterstain (blue) to visualize plaques.
Several PCR methods that can detect and differentiate Oka-vaccine strain from wild-type strains have been described previously (18,20, 26). Nuclei were visualized with DAPI. In addition, the high-throughput nature of this model renders it ideal for testing compounds with potential antiviral functions. The current VZV vaccine strain still retains the capacity to establish latency within the DRG and subsequently reactivate (17, 20). Although infrequent, a number of cases have recently been reported involving severe complications following reactivation of the vaccine strain (7, 22, 27). However, as noted previously (13, 14, 26) the application of this method for Japanese strains and probably some other Asian regions has been limited due to the circulation of strains related to Oka that cannot be distinguished from the Oka strain by using the ORF 38 marker. Data shown are the mean ± SEM.
↵*Corresponding author. Mailing address: Department of Infectious Diseases and Immunology, University of Sydney, Blackburn Building, Room 601, Sydney 2006, New South Wales, Australia. Phone: 61 2 93516867. This mutation, which introduces a newSmaI restriction site into the Oka vaccine strain, formed the basis for the development of the diagnostic test described here. By immunocytochemistry, we observed many Brn3a+/peripherin+ cells (Figure 2D), which we quantified to account for at least 15% of each culture by flow cytometry (Figure 2E).