PRODUCTIVE VARICELLA ZOSTER VIRUS INFECTION OF CULTURED INTACT HUMAN GANGLIA ACCEPTED

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1 JVI Accepts, published online ahead of print on 4 April 2007 J. Virol. doi: /jvi Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. PRODUCTIVE VARICELLA ZOSTER VIRUS INFECTION OF CULTURED INTACT HUMAN GANGLIA 1,2 Kavitha Gowrishankar, 1 Barry Slobedman, 1 Anthony L. Cunningham, 1 Monica 1 Miranda-Saksena, 1 Ross A. Boadle, 1,2 Allison Abendroth 1 Center for Virus Research, Westmead Millennium Institute and 2 Department of Infectious Diseases and Immunology, University of Sydney, NSW, Australia Running title: VZV infection of cultured intact human ganglia Abstract word count: 100 Text word count: 1557 Corresponding author: Allison Abendroth, Dept Infectious Diseases and Immunology, University of Sydney and Centre for Virus Research, Westmead Millennium Institute P.O. Box 412, Westmead, 2145 NSW, Australia. Phone: Fax: allisona@med.usyd.edu.au

2 ABSTRACT Varicella zoster virus (VZV) is a species specific herpesvirus which infects sensory ganglia. We have developed a model of infection of human intact explant dorsal root ganglia (DRG). Following exposure of DRG to VZV, viral antigens were detected in neurons and non-neuronal cells. Enveloped virions were visualised by transmission electron microscopy in neurons, non-neuronal cells and within the extracellular space. Moreover, in contrast to remaining highly cell associated during infection of cultured cells such as fibroblasts, cell-free VZV was released from infected DRG. This model enables VZV infection of ganglionic cells to be studied in the context of intact DRG.

3 Varicella zoster virus (VZV) is the aetiological agent for varicella (chicken pox) and herpes zoster (shingles) (1, 3). During primary varicella infection, VZV accesses nerve axons to reach sensory ganglia, where it establishes latency (4, 5, 7, 15, 16). Reactivation from latency results in new infectious virus and axonal transport of VZV to the skin to cause herpes zoster. During herpes zoster neural and dermoepidermal inflammation occurs, resulting in neuropathic pain and the typical dermatomal rash (8). The complex aberrant repair processes that occur during herpes zoster can result in chronic neuropathic pain (post herpetic neuralgia; PHN), which can last for years after resolution of the rash (26). The high species specificity of VZV has complicated the development of small animal models that mimic productive infection (1, 17), although advances in tissue culture techniques enabled the development of SCID-hu mouse models utilizing grafted human tissue (17, 18, 20, 21). Grafting of neural cells has been used to examine infection of neurons and glial cells (2). In a variation of this model, intact human fetal DRG have been grafted into SCID-hu mice to show that after initial productive infection, VZV persisted in a form consistent with the establishment of latency (29). We have previously shown that single cell preparations of neurons from dissociated human DRG support virus replication and that unlike productive infection of human fibroblasts (HFs), infected neurons are resistant to apoptosis (13). We also provided evidence that the VZV ORF 63 gene product confers resistance to apoptosis during neuronal infection (12). In an extension of these studies, we sought to develop a human explant ganglia model as a means to study features of VZV interactions with ganglionic cells within the context of the intact ganglia.

4 Human fetal spinal tissue (14-20 weeks gestation) was obtained from the Human Fetal Tissue Distribution Centre (Prince of Wales Hospital, NSW, Australia) following informed consent and with approval by the University of Sydney Human Research Ethics Committee. Individual intact DRG were isolated from fetal spinal tissue and cultured on glass coverslips as previously described (24). Axons typically developed at day 2 post explant and only DRG with extensive axonal growth were used for infection (Figure 1A). Due to the highly cell associated nature of VZV in vitro (27), a cell associated inoculation method was utilised. Single DRG explants each cultured in 700 µl of neuronal culture media (DMEM with 0.5% fetal calf serum, 100ng/ml nerve growth factor, 100 U/ml penicllin/streptomycin, 2mM L-glutamine, B-27 Supplements, GIBCO, CA) were incubated with an inoculum consisting of 100 µl of media containing 1 X 10 5 VZV strain Schenke infected HFs at CPE 2+, or an equivalent number of mock infected HFs. The inoculum was layered on top of the explant, taking care not to disturb the ganglion, with a resulting total volume of 800 µl per well. DRG were collected at 0, 24, 48, 72 and 96 hours post infection (PI), fixed in 10% formalin and paraffin embedded. DRG incubated with uninfected HFs were collected in parallel at each time point. Analysis of 5µm sections stained with hematoxylin revealed neurons, and non-neuronal cells such as satellite cells, with sensory neurons being readily distinguished by their large size and centrally located nucleus (Figure 1B, C). In addition, immunohistochemical (IHC) staining was performed for ganglionic cell markers. To detect proteins in formalin fixed sections, antigens were unmasked using 0.01M citrate buffer ph 6 prior to incubation with primary antibodies to the neural cell adhesion molecule (NCAM; monoclonal mouse anti-human antibody, Chemicon Inc, CA) or to S100 proteins, which are expressed on satellite cells and some neurons (2, 9). The antibody used to detect S100 was a rabbit

5 anti-cow S100 polyclonal antibody (Dako). As described by the manufacturer, this antibody reacts strongly with human S100B, weakly with S100A1, very weakly with S100A6 and does not react with other S100 proteins such as S100A2, A3 and A4. Bound antibodies were detected using HRP and DAB. NCAM-positive neurons were readily detected, as were surrounding smaller satellite cells expressing S100 (Figure 1D, E). In both mock- and VZV infected ganglia, the number of S100-expressing cells increased over time post explant, consistent with studies of mouse and rat sensory ganglia reporting that the process of explanting ganglia can induce satellite cell proliferation (6, 28). VZV glycoproteins are not expressed at detectable levels during latency and so their detection is a useful indicator of productive infection (10). To assess the extent of infection, DRG sections were examined for VZV antigens by immunofluorescence (IFA) staining with either mouse monoclonal antibody to VZV glycoprotein I (Chemicon Inc, CA) or a human hyper immune serum that recognizes predominantly glycoproteins (kindly provided by A. Arvin, Stanford University), followed by secondary AlexaFluor-594 antibodies. In addition to IFA staining, sections were also subjected to IHC with mouse monoclonal antibody to VZV glycoprotein B. Viral glycoproteins were not detected at 24 hours PI, but by 48 hours PI, distinct individual VZV-positive neurons scattered throughout the DRG were identified (Figure 2 A-C). Viral antigen positive cells were also detected around the edge of the DRG body, likely representing infection from direct contact with the infected HF inoculum. The presence of discreet VZV positive neurons deep within ganglia at 48 hours PI indicated that infection of these cells may have occurred via transport of

6 virus through neuronal axons that reached beyond the body of the ganglia into the culture media. At 72 and 96 hours PI, infection was much more widespread, with a majority of neurons and non-neuronal cells being VZV antigen-positive, indicating that most DRG cells support viral replication (Figure 2D and data not shown). No staining was observed in mock infected DRG or VZV infected DRG stained with isotype control antibodies (Figure 2E and data not shown). Comparable results were obtained from four replicate experiments using ganglia from different fetal samples. The increase in number of infected cells over time suggested that VZV productively infected and spread within explanted DRG. We next used transmission electron microscopy (TEM) to examine DRG for the presence of VZV particles. DRG were collected at 48, 72 and 96 hours PI and processed for TEM as described previously (24). Viral capsids or virions were readily identified in cells throughout DRG cells in cell nuclei and cytoplasms, with the highest number detected at 96 hours P.I. (Figure 3). Neurons containing virions were confirmed by their characteristic morphology including microtubules in the cytoplasm and adjoining axonal processes. Infected neurons in the intact DRG did not show ultrastructural changes indicative of apoptosis, which is consistent with our previous findings that infection of neurons from dissociated DRG are resistant to apoptosis (12, 13). In addition to being detected intracellularly, enveloped virions were also detected in the extracellular space (Figure 3 C, D). VZV remains highly cell-associated and is not released during productive infection of cultured cells such as HFs and explanted skin tissue infected in vitro (25, 27). However, cell-free VZV is detectable in vesicular fluid from varicella and herpes

7 zoster skin lesions (22) as well as in infected human skin xenografts in SCID-hu mice (21). To determine whether VZV virions remained cell-associated or were released from infected DRG explants, pooled culture supernatants from 8 individually cultured, VZV- or mock infected DRG were collected at 24, 48, 72, 96, 120 hours PI. The supernatants were clarified by centrifugation at 10,000g for 10 min before being inoculated onto HF monolayers for the detection of cell free virus by plaque assay. In parallel, supernatants from infected HFs were similarly assessed for the presence of cell-free virus. Plaques were not detected from infected HF supernatants at any time point, confirming the highly cell-associated nature of VZV infection of these cells. In contrast, plaques were detected from infected DRG supernatants demonstrating release of infectious virus (Figure 4). The presence of cell-free virus peaked at 96 hours PI. Comparable results were obtained in an additional three independent replicate experiments. The decline in VZV released at 120 hours PI may have been due to a general loss of DRG cell viability due to culture conditions, as the histology of both mock and infected DRG had begun to deteriorate by this time post explant. In addition to the detection of infectious cell-free virus release the number of infected ganglionic cells was determined by infectious center assay. Infected DRG harvested at 48 and 96 hours PI were dissociated with collagenase and dispase to generate single cell suspensions, which were washed in PBS and inoculated onto HF monolayers. The number of infectious centers increased over time from approximately 8000 at 48 hours PI to approximately at 96 hours PI, confirming the spread of productive infection within DRG.

8 This study provides a model whereby the interaction of VZV with ganglionic cells which play a critical role in viral pathogenesis can be studied in the context of intact DRG, using in vitro cell culture techniques. The relative contributions of neuronal and non-neuronal cells to the observed release of cell-free virus remains an important component of future work to better understand the interaction of VZV with these cells. These results provide the first evidence of VZV productive infection and release of infectious cell-free virus from cultured human DRG. Extensive neuron to neuron spread during reactivation within ganglia in vivo has been suggested in herpes zoster (16), and we observed a similar spread of virus in experimentally infected DRG. These features of DRG infection can now be studied in further detail to better define the molecular mechanisms that underlie VZV infection of ganglionic cells. For example, this model provides a means to rapidly test viral gene mutant viruses and new candidate vaccine strains containing targeted gene disruptions to define viral genes that may play critical roles in VZV neurotropism and to examine in detail the outcome of infection of both neurons and non-neuronal cells with respect to apoptosis and cell function. Infection of cultured intact DRG also enables both anterograde and retrograde axonal VZV transport to be examined for the first time, particularly when combined with two-chamber culture plates, as we have done previously to study herpes simplex virus type 1 (HSV-1) axonal transport (11, 23, 24). In addition, it will now be possible to directly compare VZV and HSV DRG infection to better determine whether fundamental differences in the nature of the dermatomal rash in herpes zoster compared to the much more highly localized lesions observed during

9 HSV reactivation are due to differences in the spread of infection within DRG by these closely related α-herpesviruses. This work was supported by Australian National Health and Medical Research Council (NHMRC) Project Grant # and # K.G is the holder of an Australian Postgraduate Award and Westmead Millennium Foundation Research Scholarship Stipend Enhancement Award.

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14 FIGURE LEGENDS Figure 1. Culture of intact human explant DRG. (A) Intact DRG cultured on glass coverslips in the presence of nerve growth factor (100 ng/ml) showing extensive axonal growth (arrows) at 48 hr post plating. (B, C) Haematoxylin staining of paraffin embedded DRG 5 µm sections showing large, centrally nucleated neurons (arrow) surrounded by smaller support cells (arrowhead) at 40 X (B) and 100 X magnification (C). NCAM (D) and S100 (E) staining within VZV infected DRG. (F) Negative control consisting of VZV infected DRG section incubated with isotype control migg2a showing no specific staining. Figure 2. VZV antigen expression in infected explant human DRG. (A) Immunofluorescent staining of DRG 48 hours PI with human VZV hyper immune serum and secondary antibody consisting of fluorescently conjugated anti-human AlexaFluor 594 (red staining). VZV antigen expression on the surfaces of distinct, scattered neurons (white arrows) and around the DRG body (green arrows). Boxed inset shows magnified image of VZV antigen positive neurons. (B) Immunofluorescent staining of DRG at 48 hours PI with mouse anti-vzv gi monoclonal antibody and secondary antibody consisting of fluorescently conjugated anti-mouse AlexaFluor 594 (red staining). Nuclear blue DAPI staining is indicated. (C-E) Immunohistochemical detection of VZV infected DRG stained with mouse anti-vzv gb monoclonal antibody at 48 hours PI (C) and 72 hours PI (D) (brown staining) or mock infected DRG (E). Black arrows indicate infected neurons. Sections were counterstained with haematoxylin (blue staining).

15 Figure 3. Transmission electron micrographs of VZV infected human explant DRG. (A) Detection of numerous VZV capsids (arrow) in a DRG cell nucleus at 96 hours PI. (B) Viral particles present within the nucleus (arrow) and cytoplasm (arrow head) are indicated. (C) Presence of virions in the cytoplasm (arrow head) and in the extracellular space between cells (black arrow). Microtubular structures (white arrow) indicative of a neuronal cell. (D) Magnified image of inset from (C), showing a fully assembled extracellular, enveloped virion (arrow). Figure 4. Detection by plaque assay of cell-free infectious VZV released from cultured human DRG. Number of plaques formed on HF monolayers after the addition of pooled culture supernatants from 8 individually infected DRG or infected HF inoculum-alone samples, over a time course of infection. Plaque assay was performed in triplicate and the mean number of plaques (± standard error of mean) from a representative experiment out of four independent experiments is shown. No cell free virus was detected from the infected HF inoculum control.

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19 Number of plaques Time post infection (hours) Infected DRG Infected HFs