key: cord-0000575-egy1d90x authors: Shindler, Kenneth S.; Chatterjee, Dhriti; Biswas, Kaushiki; Goyal, Ashish; Dutt, Mahasweta; Nassrallah, Mayssa; Khan, Reas S.; Sarma, Jayasri Das title: Macrophage-Mediated Optic Neuritis Induced by Retrograde Axonal Transport of Spike Gene Recombinant Mouse Hepatitis Virus date: 2011-06-01 journal: Journal of Neuropathology & Experimental Neurology DOI: 10.1097/nen.0b013e31821da499 sha: 6a39a657c4fddc7bfb9943d9df7e7c994ada4c88 doc_id: 575 cord_uid: egy1d90x Following intracranial inoculation, neurovirulent mouse hepatitis virus (MHV) strains induce acute inflammation, demyelination and axonal loss in the CNS. Prior studies using recombinant MHV strains that differ only in the spike gene, which encodes a glycoprotein involved in virus-host cell attachment, demonstrated that spike mediates anterograde axonal transport of virus to the spinal cord. A demyelinating MHV strain induces optic neuritis, but whether this is due to retrograde axonal transport of viral particles to the retina, or if it is due to traumatic disruption of retinal ganglion cell axons during intracranial inoculation is not known. Using recombinant isogenic MHV strains, we examined the ability of recombinant MHV to induce optic neuritis by retrograde spread from the brain through the optic nerve into the eye following intracranial inoculation. Recombinant demyelinating MHV induced macrophage infiltration of optic nerves, demyelination and axonal loss whereas optic neuritis and axonal injury were minimal in mice infected with the non-demyelinating MHV strain that differs in the spike gene. Thus, optic neuritis was dependent on a spike glycoprotein-mediated mechanism of viral antigen transport along retinal ganglion cell axons. These data indicate that MHV spreads by retrograde axonal transport to the eye and that targeting spike protein interactions with axonal transport machinery is a potential therapeutic strategy for CNS viral infections and associated diseases. Neurotropic mouse hepatitis virus (MHV) infection in mice causes meningoencephalitis, myelitis, and demyelination, with relative axonal preservation. Recent studies have additionally demonstrated that neurotropic MHV strains can also induce axonal loss (1, 2) ; direct virus-mediated axonal damage can occur concurrently with and independently of demyelination (2) . Thus, neurovirulent MHV strains provide useful tools for studying the neuroinflammation, demyelination, and axonal loss and as a virus-induced model of multiple sclerosis. Recombinant MHV strains RSA59 (demyelinating strain; DM) and RSMHV2 (nondemyelinating strain; NDM) are isogenic except for the spike gene, which encodes the host attachment spike glycoprotein. Studies of these strains have elucidated mechanisms of axonal loss and demyelination (3) . RSA59 and RSMHV2 both cause hepatitis, encephalitis, and meningitis after intracranial inoculation. However, they differ in their ability to induce macrophage infiltration and subsequent demyelination and axonal loss in spinal cord (2) . There is a lack of viral antigen spread and subsequent inflammation extending into spinal cord white matter after intracranial infection with the NDM strain, whereas there is extensive macrophage-mediated white matter pathology secondary to DM strain infection. Thus, the spike protein plays a critical role in anterograde axonal transport of viral particles, an important mechanism mediating axonal damage and demyelination (2, 4) . Because both strains cause encephalitis after transcranial inoculation, the differences in spike protein between DM and NDM strains do not impair viral entry; however, differential neural cell tropism may contribute to the mechanism of demyelination (2, 4, 5) . Infection of brain neurons and oligodendrocytes occurs on inoculation with either strain, whereas in the spinal cord, oligodendrocyte infection is only seen with the DM strain. This is likely due to the route by which the virus gains access to white matter, that is, spinal cord infection does not occur as a result of direct trauma, whereas transcranial inoculation results in traumatic disruption of the brain gray-white matter interface. Viral particles that would require anterograde axonal transport from infected neurons to reach myelin are able to gain direct access to the myelin sheath and spread proximally to oligodendrocyte cytoplasm. Anterograde axonal transport and spread of virus from neurons to oligodendrocytes have been documented, but retrograde axonal spread of virus from nerve ending to neural cell body also needs to be considered. Earlier studies suggested that MHV strains may spread via retrograde axonal transport (6, 7) , but the molecular mechanisms mediating such transport are not well defined. In the optic nerve, the parental demyelinating strain MHV-A59 causes inflammation, demyelination, and axonal loss (ie, optic neuritis), in contrast to the nondemyelinating MHV-2 strain (8) . Whether MHVinduced optic neuritis is dependent on retrograde axonal transport of viral particles or is due to local traumatic disruption of the intracranial portion of retinal ganglion cell (RGC) axons during inoculation is not known. Moreover, the immune response in MHV recombinant strain optic neuritis has not been well characterized. Here, we compared the incidence and phenotype of optic neuritis after inoculation with RSA59 and RSMHV2 and assessed the ability of spike protein to facilitate retrograde axonal transport and induce optic neuritis. Recombinant isogenic DM strain of MHV (RSA59) and NDM strain (RSMHV2) have been described in previous studies (4, 9) . RSA59 and RSMHV2 strains of MHV are isogenic except for the spike gene, which encodes an envelope glycoprotein that mediates many biological properties of MHV including viral attachment to host cells and virus-cell and cell-cell fusion (10) . These recombinant strains also express enhanced green fluorescence protein (EGFP) (4, 9) . MHV-free, C57BL/6 (B6) mice (Jackson Laboratory, Bar Harbor, ME) were inoculated intracranially at 4 weeks of age with 50% LD 50 dose of RSA59 strain (20,000 plaque forming units) or RSMHV2 (100 plaque forming units), as described previously (4) . Mice were monitored daily for signs of disease. Mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution. Animals were killed (3Y5 mice per group) at day 3, days 5 to 7, and day 30 post inoculation (pi). All experimental procedures adhered to guidelines of, and were approved by, the Institutional Animal Care and Use Committee. Mice were killed at days 5 to 7 pi (peak of inflammation) or at day 30 pi (during chronic demyelination) and were perfused transcardially with phosphate-buffered saline followed by phosphate-buffered saline containing 4% paraformaldehyde. Brain, spinal cord, eyes, and optic nerve tissues were collected, postfixed in 4% paraformaldehyde overnight, and embedded in paraffin; sections were then stained with hematoxylin and eosin (H&E) to evaluate inflammation and Luxol fast blue to evaluate demyelination. Experiments were repeated at least 3 times with 3 to 5 mice. Areas of demyelination and inflammation were quantified as previously described (8, 11) . All slides were coded and read in a blinded fashion. To confirm expected virulence of the strains used, livers from the infected mice were embedded in paraffin, sectioned at 5 Km, and stained with H&E (5, 12) . The degree of optic nerve inflammation was scored by 2 blinded investigators on a 0-(no inflammation) to 4-point (severe inflammation) scale, as described (8, 11) , during the time of peak inflammation (days 5Y7 pi). Any amount of inflammation (score of 1Y4) was considered positive for optic neuritis. Serial sections from optic nerves were stained by the avidin-biotin-immunoperoxidase technique (Vector Laboratories, Burlingame, CA) using 3, 3 ¶ diaminobenzidine as substrate, and antibodies against lymphocytic cell markers, antiviral nucleocapsid antiserum, or the axonal marker antineurofilament antiserum as primary immunoglobulin G antibodies. The sources and dilution of primary antibodies are listed in the Table. Control slides from mock-infected mice were incubated in parallel. Fixed sections of optic nerve, brain, and eyes from mice at 3 or 6 days pi were stained by the previously described immunohistochemical methods using antiviral nucleocapsid antiserum. To visualize viral antigen by EGFP expression directly, frozen sections from infected mice were examined by fluorescence microscopy. Viral antigen was also assayed by Western blotting using antiYhepatitis virus nonstructural protein 9 monoclonal antibody (Rockland, Inc., Gilbertsville, PA), according to the manufacturer's instructions. Briefly, protein was extracted from the optic nerve and retina using RIPA buffer (Sigma, St. Louis, MO) in the presence of a complete protease inhibitor cocktail (Pierce, Rockford, IL) at 4-C. Protein concentration was determined with a Micro BCA Protein Assay Kit (Pierce). Protein (30 Kg) was electrophoresed on 4% to 15% SDS-PAGE, transferred to cellulose membrane, blocked, and probed with 1:1000 dilution of primary antibody. Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined as a loading control using anti-GAPDH (Sigma) diluted 1:10,000. After incubation with horseradish peroxidaseYconjugated secondary antibodies, signals were developed with enhanced chemiluminescence agent (GE Healthcare, Buckinghamshire, UK), and intensity was determined using the NIH Image J program. Longitudinal optic nerve sections were stained with antineurofilament antibody, and areas of axonal staining were quantified as described (11) . Briefly, 3 photographs were taken at 40Â magnification of each stained nerve at predefined locations (one each of the proximal, central, and distal portion of the nerve) covering a total area of 38,500 Km 2 of each nerve. The amount of tissue within this area that stained positively for neurofilament was calculated using ImagePro Plus 6.0 (Media Cybernetics, Silver Spring, MD) software. Data shown represent the cumulative area of positive staining/nerve. Comparisons of optic neuritis incidence, demyelination, and axonal density were analyzed by one-way analysis of variance followed by Tukey multiple comparison test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Data represent mean (SD) percentage of eyes that developed optic nerve inflammation or demyelination or the mean (SD) density of axonal staining. As in prior studies, RSA59-infected mice showed meningitis, encephalitis, myelitis, and concurrent axonal loss and demyelination as early as day 5 pi with an increase at day 30, whereas RSMHV2 showed only meningitis, encephalitis, and myelitis with no significant demyelination or axonal loss (data not shown) (2) . The livers of both strains showed moderate to severe hepatitis (data not shown), thereby confirming virulence. As expected, mice infected with parental demyelinating strain MHV-A59 at days 5 to 7 pi had optic nerve inflammation ( Fig. 1C) , whereas optic nerves from mice infected with parental nondemyelinating strain MHV-2 did not (Fig. 1B) ; their optic nerves appeared similar to those of mock-infected mice (Fig. 1A) (8) . The inability of MHV-2 to induce optic neuritis was expected because MHV-2 does not infect the brain parenchyma and is unable to induce encephalitis (13, 14) . By contrast, the RSMHV-2 strain induces encephalitis (2, 4, 5) . RSA59 induced optic neuritis similar to the parental MHV-A59 strain at days 5 to 7 pi (Fig. 1F) , whereas most eyes of RSMHV2-infected mice exhibited no optic nerve inflamma-tion (Fig. 1D) ; however, there was mild inflammation in a few optic nerves (Fig. 1E) . MHV-A59 induced optic neuritis in a mean of 55.6% of optic nerves in 4 experiments, whereas MHV-2 induced optic neuritis in only 3.7% of optic nerves (Fig. 1G) ; this result is similar to the incidence in prior studies (8) . RSA59infected mice developed optic neuritis with a high incidence (68.5%) similar to that in MHV-A59-infected mice, and significantly higher than the incidence in RSMHV-2 infected mice (25.1%) (Fig. 1H) . Serial sections from RSA59-and RSMHV2-infected mice were stained with anti-CD45 (leukocyte common antigen; LCA), antiYIba-1 (microglia/macrophage marker), anti-CD3 (T-cell marker), or anti-CD19 (B-cell marker) (Table) . LCA staining confirmed the presence of infiltrating inflammatory cells in optic nerves from RSA59-infected mice, whereas few LCA-positive cells were found in those of RSMHV2-infected mice (not shown). Among the LCA-positive inflammatory cells, the majority in the RSA59-infected mice were Iba-1+ microglia/macrophages (Fig. 2C) . The optic nerves of some control mice (Fig. 2) and RSMHV2-infected mice (Fig. 2B ) also had scattered Iba1+ cells that likely represent resident microglia, but there were far fewer than in the RSA59-infected samples. This result was confirmed by 2 blinded investigators. Significantly more optic nerves from RSA59-infected mice had increased Iba1+ cells versus nerves from RSMHV2-infected mice (14/23 total nerves from 4 experiments versus 4/20, respectively; p = 0.0124 by Fisher exact test). Few or no CD3-positive T cells were present in optic nerves of RSA59-infected mice (Fig. 2D ) and no CD19stained B cells were observed (Fig. 2E ). Spleen sections from mock-infected mice stained with either anti-CD3 (Fig. 2F ) or anti-CD19 antibodies (not shown) served as positive controls. Thus, there was an increase of Iba-1Ypositive cells and few CD3-positive cells in optic nerves of RSA59-infected mice. Optic nerves from RSA59-infected mice had areas of demyelination detected by Luxol fast blue staining both at day 7 and day 30 pi (Fig. 3) , whereas no demyelinating plaque was observed in day 7 pi RSMHV2 mouse optic nerve and little or no demyelination was observed at day 30 pi. These data are consistent with the previous result obtained from parental demyelinating strain MHV-A59 and nondemyelinating strain MHV-2 (8). No axonal loss was identified at 5 to 7 days pi in optic nerves of RSA59-or RSMHV2-infected mice (Figs. 4AYC). At day 30 pi, RSMHV2-infected mice continued to show no axonal loss (Fig. 4D) , whereas optic nerves from RSA59infected mice showed regions of mildly reduced axonal staining, with focal areas of axon loss intermixed with areas of normal axons (Figs. 4E, F) . Quantification of the area of axonal staining across all sections of optic nerves of RSA59infected mice demonstrated a small but significant decrease compared with optic nerves of either RSMHV2-infected or mock-infected control mice (p = 0.0306) (Fig. 4G ). The optic nerve inflammation, demyelination, and axonal loss observed after RSA59 infection, but not after RSMHV2 infection, demonstrate that the MHV-A59 spike protein is required for the induction of optic neuritis. This suggests that the spike protein may mediate retrograde axonal transport of the virus, allowing it to travel from brain regions containing RGC axonal projections along the optic nerve. Viral antigen was consistently detected within thalamic neurons in RSA59-infected mouse brains (Figs. 5C, D) , as well as in some RSMHV2-infected brains (Fig. 5B) . RSA59 viral antigen was also detected in the superior colliculi in some animals (data not shown). Light diffuse staining detected in optic nerves from RSA59-infected mice (Fig. 5G ), but not RSMHV2-infected mice (Fig. 5F ), suggests that only the RSA59 viral antigen is transported to the optic nerves. To further investigate this, the optic nerves were isolated 3 or 6 days pi and frozen sections were examined for the presence of EGFP. Viral antigenY positive EGFP signal was observed in optic nerves from RSA59-infected mice (Figs. 5I, J) but not RSMHV2-infected mice (Fig. 5H) . The punctate EGFP signal in optic nerve sections observed by fluorescent microscopy in RSA59-infected mice (Fig. 5J ) further suggests axonal transport through the optic nerve and was seen as early as 3 days pi. To determine whether viral antigen is transported all the way to the RGC cell bodies, whole eyes were isolated and sectioned. Viral antigen immunostaining of retinal sections demonstrated no viral antigen in cells of the RGC layer of RSMHV2-infected mice (Fig. 6A) , whereas RSA59 viral antigen was detected in some cells within the RGC layer (Figs. 6B, C) at day 6 pi. Overall, viral antigen staining was detected in 60% of eyes from RSA59-infected mice, with 15 serial cross sections examined from each of 5 eyes. The timing of viral antigen spread and relative levels of antigen in retina and optic nerve were determined in protein extracts isolated at days 3 and 6 pi. Western blot analysis demonstrated presence of viral antigen in retinal tissue from RSA59infected mice at day 6 pi but not at day 3 and no viral antigen was detected in RSMHV2-infected mice (Fig. 6D ). Viral antigen was detected in protein extracts from optic nerves of RSA59-infected mice earlier than it was detected in retina. Antigen was detected on Western blots at both days 3 and 6 pi (Fig. 6E) . These results suggest that the viral antigen was able to enter the RGC axons and was transported retrograde to the cell bodies within the eye. RSA59 induces optic neuritis at comparable levels of severity and incidence as that seen in mice infected with its parental strain MHV-A59 (8), whereas RSMHV2 has a limited ability to induce optic nerve inflammation. Accordingly, nerves from RSA59-infected mice at the chronic stage showed significantly decreased axonal density compared with nerves from RSMHV2-infected mice. This differential ability of MHV strains to induce optic neuritis and axonal injury is dependent on spike glycoprotein mediated retrograde transport of viral antigen along RGC axons. Prior studies demonstrated that RSA59 and RSMHV2 both can cause meningitis and encephalitis, but RSMHV2 did not induce subsequent demyelination and axonal loss in spinal cord. Our current results demonstrate that in addition to demyelination and axonal loss, RSMHV2 also has limited ability to cause significant optic nerve inflammation and RGC infection. However, RSMHV2 did induce some optic nerve inflammation, unlike the parental strain MHV2. This difference may be due to the ability of RSMHV2 to enter neurons in the brain parenchyma and replicate, as demonstrated previously (2, 4, 5) and shown again here, leading to a higher viral load that may allow some diffusion of the virus at an undetectable level. Alternatively, the higher viral load might trigger a more diffuse central nervous system (CNS) inflammatory response associated with mild, nonspecific inflammation in some optic nerves. MHV2, on the other hand, does not enter the brain parenchyma, and infection with it results in meningitis without inducing encephalitis (3). Prior studies demonstrated that one mechanism limiting the ability of the NDM strain to induce demyelination and axonal loss in the spinal cord involves impaired interneuronal spread of viral particles and defective translocation of viral antigen from gray matter to white matter (2) . Evaluation of axonal loss and demyelination in the spinal cord demonstrated that DM MHV infection begins in the neuronal cell body, propagates to the axon, and subsequently induces axonal degeneration and demyelination. The propagation of viral antigen from gray to white matter is dependent on anterograde axonal transport of virus particle mediated by the spike protein (2, 4) . Spike proteinYmediated axonal transport as an underlying mechanism is further supported by the current studies and demonstrates that it also plays a role in mediating retrograde axonal transport. It is especially intriguing that MHV uses similar molecular mechanisms to interact with cellular axonal transport machinery, suggesting that targeted disruption of this protein function might prevent all pathogenic spread of the virus. Although it has been reported previously that neurotropic MHV strains can spread within neurons in a retrograde direction, the molecular interactions mediating this spread have not been fully examined (6, 7) . In cultured neurons, viral interaction with the microtubule network in neuronal processes has been shown to be important; based on cross-interaction of antimicrotubule antibodies and the nucleocapsid protein (N), it is possible that such interactions might be involved in axonal transport mechanisms (15) . However, in view of the fact that the DM and NDM strains used in the present study differ only in spike protein and contain identical N proteins, our data suggest more of a role for the spike protein. How the DM strain of MHV initially infects neurons requires further study. One possible explanation could be that traumatic disruption to the nerve endings in the brain allows access of the virus through the damaged axolemma; or the virus may be capable of directly infecting intact axons either in the brain, or after diffusing into the optic nerve in the cerebrospinal fluid. Alternatively, virus may be engulfed into the axons at synaptic terminals. Although direct infection after diffusion in cerebrospinal fluid could potentially explain the presence of viral antigen in optic nerves, the fact that antigen is detected in RGC bodies in the retina demonstrates that retrograde transport does occur. Although the presence of viral antigen in RGCs alone cannot exclude hematogenous spread as an alternate mechanism from retrograde transport, the timing of its spread (ie, detected only at day 6 pi in the retina versus day 3 pi within optic nerves and within liver via hematogenous spread), the lack of hematogenous spread to the CNS after systemic infection with equivalent doses of MHV-A59 (6) , and the presence of viral antigen in areas of the brain containing RGC projections all suggest that retrograde transport is the more likely mechanism of viral spread. Retrograde transport may follow fusion of the spike protein with axonal membranes followed by loss of most of the viral structural proteins and then binding of the nucleocapsid via one or more viral proteins to the retrograde molecular motors. Such a mechanism has been seen in other neurovirulent viruses that follow a retrograde direction of axonal transport (16, 17) . Although it is not common that a single virus can use both directional transport mechanisms, this is not the first virus to demonstrate such properties. For example, herpesviruses can be transported rapidly along microtubules in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterograde in the opposite direction (18, 19) . Axonal transport is an important strategy used by several neurovirulent viruses, including herpes simplex, rabies, polio, influenza, and Borna disease viruses, which can be transported in axons either in anterograde or in retrograde direction (20Y22). In some instances, viruses can be transported within axons for a long distance, yet this journey occurs within the cell. Therefore, the virus cannot be inactivated by neutralizing antibody during its transit and may spread in the CNS without inducing an antivirus immune response while it is within the cell. When viruses can spread only within axons and/or via direct cell-tocell contact, they could potentially escape the attack of antiviral drugs or neutralizing antibodies. In the current studies, optic nerve inflammation induced by DM strain RSA59 consisted of predominantly macrophages/microglia, similar to immune responses in spinal cord (2) and without the marked T-cell infiltration seen in other demyelinating disease models (23Y26). Iba-1 staining was diffuse and was observed in cell bodies as well as cell processes, as has been seen previously with this macrophage/microglia marker (27, 28) . Although Iba-1 cannot distinguish between macrophages and microglia, we suspect most labeled cells represent infiltrating macrophages based on the overall increase in the number of cells observed. Macrophages have been shown to play both proinflammatory and anti-inflammatory roles in optic nerves (29) , and we suggest that the DM strain viral antigen might be moving within axons to avoid being cleared by infiltrating macrophages. It is known that some viruses make use of the microtubules and/or the actin cytoskeleton for axonal transport (30Y32). Our studies have shown that a major mechanism of both retrograde and anterograde axonal transport of neurovirulent MHV is mediated by spike protein and further experiments will focus on identifying the molecular mechanisms by which the DM virus interacts with the axonal transport system and whether specific interventions targeting the transport system can delay or prevent the DM strain-induced axonal loss and demyelination. Analyzing the underlying principles of MHV axonal transport will be helpful in the design of viral vectors to be used in research, in human gene therapy, and in the identification of new antiviral therapies. Such therapies may also have the potential to prevent CNS demyelinating diseases that might be triggered initially by viral infection. Axonal damage is T cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus Mechanisms of primary axonal damage in a viral model of multiple sclerosis A mechanism of virus-induced demyelination Demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose and route of inoculation Effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain Experimental optic neuritis induced by a demyelinating strain of mouse hepatitis virus Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system Coronavirus spike proteins in viral entry and pathogenesis Inflammatory demyelination induces axonal injury and retinal ganglion cell apoptosis in experimental optic neuritis Murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis Sequence analysis of the S gene of recombinant MHV-2/A59 coronaviruses reveals three candidate mutations associated with demyelination and hepatitis Mouse hepatitis virus type-2 infection in mice: An experimental model system of acute meningitis and hepatitis Distribution and trafficking of JHM coronavirus structural proteins and virions in primary neurons and the OBL-21 neuronal cell line Double-labeled rabies virus: Live tracking of enveloped virus transport Herpes simplex virus type 1 glycoprotein E mediates retrograde spread from epithelial cells to neurites Anterograde spread of herpes simplex virus type 1 requires glycoprotein E and glycoprotein I but not Us9 Herpesviruses use bidirectional fastaxonal transport to spread in sensory neurons The cycle of human herpes simplex virus infection: Virus transport and immune control Limited trafficking of a neurotropic virus through inefficient retrograde axonal transport and the type I interferon response Inside-out versus outside-in models for virus induced demyelination: Axonal damage triggering demyelination Experimental autoimmune encephalomyelitis (EAE) mediated by T cell lines: Process of selection of lines and characterization of the cells A pathogenic role for myelinspecific CD8(+) T cells in a model for multiple sclerosis Viral infection triggers central nervous system autoimmunity via activation of CD8 + T cells expressing dual TCRs Theiler's virus infection induces a predominant pathogenic CD4 + T cell response to RNA polymerase in susceptible SJL/J mice Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain Neurofibromatosis-1 heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth Myelin-phagocytosing macrophages in isolated sciatic and optic nerves reveal a unique reactive phenotype Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport Rapid actin-dependent viral motility in live cells Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera The authors thank the Du Pre Foundation for support of AG.