One proline deletion in the fusion peptide of neurotropic mouse hepatitis virus (MHV) restricts retrograde axonal transport and neurodegeneration.

Mouse hepatitis virus (MHV; murine coronavirus) causes meningoencephalitis, myelitis, and optic neuritis followed by axonal loss and demyelination. This murine virus is used as a common model to study acute and chronic virus-induced demyelination in the central nervous system. Studies with recombinant MHV strains that differ in the gene encoding the spike protein have demonstrated that the spike has a role in MHV pathogenesis and retrograde axonal transport. Fusion peptides (FPs) in the spike protein play a key role in MHV pathogenesis. In a previous study of the effect of deleting a single proline residue in the FP of a demyelinating MHV strain, we found that two central, consecutive prolines are important for cell-cell fusion and pathogenesis. The dihedral fluctuation of the FP was shown to be repressed whenever two consecutive prolines were present, in contrast to the presence of a single proline in the chain. Using this proline-deleted MHV strain, here we investigated whether intracranial injection of this strain can induce optic neuritis by retrograde axonal transport from the brain to the retina through the optic nerve. We observed that the proline-deleted recombinant MHV strain is restricted to the optic nerve, is unable to translocate to the retina, and causes only minimal demyelination and no neuronal death. We conclude that an intact proline dyad in the FP of the recombinant demyelinating MHV strain plays a crucial role in translocation of the virus through axons and subsequent neurodegeneration.

ZIKA virus infection causes persistent chorioretinal lesions.

Zika-infected patients can have eye involvement ranging from mild conjunctivitis to severe chorioretinal lesions, however the possible long-term sequelae of infection and timeline to recovery remain unknown. Here we describe the partial recovery of chorioretinal lesions in an immunocompetent patient diagnosed with bilateral posterior uveitis associated with Zika infection and show that some lesions resolved with focal atrophy evident as pigmentary changes on funduscopy. To better understand the progression of the lesions and correlate the changes in fundus imaging with local viral load, immune responses, and retinal damage, we developed a symptomatic mouse model of ocular Zika virus infection. Imaging of the fundus revealed multiple hypopigmentary patches indicative of chorioretinal degeneration as well as thinning of the retina that mirror the lesions in patients. Microscopically, the virus primarily infected the optic nerve, retinal ganglion cells, and inner nuclear layer cells, showing thinning of the outer plexiform layer. During acute infection, the eyes showed retinal layer disorganization, retinitis, vitritis, and focal choroiditis, with mild cellular infiltration and increased expression of tumor necrosis factor, interferon-γ, granzyme B, and perforin. Focal areas of gliosis and retinal degeneration persisted 60 dpi. The model recapitulates features of ZIKA infections in patients and should help elucidate the mechanisms underlying the damage to the eyes and aid in the development of effective therapeutics.

Long-term consequences of topical dexamethasone treatment during acute corneal HSV-1 infection on the immune system.

Herpes simplex virus type 1 (HSV-1) is a leading cause of neurotrophic keratitis (NTK). NTK is characterized by decreased corneal sensation from damage to the corneal sensory fibers. We have reported on the regression of corneal nerves and their function during acute HSV-1 infection. That nerve loss is followed by an aberrant process of nerve regeneration during the latent phase of infection that lacks functional recovery. We recently showed the elicited immune response in the infected cornea, and not viral replication itself, is part of the mechanism responsible for the nerve degeneration process after infection. Specifically, we showed infected corneas topically treated with dexamethasone (DEX) significantly retained both structure and sensitivity of the corneal nerve network in comparison to mice treated with control eye drops, consistent with decreased levels of proinflammatory cytokines and reduced influx of macrophages and CD8+ T cells into the cornea. This study was undertaken to analyze the long-term effect of such a localized, immunosuppressive paradigm (DEX drops on the cornea surface during the first 8 d of HSV-1 infection) on the immune system and on corneal pathology. We found the profound immunosuppressive effect of DEX on lymphoid tissue was sustained in surviving mice for up to 30 d postinfection (p.i.). DEX treatment had prolonged effects, preserving corneal innervation and its function and blunting neovascularization, as analyzed at 30 d p.i. Our data support previously reported observations of an association between the persistent presence of inflammatory components in the latently infected cornea and structural and functional nerve defects in NTK.

Evaluation of lateral spread of transgene expression following subretinal AAV-mediated gene delivery in dogs.

Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV) vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP) using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F) capsid mutant), self-complementary (sc) AAV2/8 (single Y-F capsid mutant) and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire "lateral spread" of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain with these AAV serotypes.

Neuromyelitis optica associated with subacute human T-lymphotropic virus type 1 infection.

Although human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy, or tropical spastic paraparesis (HAM/TSP), is usually considered as a progressive myelopathy, a subacute variant has been described. It is unusual for optic neuritis (ON) to be associated with an HTLV-1 infection. Neuromyelitis optica (NMO) is characterised by severe attacks of acute transverse myelitis and ON of unknown aetiology. We report a 61-year-old Afro-Caribbean male patient with subacute HAM/TSP associated with bilateral ON that occurred 5years previously. To our knowledge this is the first report of recurrent NMO syndrome associated with HTLV-1 infection.

Anterograde spread of herpes simplex virus type 1 requires glycoprotein E and glycoprotein I but not Us9.

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.

Sequential leukemic infiltration and human herpesvirus optic neuropathy in acute lymphoblastic leukemia.

Leukemic infiltration is a common cause of optic disk swelling in a patient with acute lymphoblastic leukemia (ALL). Recurrence of optic disk swelling in a patient with previous leukemic infiltration carries a grave prognosis when it is associated with recurrent central nervous system disease. We report a case of recurrent swelling of an optic disk in a patient with T-cell ALL who had previously been treated for CNS relapse with optic nerve involvement. In this case the swelling was associated with cytomegalovirus infection and resolved following treatment with antiviral therapy.

Safety in nonhuman primates of ocular AAV2-RPE65, a candidate treatment for blindness in Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV-RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2.RPE65 in RPE65-mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vectorrelated toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2.RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2.RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina.

Herpes simplex virus type 1 glycoprotein e is required for axonal localization of capsid, tegument, and membrane glycoproteins.

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promotes cell-to-cell spread at basolateral surfaces of epithelial cells, but its activity in neurons is less clear. We used the mouse retina infection model and neuronal cell cultures to define the spread phenotype of gE mutant viruses. Wild-type (WT) and gE-null (NS-gEnull) viruses both infected retina ganglion cell neurons; however, NS-gEnull viral antigens failed to reach the optic nerve, which indicates a defect in axonal localization. We evaluated two Fc receptor-negative gE mutant viruses containing four amino acid inserts in the gE ectodomain. One mutant virus failed to spread from the retina into the optic nerve, while the other spread normally. Therefore, the gE ectodomain is involved in axonal localization, and the Fc receptor and neuronal spread are mediated by overlapping but distinct gE domains. In the retina infection model, virus can travel to the brain via the optic nerve from presynaptic to postsynaptic neurons (anterograde direction) or via nerves that innervate the iris and ciliary body from postsynaptic to presynaptic neurons (retrograde direction). WT virus infected the brain by anterograde and retrograde routes, whereas NS-gEnull virus failed to travel by either pathway. The site of the defect in retrograde spread remains to be determined; however, infection of rat superior cervical ganglia neurons in vitro indicates that gE is required to target virion components to the axon initial segment. The requirement for gE in axonal targeting and retrograde spread highlights intriguing similarities and differences between HSV-1 and pseudorabies virus gE.

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo.

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.

SOD2 gene transfer protects against optic neuropathy induced by deficiency of complex I.

Mutations in genes encoding the NADH ubiquinone oxidoreductase, complex I of the respiratory chain, cause a diverse group of diseases. They include Leber hereditary optic neuropathy, Leigh syndrome, and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. There is no effective treatment for these or any other mitochondrial disorder. Using a unique animal model of severe complex I deficiency induced by ribozymes targeted against a critical complex I subunit gene (NDUFA1), we attempted rescue of the optic nerve degeneration associated with Leber hereditary optic neuropathy. We used adenoassociated virus to deliver the human gene for SOD2 to the visual system of disease-induced mice. Relative to mock infection, SOD2 reduced apoptosis of retinal ganglion cells and degeneration of optic nerve fibers, the hallmarks of this disease. Rescue of this animal model supports a critical role for oxidative injury in disorders with complex I deficiency and shows that a respiratory deficit may be effectively treated in mammals, thus offering hope to patients.

Axonal transport and sorting of herpes simplex virus components in a mature mouse visual system.

The time course for delivery and transport of two major proteins of herpes simplex virus (HSV) has been determined for mature mouse retinal ganglion cell axons in vivo. Twenty-four hours after intravitreal injection of HSV, valacyclovir was introduced into the drinking water of the mice to inhibit subsequent viral replication. Without treatment, viral spread and replication in periaxonal glial cells confound study of axonal transport. At 2 to 5 days after infection, the animals were sacrificed and contiguous segments of the optic pathway were removed. Immunofluorescence microscopy indicated that the number of infected astrocytes was reduced in the proximal optic nerve and eliminated in the optic tract. Western blots of the retina with antibodies for envelope and capsid components, glycoprotein D (gD) and VP5, respectively, revealed that both components were expressed in retinal homogenates by 2 days. Results of reverse transcription-PCR indicated that there was no gD mRNA present in the treated optic tract 5 days after infection. Therefore, we conclude that gD is transcribed from viral mRNA in the retinal ganglion cell bodies. The gD accumulated in the proximal ganglion cell axon by 2 days and reached the most distal segment after 3 days. The VP5 first appeared in the proximal axons at 4 days, about 48 h after the appearance of gD. Thus, gD entered the axon earlier and independent of VP5. These finding confirm the subassembly model of viral transport in neurons and suggest that there is a 4- to 5-day window for initiation of effective antiviral treatment with valacyclovir.

In vivo egress of an alphaherpesvirus from axons.

Many alphaherpesviruses establish a latent infection in the peripheral nervous systems of their hosts. This life cycle requires the virus to move long distances in axons toward the neuron's cell body during infection and away from the cell body during reactivation. While the events underlying entry of the virion into neurons during infection are understood in principle, no such consensus exists regarding viral egress from neurons after reactivation. In this study, we challenged two different models of viral egress from neurons by using pseudorabies virus (PRV) infection of the rat retina: does PRV egress solely from axon terminals, or can the virus egress from axon shafts as well as axon terminals? We took advantage of PRV gD mutants that are not infectious as extracellular particles but are capable of spreading by cell-cell contact. We observed that both wild-type virus and a PRV gD null mutant are capable of spreading from axons to closely apposed nonneuronal cells within the rat optic nerve after intravitreal infection. However, infection does not spread from these infected nonneuronal cells. We suggest that viral egress can occur sporadically along the length of infected axons and is not confined solely to axon terminals. Moreover, it is likely that extracellular particles are not involved in nonneuronal cell infections. Taking these together with previous data, we suggest a model of viral egress from neurons that unifies previous apparently contradictory data.

Optic chiasm, optic nerve, and retinal involvement secondary to varicella-zoster virus.

Immunocompromised patients are known to be at risk for varicella-zoster virus reactivation, often in atypical manners. We describe a 30-year-old man with simultaneous involvement of the retina, optic chiasm, and optic nerve with varicella-zoster virus who had a bitemporal visual field defect.

Reporter expression persists 1 year after adeno-associated virus-mediated gene transfer to the optic nerve.

OBJECTIVE: To determine the foci and duration of protein expression following virus-mediated gene transfer to the optic nerve. METHODS: A cytomegalovirus (CMV) promoter was linked to a lacZ-SV40 polyA reporter gene or a humanized green fluorescent protein (hgfp) reporter gene, then inserted into a bacterial plasmid containing adeno-associated virus (AAV) terminal repeat sequences. The CMV-lacZ or the CMV-hgfp construct were injected into the vitreous cavity of strain-13 guinea pigs. Controls consisted of eyes injected with AAV without the promoter and reporter elements or eyes that received no injections. The eyes and optic nerves were processed for beta-galactosidase immunohistochemistry and hgfp fluorescence analyses. Cellular transduction at the messenger RNA (mRNA) level was evaluated by in situ reverse transcription-polymerase chain reaction. RESULTS: Weekly fundus photography, done for 1 month, documented the absence of any ocular abnormality due to the viral injections. No in vivo hgfp fluorescence of the retina was visualized. Beta-galactosidase histochemical analysis of eye cups that received the lacZ gene construct showed blue lacZ staining of the optic nerve head at 2 weeks. Light microscopy revealed the blue beta-galactosidase reaction product in fibers, glial cells, and blood vessels of the optic nerve head and retrobulbar nerve. Histochemistry showed absence of beta-galactosidase in the optic nerve at 3 to 12 months, but immunochemistry showed the persistence of beta-galactosidase in fibers, glial cells, and blood vessels as late as 1 year after a single ocular injection. In the retina, histochemical staining showed evidence of lacZ at 3 months, but not later. In situ reverse transcription-polymerase chain reaction revealed brown lacZ mRNA reaction product in ganglion cells of the retina. Control eyes that received AAV without the promoter and reporter elements and the eyes that received no viral injections and were processed for beta-galactosidase showed no reporter gene expression in any ocular tissue or cell type. CONCLUSIONS: Viral-mediated gene transfer can be successfully accomplished in the optic nerve. Further evaluation is needed to determine whether the level of protein expression at 1 year after injection, which is clearly reduced relative to shorter postinjection time, is sufficient for therapeutic purposes. CLINICAL RELEVANCE: We have previously shown that gene therapy with catalase suppressed experimental optic neuritis at 1 month after injection. Viral-mediated gene transfer may be a powerful technique for the treatment of optic neuropathies, particularly for recurrences of optic neuritis, if long-term expression of transduced protein can be demonstrated in the optic nerve.

Herpes simplex encephalitis and bilateral acute retinal necrosis syndrome after craniotomy.

PURPOSE: Acute retinal necrosis (ARN) syndrome is associated with members of the herpes virus family, but the mechanisms of infection remain unclear. The purpose of this study is to report a unique case of acute retinal necrosis syndrome associated with herpetic encephalitis in order to elucidate possible factors involved in herpetic central nervous system disease. METHOD: Case report. RESULTS: A 64-year-old woman who developed acute herpes simplex virus encephalitis associated with bilateral acute retinal necrosis syndrome after craniotomy for resection of a suprasellar craniopharyngioma is presented. The results of lumbar puncture, magnetic resonance imaging, and ophthalmologic examination are consistent with herpetic infection. The origin of acute retinal necrosis syndrome and the association of acute retinal necrosis syndrome with encephalitis are reviewed. CONCLUSIONS: After craniotomy, we hypothesize reactivation of previously latent herpes simplex virus in the area of the inferior frontal lobe and optic chiasm. Reactivated virus may have migrated to the retina by axonal transport, through the optic nerves, to produce the acute retinal necrosis syndrome.

Optic neuritis and human T-lymphotropic virus type 1-associated myelopathy: a case report.

A 50-year-old woman with human T-lymphotropic virus type 1-associated myelopathy developed sudden loss of vision in her left eye. The patient had a relative afferent pupillary defect and a large central scotoma on visual field examination, but no abnormal findings were apparent upon slitlamp microscopy and funduscopic examination. A diagnosis of retrobulbar optic neuritis was made. We discuss the possible association of human T-lymphotropic virus type 1 infection.

AIDS-related optic neuropathy: a histological, virological and ultrastructural study.

BACKGROUND: Clinical and histopathological evidence of optic nerve axonal loss has been reported in AIDS patients without retinitis. The study was carried out to investigate the possible involvement of HIV-infected cells in the development of optic nerve degeneration. METHODS: Optic nerves were obtained from eight AIDS patients and four normal controls. These nerves were morphologically and immunohistochemically analyzed. Additionally, using PCR amplification techniques, the retina and optic nerve samples obtained from three HIV-seropositive patients and one control were examined for the presence of HIV and cytomegalovirus antigens. RESULTS: We noted various stages of axonal degeneration in the optic nerves obtained from AIDS patients in whom there was an absence of retinal findings. Characteristic glial changes involving hypertrophic astrocytes, vacuolated oligodendrocytes, and mononuclear phagocyte series cells were also seen in the AIDS optic nerves. HIV DNA was present in at least four of five optic nerves but in only one of five retinas. Control specimens were each negative for all cytomegalovirus and HIV antigens. CONCLUSIONS: Degeneration in the optic nerve may be mediated by HIV-infected macrophages rather than by direct viral infection of neurons. Axonal degeneration due to AIDS at the level of the optic nerve can occur independently of retinal infection.