C. Shimeld

Immune cell infiltration and persistence in the mouse trigeminal ganglion after infection of the cornea with herpes simplex virus type 1

Following inoculation of the mouse cornea with herpes simplex virus type 1 (HSV-1), the spread of virus was investigated and the types of immune cell infiltrating the trigeminal ganglion (TG) were identified in low temperature paraffin wax sections. Virus antigen was first found on day 3 and was absent after day 14. Early presentation of antigen to T cells may occur since increased expression of major histocompatibility complex (MHC) class II antigens, including de novo expression on satellite and Schwann cells, was detected in foci of such antigen on day 3. A second large peak of such expression was detected on day 10 together with increasing numbers of B and T cells. Large numbers of these lymphocytes and extensive expression of MHC class II were seen in the TG well into the phase of virus latency; the significance of this is discussed.

Cytokine production in the nervous system of mice during acute and latent infection with herpes simplex virus type 1.

Immunocytochemistry on serial paraffin sections was used to monitor the production dynamics of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha) and viral antigens in the trigeminal ganglion (TG) and the central side of the dorsal root entry zone (DRE) of mice, following infection of the cornea with herpes simplex virus type 1. In normal TG, scattered satellite cells were TNF-alpha+ and in the DRE, TNF-alpha+ and/or low numbers of IL-6+ cells were detected. On day 3 after infection, foci of TG neurons with viral antigens were surrounded by large numbers of TNF-alpha+ and/or IL-6+ cells and low numbers of IFN-gamma+ cells. IL-2+ and/or IL-4+ cells appeared later, when viral antigens had almost cleared. In the TG, the most striking changes occurred with TNF-alpha, with respect to its source (satellite cells, Schwann cells and infiltrating cells) and the extent and long duration of its production. TNF-alpha was the predominant cytokine throughout acute and latent infection and even by day 30, numbers of satellite cells expressing this cytokine were three times higher than those in normal ganglia. Moreover, in the DRE, TNF-alpha was the only cytokine detected during virus clearance and again, its production continued, along with that of IL-6, on days 20 to 30, in both infiltrating cells and astrocytes. Thus, cytokines, particularly TNF-alpha and perhaps IL-6, from infiltrating cells and resident glial cells may have a role both in virus clearance and in normal homeostatic mechanisms in the nervous system such as repair and protection of neurons from damage.

Reactivation of latent infection and induction of recurrent herpetic eye disease in mice.

During primary ocular infection of mice with herpes simplex virus type 1 (HSV-1) strain McKrae, dendritic corneal ulcers developed and many eyes became permanently damaged. When primary infection had subsided, latent infection was detected in the three parts of the trigeminal ganglion and in the superior cervical ganglion. Such latently infected mice were treated with cyclophosphamide, dexamethasone and u.v. irradiation, or cyclophosphamide and dexamethasone alone. After treatment with immunosuppressive drugs and u.v. irradiation infectious virus was isolated from the ophthalmic part of the trigeminal ganglion, and in eyelids and eyewashings; recurrent herpetic eye disease was seen but only in eyes undamaged by primary infection. After treatment with cyclophosphamide and dexamethasone alone there was a lower incidence of virus isolated from eyewashings and no recurrent disease was seen. There was a good correlation between the pattern and distribution of recurrent lesions and the distribution of cells stained due to the presence of virus antigens.

Spread of virus and distribution of latent infection following ocular herpes simplex in the non-immune and immune mouse.

In both non-immune and immune mice infected with herpes simplex virus the incidence of latent infection of the trigeminal ganglion was related to the severity of ocular virus infection. During primary infection, virus was shown to travel via the ophthalmic part of the ganglion to reach the brainstem, from where centrifugal spread resulted in latent infection of neurones in the trigeminal ganglion which did not serve the site of inoculation. Primary infection also resulted in latent infection of the superior cervical ganglion. Shedding of virus occurred rarely in the tears of animals which had recovered from primary disease. In immune mice, spread of virus resulted in a much lower incidence of latent infection and that occurred only in ophthalmic neurones.

Herpes simplex virus isolation in chronic stromal keratitis: human and laboratory studies

The corneal discs of 41 patients with scarring reminiscent of herpetic infection were organ cultured for HSV isolation. Of the 41 patients, 34 had a definite history of herpetic keratitis, from 10 of whom (29.4%) HSV was isolated. There were no clinical features which distinguished between these groups; there was however an indication that those from whom HSV was not isolated had been previously treated with substantial amounts of topical acycloguanosine. In three patients of 12 patients when the disc was separated into 7 parts using a punch technique, virus was isolated exclusively from those portions demonstrating clinical scarring. Electron microscopy (EM) demonstrated HSV particles in stromal cells in the cultured corneas of seven patients. In two of the patients no virus was detected prior to culture with EM. In one patient HSV antigen was not found using peroxidase-antiperoxidase (PAP) staining prior to subsequently positive organ culture. Studies were made to determine how HSV accedes to the corneal stroma using a murine model in which keratitis occurs by zosteriform spread of HSV following inoculation of the snout. Preliminary evidence using PAP staining indicates that the virus reaches the stroma at the same time as the epithelium, via the sensory nerves. Evidence of HSV persistence in anterior segments was obtained in the same model, in contrast to which no virus could be isolated following direct inoculation into the cornea. It is speculated that for virus to set up a longterm association with the stromal keratocyte, it must be introduced via the sensory nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and immune cell infiltration

The corneas of latently infected mice were UV irradiated to induce reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG). On days 1 to 4 after irradiation, TG were removed, serially sectioned and double stained to identify immune cells and virus antigens. Virus antigen was detected in small numbers (most commonly one) of neurons per ganglion as early as day 1, confirming the rapidity of reactivation and the neuron as the likely site of this event. The immune response was also rapid and effective since virus antigen was identified in immune cells at day 1 and by day 4 all samples were negative. The predominant infiltrating cells on days 1 and 2, when virus antigen was present and being cleared, were T cells, both CD4+ and CD8+. Later, large numbers of B cells appeared, suggesting that local antibody production may also be involved in controlling the reactivated infection. The observations suggest that a significant proportion of reactivation events do not result in disease of the eye or shedding of virus in the tear film. However, they also suggest that as little as one reactivating neuron in the ganglion may be sufficient to lead to such disease and/or shedding.

Isolation of herpes simplex virus from corneal discs of patients with chronic stromal keratitis.

Herpes simplex virus (HSV) was isolated from the corneal discs of six patients with herpes simplex keratitis, which were removed during penetrating keratoplasty and then cultured in vitro. The demonstration of persistent infection in stromal keratitis, even when the condition is quiescent, emphasizes the need for antiviral agents in treatment. The findings support the possibility that latent infection may become established at a peripheral site.

Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system

Mice were treated with serum containing antibodies to herpes simplex virus type 1 (HSV-1) or normal serum, 1 day before inoculation on the cornea with HSV-1 strain McKrae. As expected, without passive immunization, mice developed high levels of serum neutralizing antibody. By contrast, in passively immunized animals, such antibody became undetectable by 29 days after inoculation of serum, in spite of the virus infection. There was no difference between passively immunized mice and those given normal serum in the duration of shedding of virus in tears and the duration and severity of corneal epithelial disease. However, non-immunized mice had a high incidence of mortality and developed disease of the iris, corneal stroma and lids, and their corneas became opaque and vascularized. In non-immunized animals, the timing of isolation of virus from nervous tissues and the sequence of appearance of virus antigens in ocular tissues indicate that the disease of deeper eye tissue was caused by virus spreading from the nervous system back to the eye. Restriction of such spread in passively immunized animals seems the likely explanation for their protection from death and severe ocular damage. Despite this restricted spread, passively immunized animals had a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion. However, in comparison with mice given normal serum, there was a far lower incidence of such infection in the other two parts of this ganglion and in the superior cervical ganglion. Since passively immunized animals have a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion and their eyes are normal, they will prove useful in studies involving induction of recurrent disease.

Tracking the Spread of a lacZ-Tagged Herpes Simplex Virus Type 1 between the Eye and the Nervous System of the Mouse: Comparison of Primary and Recurrent Infection

ABSTRACT The spread of herpes simplex virus type 1 (HSV-1) during primary ocular infection and after reactivation of latent infection in the trigeminal ganglion (TG) was examined in the mouse using a genetically modified virus containing the lacZ reporter gene under the control of the immediate-early 110 promoter. Whole tissue mounts of the eye and lids, their sensory nerves, and TG with the attached dorsal root entry zone (DRE) into the central nervous system (CNS) were stained for β-galactosidase. Sixteen hours after inoculation of the cornea by scarification, staining was found in the scarified epithelium of the cornea and in the unscarified conjunctiva. By 24 h, staining was also seen in a few TG neurons and by 96 h their number had greatly increased and their distribution was more widespread. Stained cells (identified as Schwann cells by their staining for glial fibrillary acidic protein [GFAP] or S-100) in the TG were first seen close to stained neurons at 40 h, and by 48 h lines of such cells extended partway toward the periphery and toward the DRE. By 72 h, these lines had reached the periphery and the DRE where the adjacent CNS was also stained. In the cornea, stained cells with the morphology and arrangement of Schwann cells were seen from 40 to 120 h. After reactivation of latent infection, 10 of 22 samples had positively stained neurons. In eight samples, corneal and lid epithelial cells were stained. No stained Schwann cells were seen in the TG; however, branched networks of such cells were present in the cornea and the lids. This detailed sequential analysis has provided new information on the involvement of Schwann cells in the pathogenesis of primary and recurrent HSV-1 disease in the TG and the cornea.

Immune cell infiltration in corneas of mice with recurrent herpes simplex virus disease

Reactivation of latent herpes simplex virus type 1 (HSV-1) infection was induced by UV irradiation of the corneas of latently infected mice. On days 1-4 after stimulation, infectious virus was sought in nervous and ocular tissue. On days 4, 7 and 10, eyes with either recurrent epithelial or stromal disease and appropriate controls were stained to identify immune cells and HSV-1 antigens. The maximum incidence of infectious virus was on day 2 when 5/10 ophthalmic parts of the trigeminal ganglion yielded HSV. Thus in this mouse model, as in humans, reactivation of virus in the trigeminal ganglion is the likely source of virus producing recurrent disease and shedding in the tear film. On day 4, when virus antigens were still present, granulocytes were the predominant infiltrating cell in corneas with either type of disease. Small numbers of T cells, dendritic cells and cells expressing MHC class II were also present. In stromal disease, the granulocyte infiltrate persisted and T cells remained sparse. In contrast, in epithelial disease, granulocyte numbers rapidly declined and both CD4+ and CD8+ T cells (present at a ratio of 1:1) increased significantly. The secondary immune response to virus antigen is more rapid and vigorous than that during primary corneal infection. Granulocytes may play a role in the initial clearance of virus, however, the other types of cells present early on provide the potential for a local secondary immune response. The high proportion of CD8+ cells in epithelial disease compared with stromal disease suggests that they may be acting as suppressors.