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.

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 beta-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.

Cytokine production in a murine model of recurrent herpetic stromal keratitis.

PURPOSE: To determine the pattern of cytokine production in the cornea and its relationship with viral antigens, in our murine model of recurrent ocular herpes simplex virus (HSV)-1 infection. METHODS: Six weeks after corneal inoculation with HSV-1, the eyes of latently infected and control mice were UV irradiated and examined for signs of disease and viral reactivation. The eyes of five mice with recurrent stromal disease and two controls were processed for immunohistochemistry on days 4, 7, 10, and 14 after irradiation. Sections were double stained for viral antigens and one of the following cytokines: interleukin (IL)-1ss, IL-2, IL-4, IL-6, IL-10, IL-12, and interferon (IFN)-gamma. RESULTS: Fifty percent of mice showed signs of recurrent stromal disease, the severity of which peaked on day 10 after UV irradiation. There was a large cellular infiltrate in the stroma of all the corneas with recurrent disease and the predominant cytokines were IL-1ss, IL-6, IL-10, IL-12, and IFN-gamma, all present in large numbers of cells on the days studied. There were very few cells producing IL-2 and IL-4. Control eyes had no significant cytokine-producing cells in the stroma. CONCLUSIONS: These observations suggest that recurrent herpetic stromal keratitis (HSK) may not be characterized by a classic T-helper (Th)1 or Th2 response. However, the large number of IFN-gamma(+) and IL-12(+) cells and the relative absence of IL-4 favors a Th1 response, and despite the numerous IL-10(+) cells, the overall balance of cytokine production appears to be proinflammatory.

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines.

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-alpha from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-alpha+ and most were also IL-6(+). Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-alpha and/or IL-6. IL-4(+) cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2(+) and/or IFN-gamma+ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-alpha and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.

Induction of mucosal immunity against herpes simplex virus type 1 in the mouse protects against ocular infection and establishment of latency.

Immune responses were assessed after intranasal immunization of mice with a mixture of herpes simplex virus type 1 (HSV-1) glycoproteins with cholera toxin and its B subunit as adjuvant. Antigen-specific serum antibodies, which were largely IgG with IgG1 the major subclass, neutralized virus in vitro with a titer equivalent to that elicited by active infection. Significant levels of antigen-specific IgA were found in mucosal fluids of the eye as well as the vagina. Lymphocytes from draining lymph nodes showed secondary proliferative responses when cultured with HSV-1 in vitro, in immunized mice only, with the production of interleukin-2, interferon-gamma, interleukin-4, and interleukin-5. After ocular challenge, immunized mice were protected against the development of severe eye disease, zosteriform spread, or encephalitis, whereas the incidence of clinical symptoms in mock-immunized mice was 83%, 74%, and 52%, respectively. Finally, the incidence of latency was reduced from 88% to 13% after intranasal immunization.

Models of Recurrent Infection with HSV in the Skin and Eye of the Mouse.

Animal models remain essential for studies of many aspects of the biology of herpes simplex virus (HSV). Such studies include basic experiments on pathogenesis (including characterization of viral mutants), tests of antiviral drugs, and methods of immunization. With reference to models of recurrent infection, high levels of recurrence and clinical disease have been achieved with guinea pigs (particularly with genital infection) and rabbits (particularly with ocular infection; reviewed in ref. 1). However, in contrast to these animals, with the laboratory mouse there are many inbred and congenic lines; a major advantage for immunological studies. To this can now be added the growing technology of transgenic and "knockout" animals. For these reasons we have expended considerable effort in developing various mouse models of infection, particularly with HSV type 1 (HSV-1).

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.

Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues.

We have successfully developed a method for the immunohistochemical detection of interleukin 2 (IL-2), IL-4, IL-6, IL-10O, IFNgamma and TNFalpha using monoclonal antibodies (MAb), in sections of mouse tissue embedded in paraffin wax. The method involved fixation in periodate-lysine-paraformaldehyde (PLP), rapid dehydration and infiltration under vacuum with paraffin wax at 54 degrees C. Comparative observations demonstrated that the method gives equivalent or better results than formaldehyde fixed, frozen sections. Since reliable controls, both positive and negative, are paramount for interpretation of immunohistochemical staining, such controls were determined. The following tissues were shown to be suitable as positive controls when using paraffin-embedding: spleen for the detection of TNFalpha, small intestine for IL-2, IL-4 and IL-10, and HSV-1 infected eyes for IL-6 and IFNgamma. We conclude that PLP fixation and low temperature paraffin-embedding is a method which provides both preservation of excellent tissue morphology and reliable immunohistochemical identification of cytokines. These attributes will be invaluable in a wide variety of experimental situations.

In vivo immunofluorescence to diagnose herpes simplex virus keratitis in mice.

BACKGROUND/AIMS: Herpes simplex virus keratitis (HSK) is the most common cause of corneal blindness in the Western world. Delay in the treatment of HSK can lead to a more significant corneal scar and topical steroid treatment in unsuspected active HSK can lead to corneal melting. Current culture techniques for herpes simplex virus (HSV) take several days and commercially available HSV laboratory based diagnostic techniques such as Herpchek vary in sensitivity. This study was conducted to assess the viability of a new, quicker, and simpler method to diagnose HSK. METHODS: Direct immunofluorescence was used in vivo in a masked study to diagnose HSK in mice using a standard slit lamp with cobalt blue illumination. Murine monoclonal fluorescently labelled antibody was applied to the cornea for 10 or 20 minutes and then washed off with phosphate buffered solution. Mice with HSK were stained with either fluorescently labelled monoclonal antibody against HSV or fluorescently labelled monoclonal antibody against cytomegalovirus. Mice with corneal abrasions of non-viral origin were given fluorescently labelled monoclonal antibody against HSV. RESULTS: Fluorescence was seen only in the mice with HSK given fluorescently labelled monoclonal antibody against HSV. This observation was confirmed upon microscopic immunofluorescent imaging of the corneal epithelial sheets. CONCLUSION: In vivo immunofluorescence may be useful in the clinical diagnosis of HSK.

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.

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.

Recurrent herpes simplex after corneal transplantation in rats.

PURPOSE: To ascertain the effect of trauma from surgery and rejection on the incidence and timing of recurrent herpes simplex virus (HSV) disease after corneal transplantation. To locate virus antigen and identify cells of the immune system infiltrating corneas with recurrent disease. METHODS: PVG rats were inoculated on the cornea with HSV-1 McKrae. Recurrent disease was induced either by ultraviolet (UV)-irradiation of the cornea or by corneal transplantation. After corneal transplantation, animals shedding virus in the tear film were killed on days 1 to 4 of shedding. Eyes were fixed, embedded, sectioned, and stained for virus antigens, infiltrating cells, major histocompatibility complex class II, and adhesion molecule molecule expression. RESULTS: In the first 15 days after corneal transplantation, 8 of 91 rats shed virus, and between days 16 and 30, an additional 3 of 60 rats shed virus (12% of total rats, comparable to the percent that shed after UV irradiation). Shedding sometimes was accompanied by punctate epithelial lesions in the recipient cornea and stromal opacity. The rejection process itself did not induce or exacerbate recurrent disease. In all corneas examined from eyes that shed virus, antigen was found in several locations at the graft-host junction, sometimes in the absence of clinical signs of disease, and frequently it extended through the stroma to the endothelium. Granulocytes were the main infiltrating cell in areas of virus antigen. CONCLUSIONS: Corneal transplantation trauma is a stimulus to recurrent disease of similar potency to UV irradiation. The graft-host junction is an area in which virus spreads easily and can reach the endothelium readily. In humans, the incidence of recurrent disease at this location may be greater than has been recognized.

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.

Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.

We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.

Ocular infection with herpes simplex virus in several strains of rat.

PURPOSE: To assess the suitability of the rat for studies of ocular infection with herpes simplex virus (HSV). METHODS: LEW, AO, DA, PVG, and (DAxLEW)F1 x LEW backcross generation rats, 7 to 9 weeks of age, were inoculated with HSV-1 McKrae. The course of primary disease was assessed by clinical observation using a slit lamp. Infectious virus was assayed in ocular and nervous tissue, and the incidence of latent infection was determined. RESULTS: LEW and AO strains were the most susceptible. All LEW rats died after an inoculum of 4 x 10(2) plaque-forming units (pfu) and developed severe corneal disease and uveitis. In contrast, all PVG rats survived 10(4) pfu, 60% survived 4 x 10(4) pfu, and eye disease was restricted to epithelial lesions, sometimes accompanied by mild stromal haze. This resolved, even in animals that developed central nervous system disease. The DA strain showed intermediate susceptibility. Resistance was dominant because disease in backcross generation (DA x LEW)F1 x LEW rats resembled that of the DA rather than the LEW strain. Resistance appeared to be linked to coat color (P < 0.001) rather than to major histocompatibility complex (MHC) type. Chronic stromal disease did not occur in survivors (DA, PVG, and hybrid strains only). CONCLUSIONS: The susceptibility of rat strains to infection of the cornea with HSV varies, and, as with mice, resistance seems to be controlled by non-MHC genes. Rats may prove useful for immunologic studies. Virus reactivation will be the subject of a future report.

Protection against ocular and cutaneous infection with herpes simplex virus type 1 by intragastric immunization with live virus.

Intragastric administration of live herpes simplex virus type 1 (HSV-1) was assessed for the induction of humoral immune responses and for protection against ocular and cutaneous challenge with virus. Mice showed no clinical abnormalities following intragastric inoculation with three different strains of virus (Miyama + GC, SC16, and P2C6, a thymidine kinase-defective mutant). Replication of virus was not detected in the oesophagus, superior cervical ganglia or coeliac ganglia of such animals and latent infection was not detected in these ganglia at later times after inoculation. Induction of a mucosal immune response was indicated by the presence of antibody (mainly IgG or IgA)-secreting cells in Peyer's patches. Intragastric immunization gave protection to some extent against ocular challenge and to a greater extent against cutaneous challenge with HSV-1. Following the latter challenge, particularly after intragastric immunization with strains SC16 and Miyama, the establishment of latency was almost completely prevented.

Non-traumatic acquisition of herpes simplex virus infection through the eye.

Primary ocular herpes is usually seen as a follicular conjunctivitis and blepharitis, with or without involvement of the cornea. It is unknown, however, to what extent asymptomatic and/or subclinical primary disease occurs, and whether primary ocular herpes follows direct droplet spread to the eye. Previous models of murine ocular herpes have used trauma (scarification) to introduce virus into the cornea, producing disease which results in significant corneal scarring. To mimic a likely route of infection in humans, a droplet containing virus was placed on the mouse eye and clinical disease recorded. At least 1 month after inoculation, serum was assayed for neutralising antibodies and the cornea, iris, and trigeminal ganglion were investigated for evidence of herpes simplex virus type 1, by cocultivation and the polymerase chain reaction. Some animals showed a severe ulcerative blepharitis with little to no involvement of the cornea, while disease was undetectable in others. The development of disease depended on the dose and strain of virus and age of the animal, with older mice appearing more resistant. Virus was isolated from the trigeminal ganglion of younger animals inoculated with higher doses of virus, after 21 days in culture, suggesting that latency had been established. Neutralising antibodies were present in most mice irrespective of the presence of recognisable clinical disease. Using primers for the thymidine kinase and glycoprotein C regions of the viral genome, herpes simplex virus type 1 DNA was found in the cornea, iris, and trigeminal ganglion of most animals and showed a good correlation with the presence of neutralising antibodies. It would thus appear that herpes simplex virus type 1 is able to accede into the cornea, iris, and trigeminal ganglion following nontraumatic application of virus onto the mouse eye. This model mimics primary ocular disease in humans and may be useful for studies on recurrent disease and the spread of ocular herpes.

Acute and latent infection of mice immunised with HSV-1 ISCOM vaccine.

The effect of immunisation with an HSV-1 antigen preparation (containing at least 6 viral glycoproteins) on primary infection with HSV and the establishment of latency, was assessed in two mouse models (involving either skin or corneal challenge with virus). The vaccine preparation, given either with Freund's complete adjuvant or aluminium hydroxide gel or in the form of immunostimulating complexes (ISCOMS), induced high ELISA antibody responses (highest with HSV as the ISCOM preparation) and low levels of neutralising antibody. In both models, immunisation with the HSV ISCOM preparation significantly reduced the incidence of zosteriform spread of virus and the severity of disease and, in some cases, the incidence of latent infection in sensory ganglia. In the eye model it was possible to show that immunisation with the HSV ISCOMS restricted the establishment of latency almost entirely to the ophthalmic part of the trigeminal ganglion. Protection from establishment of latency correlated with prechallenge antibody levels.

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.

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.