Herpes Simplex DNA in Tears of Atypical Dendritic Keratitis and Multiple Punctate Subepithelial Stromal Opacity: A Case Report.

PURPOSE: To report an atypical presentation of herpes simplex virus (HSV) keratitis followed up using expression levels of HSV DNA in tears. METHODS: A 22-year-old Japanese woman with hyperemia and foreign body sensation in her left eye was diagnosed with atypical dendritic keratitis. A slit-lamp examination at presentation indicated the presence of a rush of dendritic lesions with a sparse branching pattern and poor development of terminal bulbs; follicular conjunctivitis was also observed. Positivity for house-dust-mite- and cedar pollen-specific IgE antibodies in her serum indicated atopic diathesis. The HSV DNA levels in her tears were measured by a real-time polymerase chain reaction. RESULTS: At the initial visit, the HSV DNA levels in tears were 6.4 × 10 copies/sample in the right eye and 1.6 × 10 copies/sample in the left eye. The keratitis improved after treatment with topical acyclovir ointment, 5 times a day for 7 days, and systemic valacyclovir 1000 mg/d for 5 days. Multiple punctate subepithelial opacities developed in her left eye on day 7, with undetectable HSV DNA in tears, bilaterally. CONCLUSIONS: We have successfully monitored the HSV DNA levels in tears using quantitative real-time polymerase chain reaction in HSV keratitis where the corneal findings progressed from atypical dendritic keratitis to multiple punctate corneal subepithelial opacities during the treatment period.

Induction of Fc and C3b receptors on rabbit corneal cells by herpes simplex virus.

The expression of receptors for Fc portion (FcR) of immunoglobulin G (IgG) and for a C3b component of complement (C3bR) by herpes simplex virus (HSV) was studied in primary cultures of rabbit corneal cells. Monolayer cultures of epithelial, stromal and endothelial cells of the rabbit cornea were infected with three strains each of type 1 (HSV-1) and type 2 HSV (HSV-2). Rosette methods were used to detect receptors by means of sheep erythrocytes sensitized with rabbit IgG for FcR and C3b-coated sheep erythrocytes for C3bR. The FcR were expressed regularly on epithelial, stromal and endothelial cells by all three strains of both HSV-1 and HSV-2. The C3bR, however, were expressed only by HSV-1 on epithelial and stromal cells. Little or no C3bR activities could be detected on endothelial cells infected with any strain of HSV-1 or HSV-2. The FcR and C3bR expressed on corneal cells were induced by HSV and were blocked by monoclonal antibody to HSV-1 glycoprotein E(gE) or glycoprotein C(gC) respectively, confirming findings of other investigators that gE acts as FcR and gC as C3bR.

Stress proteins accumulate in cultured retinal glial cells during herpes simplex viral infection.

The production of stress- or heat-shock proteins (SP) which are defined by three monoclonal antibodies (TI56, TG5E and TG7A) were examined in cultured retinal glial cells with and without herpes simplex virus (HSV) infection. Indirect immunofluorescence showed that 80-90% of uninfected cells reacted with anti-glial fibrillary acidic protein (GFAP) and that 10-20% of uninfected cells were weakly labelled with anti-SP antibodies. By 6 hr after HSV infection, the proportion of GFAP labelled cells decreased to 60-70% whereas cells strongly expressing SP antigens were demonstrated. At 24 hr, GFAP+ cells were markedly reduced in number and immunolabelling with anti-SP antibodies was evident in approximately 50% of cells, directly demonstrating the accumulation of SP in cultured retinal cells after HSV infection. Double labelling with GFAP/TI56 indicated that 30% of GFAP+ cells were labelled with TI56 and 30-50% of TI56+ cells were also GFAP+, despite the abrupt loss of GFAP+ cells during HSV infection. These results indicate that SP normally expressed at low level are significantly upregulated in retinal glial cells following HSV infection.

Reevaluation of human alpha interferons against herpesvirus infection of the rabbit eye.

Because of reported differences in potency, recombinant DNA-derived human alpha interferons (IFNs) were reevaluated for use against acute Herpes Simplex Virus type 1 (HSV-1) infection of the rabbit eye. The IFNs used topically were IFN-alpha 2, (IFN-alpha 2) and a consensus of known human IFN-alpha s, designated IFN-alpha Con1. Prophylactic treatment with IFN-alpha Con1 at 1 or 15 X 10(6) U/eye/day beginning 48 hours before HSV-1 inoculation and therapeutic treatment with 5 or 15 X 10(6) U/eye/day beginning 4 hours after inoculation with either IFN-alpha Con1 or IFN-alpha 2 appeared to prevent or significantly reduce the development of corneal epithelial involvement. The effects were dose dependent with no evidence for decreased activity at the higher dose. The duration of HSV-1 shedding into tear film was not significantly reduced.

Analysis of glycoproteins expressed by isolates of herpes simplex virus causing different forms of keratitis in man.

An in vitro analysis of glycoprotein produced by nine human ocular isolates of HSV-1 is reported. The source of the isolates was; three patients with recurrent dendritic keratitis, three with chronic stromal disease and three with primary keratoconjunctivitis. Virus strains were labelled with the radioactive precursors (35S) methionine and (14C) glucosamine. Radiolabelled viral glycoproteins were subsequently analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. Viral glycoproteins were further characterised by immuno-precipitation with polyclonal and monoclonal antibodies to HSV. The stromal isolates excrete larger amounts of 'soluble' precursor glycoprotein D than those in the other two disease categories. It is possible that the immune response to glycoprotein D is in part responsible for the severity of stromal disease.

Interferon production in inbred mice during herpetic eye disease.

Low levels (less than 5 units/eye) of interferon (IFN) were detected in the eyes of BALB/c and C57BL/6 mice one to five days after instillation of 10(7) pfu/eye of herpes simplex virus type 1 (HSV) onto scarified corneas. This dose of virus produced herpetic keratitis characterized by dendritic epithelial lesions one day post infection in both strains of mice. The disease progressed to severe necrotizing stromal keratitis in the eyes of all BALB/c mice, but only three of 10 eyes of C57BL/6 mice by 21 days after infection. Footpad immunization 30 days prior to ocular infection protected both strains from stromal disease, but did not enhance IFN production in the eye. At lower inoculating doses of virus (less than or equal to 10(5) pfu/eye), C57BL/6 mice showed greater resistance to stromal disease and produced less virus over a shorter period of time than BALB/c mice. No IFN was detected at any time after infection with doses of virus less than 10(7) pfu/eye, nor was IFN detected in plasma of any infected mice. The failure to detect high levels of IFN in homogenates of eyes did not reflect an inability of ocular tissues to produce IFN since IFN-beta was detected as early as two hours after topical treatment with the potent IFN inducer, carboxymethylacridanone (CMA). The two mouse strains produced similar levels of IFN in the eye in response to CMA. These data indicated that the relative resistance of mice to HSV eye infection was not related to the rapid local production of IFN, nor was resistance related to systemic IFN production in plasma or spleen.

Effect of neuraminidase on Fc and C3b receptors on rabbit corneal cells infected with herpes simplex virus.

The effect of neuraminidase on Fc receptors (FcR) and C3b receptors (C3bR) was studied in epithelial, stromal and endothelial cells of the rabbit cornea infected with type 1 (HSV-1) and type 2 herpes simplex virus (HSV-2) in vitro. FcR were induced on epithelial, stromal and endothelial cells of the rabbit cornea by both HSV-1 and HSV-2, but their activities were not enhanced by neuraminidase. On the other hand, the treatment of HSV-infected corneal cells with neuraminidase resulted in the enhancement of C3bR activities on epithelial, stromal and endothelial cells infected with HSV-1, and the enhancing effect of neuraminidase was more pronounced on corneal endothelial cells. A similar neuraminidase treatment had no significant effect on C3bR activities on the corneal cells infected with HSV-2.

Arachidonic acid metabolism to eicosanoids in herpes virus-infected rabbit cornea.

The metabolism of the polyunsaturated fatty acid, arachidonic acid (20:4, n-6) in rabbit cornea with varying severities of herpes simplex viral infection was investigated. The results indicate an active synthesis of the lipoxygenase and cyclooxygenase reaction products of arachidonic acid in central cornea and corneal-scleral rim. Stimulation of 12-hydroxyeicosatetraenoic acid (12-HETE) production in herpes-infected cornea was correlated positively with the severity of infection. Other eicosanoids were increased maximally in moderately infected corneas. The stimulation of eicosanoid synthesis was more evident in central cornea as compared to corneal-scleral rim. Herpes infection also caused a decline in the incorporation of radiolabeled arachidonic acid into membrane glycerolipids. These data indicate that the production of eicosanoids from arachidonic acid is stimulated in herpes-infected cornea. The stimulation may reflect the presence of phagocytic cells in the infected cornea, an enhanced capacity of the cornea itself to produce eicosanoids, or a combination of these effects. Decreased acylation of membrane lipids may be the result of infection-induced activation of fatty acid release mechanisms, which would lead to degradation of cell membranes. The presence of lipoxygenase reaction products in the herpes-infected cornea introduces a new factor for consideration in the design of therapeutic regimens for this disease.

Therapy with 9-beta-D-arabinofuranosyladenine (ARA-A) and 2'-deoxycoformycin increases the ARA-A content of ocular tissues.

The amount of 9-beta-D-arabinofuranosyladenine (ARA-A) and 9-beta-D-arabinofuranosylhypoxanthine (ARA-Hx) present in ocular tissues of rabbits was determined following therapy with ARA-A alone and when ARA-A was used in combination with 2'-deoxycoformycin (dCF). Topical therapy was initiated three days after infection of the corneas of rabbits with herpes simplex virus type 1. Ocular tissues were harvested after two days of therapy and analyzed by high performance liquid chromatography. Combination topical therapy with ARA-A and dCF significantly increased the tissue content of ARA-A in all tissues examined except retina, as compared to therapy with ARA-A alone. The ARA-A content of the two ocular tissues most often subject to acute herpes infections, the conjunctiva and cornea, was increased from 29.9 +/- 11.7 to 144.0 +/- 53.3 pmoles/mg dry weight and from 15.4 +/- 6.1 to 231.8 +/- 30.8 pmoles/mg dry weight, respectively. Except for the aqueous humor, the total arabinoside content of each tissue was not significantly altered by combination therapy, merely the ratio of ARA-A to ARA-Hx was changed. These studies demonstrate that combination topical therapy with ARA-A and an inhibitor of ARA-A catabolism, dCF, can effectively result in elevated amounts of ARA-A in ocular tissues.

Penetration of 14C-methisoprinol into rabbit eyes with experimentally induced herpetic keratitis.

The authors studied the penetration of 14C-labelled methisoprinol into ocular tissues (cornea, aqueous humour and iris) in rabbits, in which herpetic keratitis had been experimentally induced. It has been demonstrated that the methisoprinol crosses the corneal barrier. The maximum concentration of the drug in the tissues examined was measured 30 min after the instillation of the drug into the conjunctival sac, and persisted for 60 min, although at a slightly lower level.

Antiproteases in herpetic keratitis.

Proteolytic enzyme inhibitors are capable of preventing decomposition of the collagen structures of the corneal stroma, and also have a regulating influence on various aspects of the inflammatory process. The content of proteolytic enzymes (alpha 1-antitrypsin and alpha 2-macroglobulin) was studied in the tears and the blood serum of patients with herpes viral keratitis, as well as in the blood serum of rabbits with experimentally-induced ophthalmoherpes. Herpetic keratitis was attended by significant changes in the content of proteolysis inhibitors, as well as in the peripheral blood neutral proteases activity, this presumably serving as a nonspecific blood protective reaction to the inflammation. In the tears of patients suffering from herpetic keratitis the inhibitors are expended for binding activated proteases in the cornea. Local application to rabbits of protein preparations with an inhibitory effect (contrykal, gordox) at the acute period of the experimentally-induced ophthalmoherpes produced a marked antiphlogistic effect.

Recombinant human interferon alpha D in HSV-1 recurrence in the rabbit.

Recombinant human interferon alpha subtype D (RIFN alpha D) was effective in reducing the shedding of herpes simplex virus type-1 (HSV-1) induced by 6-hydroxydopamine iontophoresis followed by topical epinephrine application in previously infected rabbit corneas. A treatment schedule of RIFN alpha D, two drops QID was superior to one drop BID. RIFN alpha A also appeared to be effective in reducing viral shedding. Rabbits treated with RIFN alpha D during two episodes of adrenergically induced HSV-1 shedding, but not during anticipated episodes of spontaneous shedding, did not show a significant reduction in shedding of virus. Interferon was present in significantly higher concentration in tear samples following treatment with RIFN alpha D as compared with RIFN alpha A.

Oral acyclovir in herpetic keratitis.

A double blind comparative study of oral acyclovir and acyclovir ophthalmic ointment was carried out on 29 patients with simple herpetic dendritic corneal ulceration. Patients were randomly allocated to the study. Healing was achieved in all fourteen patients treated with acyclovir ointment, and in fourteen out of fifteen patients treated with oral acyclovir. The mean healing time of 5.6 days was identical for both patient groups. There was no significant systemic or local side effects recorded in either group. The level of acyclovir in tear fluid was measured, and in patients receiving oral acyclovir the level was in excess of the ED50 of herpes simplex virus type 1.

Analysis of viral DNA in isolates from patients with recurrent herpetic keratitis.

Isolates were obtained from 10 patients with recurrent herpes simplex type 1 infection of the eye, lids, or mouth. The viral DNA of successive isolates from each patients was analyzed by restriction endonuclease fingerprinting. Evaluation of the DNA banding patterns of all isolates by means of two different enzymes, performed in a masked fashion, demonstrated that all of the isolates from any one patient had the same genetic makeup. These results indicate that recurrent herpes infections of the eye and face in humans are caused not by unrelated, serial infections but rather by reactivation of the same latent virus that remains in the ganglion over a period of years.

Schirmer test values and lysozyme content of tears in acute dendritic keratitis.

In acute dendritic keratitis, the lacrimal flow is increased in the affected eye but not in the normal fellow eye. After recovery the tearflow returns to normal. The lysozyme concentration in the affected and the normal fellow eye do not differ statistically significantly nor do they differ significantly from values of an age- and sex-matched control group. There is no correlation between tearflow and lysozyme concentration. These findings support the view of a constant composition of stimulated tear fluid, irrespective of tear volume produced.

Corneal sensitivity and substance P in experimental herpes simplex keratitis in mice.

Experimental herpes simplex keratitis in the mouse produced a rapid fall in both corneal sensitivity and levels of corneal substance P (SP). This finding supports the association of SP with sensory neurones and shows that such levels can be used as an indication of damage to neurones resulting, for example, from infection with HSV. However, the delay in recovery of SP compared to the more rapid and complete recovery of sensitivity suggests that SP in the cornea is not directly involved in mediation of the blink reflex.

Lipofuscin in rabbit skin: Its occurrence after ocular herpes simplex infection.

Systemic infection, induced by intraocular inoculation of type 1 herpes simplex virus (HSV 1) in young rabbits, is accompanied by the appearance of autofluorescent pigments precisely in the midportion of their fur follicles. Histochemical and solubility reactions of the pigment led to its characterization as a lipofuscinlike pigment. Follicle fluorescence correlated with the severity of clinical symptoms; it was present in 100% of the follicles of rabbits that died but was found in only 30% to 50% of follicles of surviving animals. Similar fluorescence was also present in 10% to 20% of the follicles of uninfected rabbits. This autofluorescent material may be formed by the peroxidation of lipids from sebaceous gland secretions, but neither HSV 1 nor antigens could be found in the skin as possible initiators of this reaction.