Presence of a large amount of herpes simplex virus genome in tear fluid of herpetic stromal keratitis and persistent epithelial defect patients.

Herpetic eye diseases exhibit various clinical manifestations making a diagnosis difficult in some patients. We quantitated herpes simplex virus (HSV) genomes in the tear fluid and aqueous humor obtained from patients with various herpetic eye diseases by real time PCR. The resulting amounts of HSV-DNA in herpetic epithelial keratitis (HEK), herpetic stromal keratitis (HSK) in active phase, and persistent epithelial defects (PED) were 3.9 x 10(6) copies (detection rate, 81.1%), 8.9 x 10(5) copies (detection rate, 59.1%), and 9.2 x 10(4) copies (detection rate, 88.9%), respectively. In the tear samples obtained from quiescent phase of HSK and endotheliitis, no HSV-DNA was detected. In the aqueous humor of uveitis patients, HSV-DNA was found 3.8 x 10(5) copies/ml (detection rate, 16.7%). Previous studies have shown that active viral replication is not directly related to the persistent epithelial defects and progressive HSK. A relatively high level of HSV-DNA, however, was detected in the tear samples of these two disease forms, although the source of the viral replication was not identified. These findings might bring new ideas about the mechanisms of developments in HSK and PED.

A-5021: a new acyclovir analogue inhibits murine herpetic keratitis.

PURPOSE: To determine the efficacy of A-5021, a new analogue of acyclovir, on murine herpetic keratitis. METHODS: Herpes simplex virus type 1 (strain CHR3) was inoculated onto bilateral scarified BALB/c corneas. Clinical scores on the corneas treated with A-5021 eyedrops were compared with those obtained from the treatment with 3% acyclovir eye ointment by slit lamp microscopy. Virus titers of the trigeminal ganglia and eyeballs were quantitated on Vero cell monolayers. Mice treated with saline or a white petroleum jelly were used as controls. RESULTS: A-5021 eyedrops significantly suppressed both corneal epithelial and stromal lesions at all concentrations used. Clinical scores on the epithelium and stroma treated with 0.1% A-5021 were equivalent to those with 3% acyclovir treatment. When compared with the non-drug-treated control mice, virus titers in the eyeballs and trigeminal ganglia in A-5021- and acyclovir-treated mice were significantly less than those in controls. CONCLUSIONS: A-5021 eyedrops, which are easily applied onto the affected cornea, ameliorated clinical scores and suppressed virus growth. It is a promising alternative treatment of herpetic keratitis.

Corneal buttons obtained from patients with HSK harbor high copy numbers of the HSV genome.

PURPOSE: To detect herpes simplex virus (HSV) genome in the cornea, we sampled the limbal corneas and scleras of the imported eye bank eyes and recipient's corneal buttons and quantitated HSV genome in them by real-time polymerase chain reaction (PCR). METHODS: Forty-four recipient corneas including 7 corneas with and 37 corneas without a history of herpetic keratitis, 70 eye bank donor limbal corneas, and 35 eye bank donor scleras were obtained. Primers for real-time PCR were synthesized using the HSV-1 and -2 common regions of the viral DNA polymerase. Primers for conventional PCR were designed to detect HSV-1 and -2 and varicella zoster virus (VZV). RESULTS: Significantly higher copy number of HSV DNA was detected in corneas with a history of herpetic keratitis 85.7% (6/7), with an average of 1.6 x 10(4) copies/mg tissue weight than in corneas without a history of herpetic keratitis 10.8% (4/37), with an average of 8.7 copies/mg tissue weight (P < 0.05, Mann-Whitney U test). HSV DNA was detected in 5.7% (4/70) of the eye bank donor corneas, with an average of 4.9 x 10(2) copies/mg tissue weight, and in 8.6% (3/35) of the donor scleras, with an average of 10.6 copies/mg tissue weight. HSV-2 and VZV-DNA were not detected in these samples. CONCLUSIONS: Real-time PCR quantitated HSV genome in the cornea even at a quiescent phase of infection. HSV genome was detected in the corneas and scleras without a past history of herpetic keratitis by this method.

Effect of oral valaciclovir on herpetic keratitis.

PURPOSE: To examine the efficacy of valaciclovir (VACV) oral formulation as an alternative to topical treatments in a case of herpetic keratitis. METHODS: The patient was a 61-year-old man who presented with dendritic keratitis in his left eye. After recognizing his difficulty in using eye ointment, we prescribed oral VACV 500 mg tablets twice daily for 7 days. We also describe our experiments with orally administered VACV in a mouse model of this disease. In this study, 143 Balb/c mice were inoculated with herpes simplex virus type 1 (HSV)-1 in each eye and treated with oral VACV 50 or 100 mg/kg twice daily, oral acyclovir (ACV) 50 mg/kg 5 times/d, 3% ACV eye ointment (ACV-O) 5 times/d, 3% ACV eye drops 5 times/d, or control for 5 days. RESULTS: After 7 days, the patient's lesion was observed healed. Corrected left visual acuity was also improved, and HSV DNA was below detectable level. In the mouse study, slit-lamp examination on days 3, 4, 5, and 7 revealed that all 5 ACV and VACV treatment groups were significantly more effective in improving symptoms of herpetic epithelial keratitis versus control (P < 0.05). Moreover, VACV 100 mg/kg was superior to other treatments. Viral titers in mouse eyeball and trigeminal ganglia were lowest in the VACV 100 mg/kg group versus other treatment groups. CONCLUSION: Our case example suggests that when frequent application, blurred vision, and foreign body sensation after ACV-O application cause difficulty for patients to follow treatment, oral VACV might be an effective and safe option for patients with herpetic keratitis.

Excimer laser photokeratectomy reactivates latent herpes simplex virus.

BACKGROUND: It has been reported that excimer laser irradiation might elicit herpes simplex virus (HSV) genome activation. We describe a clinical case in which HSV DNA sequences were detected quantitatively after phototherapeutic keratectomy (PTK). CASE: A 90-year-old woman underwent excimer laser photokeratectomy for bilateral band-shaped keratopathy. Tear film was collected from both eyes using a Schirmer's strip before and 3 and 7 days after phototherapeutic keratectomy. OBSERVATIONS: HSV-DNA was quantified by a real-time polymerase chain reaction assay. HSV-DNA was detected only on the third day postoperatively in both eyes. The amount of viral DNA was 2.0 x 10(5) (OD) and 1.3 x 10(5) (OS) copies/sample, respectively. CONCLUSIONS: Excimer laser photokeratectomy stimulated viral shedding in the tear film. Ophthalmologists should be aware that laser irradiation can reactivate latent HSV.

Microarray analysis in the HSV-1 latently infected mouse trigeminal ganglion.

PURPOSE: To review our previous studies regarding alterations in gene expression in HSV-1 latently infected mouse trigeminal ganglia (TGs) following treatment with immunosuppressants and hyperthermia. METHODS: Uninfected and HSV-1 latently infected mice were treated with immunosuppressants or heat stressed (43 degrees C for 10 minutes). In the immunosuppressant study, 4 groups of animals were examined: (1) uninfected, not treated; (2) uninfected, drug-treated; (3) latently infected, not treated; and (4) latently infected, drug-treated. In the hyperthermia study, TG from 6 groups of mice were studied: (1) uninfected, not stressed; (2) uninfected, heat-stressed; killed at 6 hours after hyperthermia; (3) uninfected, heat-stressed, killed at 24 hours after hyperthermia; (4) latently infected, not stressed; (5) latently infected, heat-stressed, killed at 6 hours after hyperthermia; and (6) latently infected, heat-stressed, killed at 24 hours after hyperthermia. PolyA mRNA from the TGs of each group was reverse-transcribed, labeled with P, incubated on a gene array membrane, and analyzed by phosphorimaging. As a comparison and to confirm microarray results, semiquantitative RT-PCR for selected genes was also performed. RESULTS: The immunosuppressive drugs significantly increased expression of two genes--calpactin 1 light chain and guanine nucleotide-binding protein alpha stimulating activity polypeptide (GNAS)--in the ganglia of uninfected mice compared with untreated, uninfected mice. Ten genes were shown to be significantly increased in the latent TGs from mice treated with the immunosuppressants compared with latently infected untreated mice. These genes were prostaglandin E2 receptor EP4 subtype (PTGER4), insulin promoter factor 1 (IPF1), glutathione S-transferase mu2, cyclin D2, peripherin, plasma glutathione peroxidase, methyl CpG-binding protein 2, retinal S-antigen, ErbB2 protooncogene, and GNAS. Eight genes were shown to be significantly decreased in the HSV-1 latent TGs treated with the drugs compared with untreated latent mice. These genes were peripheral myelin protein 22, decorin, transcription factor AP-1, dystroglycan 1, myelin protein zero, mitogen-activated protein kinase 3, prothymosin beta4, and brain lipid-binding protein. The results obtained by semiquantitative RT-PCR results were similar to those obtained by microarray analysis. Six hours after heat stress, the genes whose expression was altered included the FK506-binding protein gene (decreased), the T-complex protein 1alpha subunit gene (increased), and the 94-kDa glucose-regulated protein gene (increased in uninfected TG, decreased in infected TG). Heat stress increased expression of the DNA excision repair protein ERCC5 gene 24 hours after the treatment. Genes previously reported to exhibit increased transcription 1 hour after heat stress did not continue to show significant transcriptional activation at 6 or 24 hours. CONCLUSIONS: Those genes whose expression is altered by immunosuppressive drug treatment may play an important role in ocular HSV-1 recurrence. Changes in gene expression in the prostaglandin pathway, a transcription factor, and an enzyme in the cell cycle are considered of special importance for HSV-1 reactivation by immunosuppression. Altered gene expression at 6 and 24 hours after heat stress was different from previously reported changes in gene expression 1 hour after hyperthermia in HSV-1 latently infected mice.

Herpes simplex virus genome quantification in two patients who developed herpetic epithelial keratitis during treatment with antiglaucoma medications.

PURPOSE: We quantitated herpes simplex virus (HSV) DNA in tear film obtained from 2 patients who developed herpetic epithelial keratitis (HEK) during treatment with latanoprost and a beta-blocker. METHODS: The patient in case 1 is a 77-year-old woman with bilateral open-angle glaucoma who had been treated with latanoprost and timolol for 11 months. She developed HEK in the right eye followed by HEK in the left eye 1 month later. Both eyes healed with administration of acyclovir. Ten months later, HEK recurred in the right eye. The patient in case 2 is a 45-year-old man with bilateral normal tension glaucoma who had been treated with latanoprost for 2 years. After concurrent treatment with nipradilol for 5 months, typical dendritic keratitis developed in the left eye. In both cases, a real-time PCR assay was used to quantify HSV-DNA in the tear film. RESULTS: In the patient in case 1, 71 copies of the HSV genome were detected in the tear film obtained from the right eye at the time of presentation with HEK. After 1 week of treatment with topical acyclovir ointment, the corneal epithelial defects healed and the number of HSV genome copies present in the tear film fell below the sensitivity limit of the assay. In the patient in case 2, 7.0 x 10 copies of the HSV genome were detected in the tear film from the left eye. After 3 days of topical acyclovir ointment, it healed and the HSV genome in the tear film became undetectable. CONCLUSIONS: Herpetic keratitis may occur during treatment with latanoprost and beta-blockers. The amount of HSV DNA detected in the tear film paralleled with the activity of the corneal lesion.

Quantitative analysis of herpes simplex virus genome in tears from patients with herpetic keratitis.

PURPOSE: Herpetic keratitis is manifested in various corneal disorders, for example, dendritic keratitis, persistent epithelial defect, disciform keratitis, and endotheliitis. In this paper, we report on the quantity of herpes simplex virus (HSV) genome in tears from patients with various types of herpetic keratitis in an attempt to understand the role of HSV in these conditions. METHODS: We collected tear samples from both eyes of 56 consecutive patients with herpetic keratitis who visited Kinki University Hospital between June 2000 and May 2002. All patients had unilateral herpetic keratitis: epithelial keratitis in 27 eyes; persistent epithelial defect in 6; active disciform stromal keratitis in 14; silent stromal keratitis in 6; and endotheliitis in 3. We measured levels of HSV genome in these tear samples using a real-time polymerase chain reaction (PCR) method. RESULTS: In epithelial keratitis, HSV DNA was detected in all 27 samples from affected eyes (6.4 +/- 4.4 x 10(5) copies/sample). In persistent epithelial defect, HSV DNA was detected in all 6 samples from affected eyes (8.5 +/- 3.3 x 10(4) copies/sample). In active disciform stromal keratitis, HSV DNA was detected in 8 of the 14 affected eyes (1.4 +/- 1.1 x 10(5) copies/sample including zero values in negative samples). HSV DNA was not detected in samples from unaffected eyes or eyes affected by silent stromal keratitis or endotheliitis. CONCLUSION: Real-time PCR is a useful method for quantifying HSV DNA in tear samples from patients with herpetic keratitis. Using this method, we demonstrate that HSV reproduction occurs in persistent epithelial defect and disciform stromal keratitis.