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Purpose: Aciclovir (ACV), valaciclovir (VACV) and famciclovir (FCV) are used for systemic infections caused by herpes virus. In Japan, only topical ACV is permitted for use against herpetic keratitis. We investigated the effectiveness of topical ACV, oral VACV and oral FCV on mouse epithelial herpetic keratitis., Methods: C57/BL76 mice were inoculated with HSV-1 McKrae strain in the cornea. Once infection was confirmed 4 days after inoculation, topical ACV, oral VACV and FCV were started and administered for 5 days. Control groups were given either topical or oral saline. On days 2, 4, 6 and 10 after medication started, tears, eyeballs, and trigeminal ganglia were examined using viral culture and real-time PCR., Results: Viral culture of tears detected no HSV in the topical ACV group on day 4 after administration start; with similar results for the oral VACV group on day 4; and the oral FCV group on day 6. Real-time PCR of the eyeballs showed significant decrease of HSV DNA copy number in the topical ACV group on days 4 and 6 compared to the topical saline group. Real-time PCR of the trigeminal ganglia showed significant decrease of HSV DNA copy number in the oral VACV group on days 4 and 6, and in the oral FCV group on day 6 compared to the oral saline group., Conclusion: We suggest that 5-day administration of topical ACV, oral VACV and oral FCV are effective for mouse epithelial herpetic keratitis and sufficiently decrease HSV amounts in the ocular surface and eyeballs.

Purpose: Trigeminal and other ganglia are known as sites of latent infection by herpes simplex virus type 1 (HSV-1). In ophthalmology, HSV-1 remains latent in the trigeminal ganglia, and becomes reactivated by several factors, including stress, thermal stimulation, or immunosuppression, and may lead to herpetic keratitis. The purpose of this study was to demonstrate HSV corneal latent infection using molecular biology and virology techniques., Methods: Six corneas obtained at penetrating keratoplasty were snap-frozen; three of them were with past history of herpetic keratitis. TaqMan Real-time PCR was used to show positive HSV DNA in the corneas. We proved negative homogenate and positive explant virologically. Using real-time RT-PCR, we showed that only latency-associated transcript (LAT) was detected and no transcriptional products of other virus genes (α, β, γ) were detected., Results: All three corneas with past history of herpetic keratitis had HSV DNA and showed negative homogenate and positive explant. LAT was detected in all three corneas. However, α, β, or γ genes were not expressed. All the results of these corneas were consistent with the conditions of corneal latency. The other three corneas without history of herpetic keratitis showed negative homogenate and negative explant. None of them had LAT., Conclusion: We have shown a possibility that HSV can latently infect the cornea aside from the ganglion.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in sensory neurons that can sometimes be reactivated. HSV-1 keratitis often recurs and can be vision threatening. Reactivation of the latent virus can be stimulated by stress, immunosuppression, trauma, adrenergic iontophoresis, and UV radiation. Healthy and asymptomatic individuals are known to shed HSV-1, and this is a major factor in the spread of the virus. We investigated the frequency of shedding of HSV-1 DNA in tears of dry eye patients and individuals with conjunctivitis. Subjects were divided into 3 groups: normal (12 eyes), dry eye (11 eyes), and conjunctivitis (15 eyes). Quantitative real-time polymerase chain reaction was used for HSV DNA detection. The incidences of HSV positivity in the normal, dry eye, and conjunctivitis groups were 1 of 12 (8.3%), 3 of 11 (27.3%), and 4 of 15 (26.7%), respectively. We have previously shown that bromfenac sodium eye drops, intramuscular adenosine monophosphate, and geldanamycin effectively lower HSV-1 recurrence rates in a mouse model. Recently, we also found that nuclear factor κ-B, an IκB kinase-β inhibitor, could be a candidate for reducing HSV-1 reactivation. We sampled recipients' corneal buttons during keratoplasty and performed polymerase chain reaction. Cytomegalovirus (CMV) DNA was detected in corneas obtained from some patients, and the copy number of the detected CMV DNA was quantified. CMV DNA-positive samples were taken from 2 of the 3 patients with ocular pemphigoid; thus, in future work, the relationship between CMV in the cornea and the incidence/onset of ocular diseases of the anterior segment needs to be evaluated.

Purpose: We detected herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV), and cytomegalovirus (CMV) DNAs in recipient corneal buttons taken at the time of penetrating keratoplasty., Methods: Twenty-seven corneal buttons were obtained from 27 patients (10 men and 17 women), 7 of whom had a history of HSV keratitis. Excised corneal buttons were immediately frozen in liquid nitrogen in the operating theater and then stored at -80°C until DNA extraction. The detection of HSV-1, HSV-2, VZV, and CMV DNAs was carried out by a nested polymerase chain reaction (PCR) method. The genome copy numbers for the nested PCR-positive samples were subsequently quantified by real-time PCR., Results: HSV-1, HSV-2, VZV, and CMV DNAs were detected in 10, 1, 9, and 2 of the 27 recipient corneal buttons, respectively. HSV-1 or HSV-2 DNAs were also detected in 5 of 7 patients with a history of HSV keratitis. Both CMV-positive patients (patients 2 and 3) had ocular pemphigoid. Among the nested PCR-positive samples, 2 HSV-1, 1 HSV-2, 1 VZV, and 1 CMV sample could be quantified by real-time PCR. Copy numbers ranged from 19 to 928 copies., Conclusions: All 4 herpesviruses, including CMV, were detected in the corneal buttons. The relationship between CMV in the cornea and ocular diseases of the anterior segment should be further evaluated.

Purpose: To examine the effects of COX inhibitors on suppressing HSV-1 reactivation in a mouse model., Methods: BALB/c mice were latently infected with HSV-1 and treated by 0.1% bromfenac Na eye drops, 0.1% pranoprofen eye drops, 0.1 mg oral etodolac 4 times/day, and saline for 4 days. After reactivating the latent HSV-1, we swabbed the mouse ocular surface for the culture of the infectious virus and assessed the viral loads in the eyes and trigeminal ganglia (TGs) using real-time PCR to determine the treatment efficacies., Results: With stimulated reactivation, 10 of 24 (41.7%), 5 of 10 (50.0%), 17 of 25 (68%), and 16 of 22 eyes (72.7%) showed positive swab results in the bromfenac Na, etodolac, pranoprofen, and saline groups, respectively; and a significant difference was seen only between the bromfenac Na and saline groups (p = 0.033). None of the three drug-treated groups showed any significant difference from the saline group in the viral DNA in the eyes and TGs (p > 0.05)., Conclusions: Bromfenac Na eye drops can suppress HSV-1 reactivation.

To compare the efficacies of valacyclovir (VCV) and acyclovir (ACV) on murine herpetic epithelial keratitis, mice inoculated with herpes simplex virus type 1(HSV-1) strain McKrae were divided into 6 treatment groups: oral VCV 50 mg/kg and 100 mg/kg, oral ACV 50 mg/kg, ACV eye ointment (EO), ACV eye drops (ED), and placebo. Keratitis scores showed that oral VCV 50 mg/kg, oral ACV, and ACV ED had equivalent efficacies, while oral VCV 100 mg/kg was as efficacious as ACV EO during acute infection. Each treatment group was further divided into the stimulated group with HSV-1 reactivation by immunosuppressant drugs and hyperthermia, and the non-stimulated group without reactivation. We assessed the virus titers in tissues by plaque assay and HSV DNA copy number in the trigeminal ganglia (TG) by real time polymerase chain reaction (PCR). Results showed that the virus titers in the tissues were lowered after reactivation, and the oral VCV group with reactivation had significantly reduced DNA copy number in the TG than the same treatment group without reactivation. In conclusion, oral VCV is as efficacious as ACV EO and significantly suppresses HSV-1 reactivation.

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.

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.

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.

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.

Purpose: Polymerase chain reaction (PCR) detects the genomic materials of etiological agents with high specificity and sensitivity. However, in herpes simplex virus type 1 (HSV-1) infection, the clinical significance of the results often poses controversy because of the subclinical viral shedding during latent infection. Quantitative PCR might provide additional information to help clinical evaluation of the results., Methods: Virus DNA was extracted from high titer stock of human HSV-1 (FK 25) by phenol/chloroform treatment. Construct (p/HSV-1) was made by inserting the glycoprotein D gene obtained from virus DNA into p(GEM-T) vector. Competitor (p/DeltaHSV-1) was made by deleting the inner 40 bp of construct (p/HSV-1) with restriction enzyme. Competitive PCR was performed using primers that amplify the glycoprotein D gene, and a template made of a 1:1 molar mixture of HSV-1 DNA and the competitor., Results: The PCR product reflected the initial template dose from 20 to 30 cycles. Minimum detection level of HSV-1 DNA was 0.01 ng., Conclusion: Competitive PCR can quantitate HSV-1 DNA.

The identification of factors involved in herpes virus latency and reactivation is critical to a better understanding of the mechanisms essential to viral neuroinvasiveness and neurovirulence. Recurrent episodes of ocular herpes infections cause irreversible corneal scarring and are the primary cause of loss of vision due to an infectious agent in industrialized countries. In this study, we examined the ability of nicotine, a compound known to be involved in stress-associated immunomodulation and recognized as one of the most frequently used addictive agents, to induce ocular shedding in rabbits latently infected with herpes simplex virus type 1 (HSV-1) strain McKrae. New Zealand white rabbits latently infected with HSV-1 at 3-4 weeks post-inoculation were randomly divided into two groups. The corneas of all rabbits were free of lesions as verified by slit lamp biomicroscopy. One group received nicotine by transdermal patch (21 mg/day) for 20 days and the other group served as the control. Reactivation data were obtained by detection of virus in tear film collected by ocular swabbing performed concurrently with the administration of nicotine. Compilation of data from three separate experiments demonstrated that 16.5% (258/1560) of the swabs taken from rabbits treated with nicotine were positive for virus, compared with 8.3% (53/639) of swabs taken from controls. Rabbits receiving nicotine exhibited a significantly (P < 0.0001) higher rate of ocular shedding than controls. The concentration of nicotine in the serum was determined at various times (0-24 hrs) after new patch replacement. Peak (average) serum level of nicotine was obtained 8 hours after patch replacement and exhibited a broad range of values (0.233 microg/mL-6.21 microg/mL). These results suggest that an initial systemic exposure to nicotine significantly increases HSV-1 reactivation. Further studies are needed to reveal any effects of nicotine dependency and nicotine withdrawal on herpesvirus reactivation.

Purpose: To assess gene expression in herpes simplex virus type 1 (HSV-1) latent mouse trigeminal ganglia (TG) at 6 and 24 hours after hyperthermic stress., Methods: Uninfected and HSV-1 latently infected mice were heat stressed (43 degrees C, 10 min). TG from six groups of mice were studied: 1) uninfected, not stressed, 2) uninfected, heat-stressed, sacrificed at 6 hours after hyperthermia, 3) uninfected, heat-stressed, sacrificed at 24 hours after hyperthermia, 4) latently infected, not stressed, 5) latently infected, heat-stressed, sacrificed at 6 hours after hyperthermia, 6) latently infected, heat-stressed, sacrificed at 24 hours after hyperthermia. Poly A(+) mRNA from the TG of each group of mice was reverse transcribed, labeled with (32)P, and incubated on a nylon gene array membrane. The genes showing the largest signal-to-control changes (varying by a factor of at least 1.27-fold) were considered to have undergone significant change in expression., Results: Six hours after heat stress the genes whose expression was altered included the FK506-binding protein gene (decreased), the T-complex protein 1 alpha 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 stress did not continue to show significant transcriptional activation at 6 or 24 hours., Conclusion: 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.

Purpose: To compare the usefulness of the in vitro quiescently infected (QIF)-PC12 cell model(30) with the in vivo rabbit eye model of latency for the study of herpes simplex virus (HSV) genes implicated in reactivation from latency., Methods: HSV-1 strains 17+/pR20.5/5 and 17+/pR20.5/5/LAT, that were previously constructed by insertion of genes encoding beta-galactosidase, green fluorescent protein (GFP) or the latency associated transcript (LAT) open reading frame in the U(S)5 region,(34) were used to examine viral growth and inducible reactivation in the two models., Results: 17+/pR20.5/5 exhibited diminished reactivation phenotype when compared with wild type 17+ in neuronal cells (i.e., QIF-PC12 cell model) and the rabbit eye model of latency. 17+/pR20.5/5/LAT, which contains the deregulated LAT gene, reactivated at wild type levels. Analysis of growth in neurally differentiated (ND)-PC12 cells demonstrated a low proportion of QIF cells expressed virus-encoded signals during the quiescent infection and a direct relationship between lytic viral growth in neuronal cells and reactivation phenotype. Even though 17+/pR20.5/5/LAT produced a more severe acute infection in the rabbit cornea, the different reactivation efficiency of 17+/pR20.5/5 and 17+/pR20.5/5/LAT in vivo and in vitro was not attributed to different viral genome copy number in the cells harboring cryptic genomes., Conclusions: We conclude that 1) viral growth in neuronal cells correlates with reactivation phenotype in vivo and in vitro, 2) 17+/pR20.5/5 is attenuated in viral growth and reactivation in both models, and 3) 17+/pR20.5/5/LAT demonstrates wild-type phenotype for reactivation in both models. Attenuation of 17+/pR20.5/5 could be the result of the disruption of U(S)5 or a second site mutation. If the attenuation is the result of U(S)5 disruption, a gene that provides anti-apoptotic functions,( 41,42) this attenuation is more than compensated for by the expression of the LAT ORF. Overall, the findings indicate that the QIF-PC12 cell model is useful for segregating phases of reactivation, and particularly studying the inductive events involved in reactivation of a cryptic viral genome in neurally differentiated cells.

Purpose: To determine alterations in expression of genes in herpes simples virus (HSV)-1 latently infected mouse trigeminal ganglia (TGs), after treatment with cyclophosphamide and dexamethasone., Methods: Scarified corneas of female BALB/c mice were inoculated with HSV-1 strain McKrae. Four weeks after inoculation, cyclophosphamide and dexamethasone were intravenously injected to induce HSV-1 reactivation. Uninfected mice were also treated with the immunosuppressants. Four groups of animals were studied: uninfected, not treated; uninfected, drug treated; latently infected, not treated; and latently infected, drug treated. PolyA+ mRNA from the TGs of each group was reverse transcribed, labeled with 32P, incubated on a 1185-gene array membrane, and analyzed by phosphorimaging. As a comparison and to confirm microarray results, semiquantitative RT-PCR was also performed for selected genes., Results: The immunosuppressive drugs significantly increased expression of two genes (calpactin 1 light chain and guanine nucleotide-binding protein alpha-stimulating polypeptide [GNAS]) in the ganglia of uninfected mice compared with those in untreated uninfected mice. Ten genes were shown to be significantly increased in the latent TGs of mice treated with 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 proto-oncogene, and GNAS. Eight genes were shown to be significantly decreased in the HSV-1 latent TGs treated with the drugs, compared with untreated latently infected mice. These genes were peripheral myelin protein 22, decorin, transcription factor AP-1, dystroglycan 1, myelin protein zero, mitogen-activated protein kinase 3, prothymosin beta 4, and brain lipid-binding protein. The results obtained by semiquantitative RT-PCR were similar to those obtained by microarray analysis., Conclusions: Those genes with expression altered by immunosuppressive drug treatment may play an important role in ocular HSV-1 recurrence. Changes in expression of genes in the prostaglandin pathway, a transcription factor, and an enzyme in the cell cycle are considered especially important in HSV-1 reactivation by immunosuppression and are reviewed.

An understanding of the cellular genes whose expression is altered during HSV reactivation will enable us to better understand host responses and biochemical pathways involved in the process. Furthermore, this knowledge could allow us to develop gene-targeted inhibitors to prevent viral reactivation. Mice latent with HSV-1 strain McKrae and uninfected control mice were subjected to hyperthermic stress (43 degrees C for 10 min) and their trigeminal ganglia (TG) collected 1 h later. Two additional groups included HSV-1 latently infected and uninfected mice not subjected to hyperthermic stress. Poly A+ mRNA was enriched from total mouse TG RNA and reverse transcribed using MMLV RT. Radioactively labeled cDNAs were analyzed by microarray analysis. A stress/toxicology array of 149 mouse genes on a nylon membrane was used. The labeled cDNAs prepared from latently infected, stressed mice demonstrated 3-fold or greater increases in certain mRNA-early response genes (ERGs) compared to cDNAs from uninfected, stressed control mice. The ERG mRNAs that showed increases included two heat shock proteins (HSP60 and HSP40), a basic transcription factor (BTF T62), a DNA repair enzyme, two kinases [MAP kinase and a stress-induced protein kinase (SADK)], an oxidative stress-induced protein, a manganese superoxide dismutase precursor-2 (SOD-2), and cyclooxygenase 2 (COX-2). The gene expression in unstressed, infected TGs was similar to the gene expression in unstressed, uninfected controls. These results suggest that there is a significant difference in the ERG expression profile in latently infected TGs undergoing stress-induced reactivation compared to uninfected TGs.