Diagnostic efficacy of real-time PCR for ocular cytomegalovirus infections.

PURPOSE: The aim of this study is to determine the efficacy of quantitative real-time PCR (qPCR) and clinical characteristics to diagnose ocular cytomegalovirus (CMV) infections. METHODS: The technical factors were assessed by the outcomes of the qPCR assay at five institutions in Japan using the WHO International Standard of cytomegalovirus. The clinical factors were assessed by examining the aqueous humor samples of 197 eyes of 197 consecutive patients suspected of CMV using the receiver operating characteristics (ROCs). RESULTS: All of the institutions had excellent detection efficacy, although the copy number ranged from 0.82 to 4.66 copies/IU. In the clinical samples, CMV was detected in 51 eyes, and the amount of CMV DNA was significantly higher for CMV retinitis. In corneal diseases, the amount of CMV DNA was significantly associated with frequency of recurrences and IOP elevations. The sensitivity and specificity of qPCR for the diagnosis was 90.0 and 98.7%, respectively. For the corneal and anterior uveitis types of CMV diseases, the area under the curve (AUC) of qPCR was 0.95 and 0.96, followed by frequency of recurrences with AUC of 0.89 and 0.82, and IOP elevations with AUC of 0.78 and 0.76. Unclassified cytomegalovirus detection, which did not meet diagnostic criteria of CMV corneal endotheliitis, anterior uveitis, or retinitis, was 4.6%, and it was significantly associated with corneal diseases and history of corneal transplantation. CONCLUSIONS: qPCR with standardization is specific and accurate; however, the inclusion and knowledge of the clinical characteristics improve the diagnostic efficacy.

Assessment of real-time polymerase chain reaction detection of Acanthamoeba and prognosis determinants of Acanthamoeba keratitis.

OBJECTIVE: To evaluate the diagnostic value of real-time polymerase chain reaction (PCR) for detecting Acanthamoeba in eyes diagnosed with Acanthamoeba keratitis (AK) by conventional tests. In addition, to determine the preoperative prognosis-determining factors in eyes with AK. DESIGN: Retrospective, cross-sectional study. PARTICIPANTS: A total of 104 eyes of 103 patients who were diagnosed with AK or with bacterial or bacteria-associated keratitis (BK) by conventional tests. METHODS: Twenty-nine eyes with AK and 75 eyes with BK were evaluated for Acanthamoeba and bacterial DNA by real-time PCR. The Acanthamoeba copy numbers, bacterial load, and clinical parameters in the patients with AK were assessed for those significantly associated with poor outcome, that is, final visual acuity of <20/50 or requiring keratoplasty, by logistic regression analysis. MAIN OUTCOME MEASURES: Acanthamoeba DNA copy number, bacterial DNA copy number, and odds ratio (OR) for poor prognosis. RESULTS: The detection of amoebic DNA was 50 times more sensitive by real-time PCR than by conventional cyst counting. The Acanthamoeba copy numbers at the first visit (mean: 4.7×10(5)±3.2×10(5) copies) were significantly correlated with the AK stage, and both were significant risk factors for a poor outcome. The Acanthamoeba DNA copy numbers at the first visit and AK stage had a significantly high risk for poor outcome (OR of Acanthamoeba DNA copy per logarithm of copy numbers: 3.48, 95% confidence interval [CI], 1.04-111.63, P<0.05; OR of AK stage: 2.8 per stage increase, 95% CI, 1.07-7.30, P<0.05, after adjustment of age). In the AK cases with poor outcome, the amoebic DNA was not reduced by more than 90% after 1 month of treatment. The weak amoebic reduction was significantly associated with advanced AK stages or previous use of steroids. Bacterial 16S rDNA was detected in 53.6% of the eyes with AK, but it was not associated with any risk for refractoriness. CONCLUSIONS: Real-time PCR was effective in detecting and managing AK. The Acanthamoeba copy number and AK stage at the first visit were significantly associated with poor outcome. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

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.

Effects of anti herpetic drugs on mice with herpetic epithelial keratitis after reactivation of herpes simplex virus type 1.

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.

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.

Case of bilateral multiple herpetic epithelial keratitis manifested as dendriform epithelial edema during primary Kaposi's varicelliform eruption.

BACKGROUND: Bilateral herpetic keratitis has been reported in patients with atopy, measles, graft-versus-host disease, and altered immune status. We report a serologically verified case of primary, simultaneous onset, bilateral, atypical epithelial herpetic keratitis that manifested as dendriform epithelial edema during a generalized dermatitis incident. CASE: A 37-year-old man with chronic atopic dermatitis developed Kaposi's varicelliform eruption and bilateral dendritic epithelial keratitis with corneal epithelial edema. OBSERVATIONS: The pathogens isolated from both eyes were identified as herpes simplex virus type 1 (HSV-1) by a direct immunofluorescence method. Serological tests obtained on three different occasions over a 5-week period verified a primary HSV infection. CONCLUSIONS: With serological verification, we report a rare case of primary, simultaneous onset, bilateral, dendritic epithelial keratitis at a very early stage with complications of generalized herpetic disease in a patient with atopic dermatitis.

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.

Virological and molecular biological evidence supporting herpes simplex virus type 1 corneal latency.

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, ß, 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, ß, 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.

Effects of antiviral medications on herpetic epithelial keratitis in mice.

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.

Effect of immunosuppression on gene expression in the HSV-1 latently infected mouse trigeminal ganglion.

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.

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.

Gene expression profiling in the HSV-1 latently infected mouse trigeminal ganglia following hyperthermic stress.

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.

Herpesvirus quiescence (QIF) in neuronal cells VI: Correlative analysis demonstrates usefulness of QIF-PC12 cells to examine HSV-1 latency, reactivation and genes implicated in its regulation.

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.

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.

Results of penetrating keratoplasty triple procedure with 25-gauge core vitrectomy.

PURPOSE: To report and compare the outcomes of penetrating keratoplasty (PKP) triple procedures [combined PKP, cataract extraction, and intraocular lens (IOL) implantation] with a 25-gauge (G) system, 20-G system, and without core vitrectomy. SUBJECTS AND METHODS: Subjects comprised the following 3 groups: the 25-G group including 12 eyes of 12 patients (4 men and 8 women) that underwent PKP with 25-G core vitrectomy, the 20-G group including 9 eyes of 9 patients (3 men and 6 women) that underwent PKP with 20-G core vitrectomy, and the non-core vitrectomy group including 9 eyes of 9 patients (1 man and 8 women) that underwent PKP without core vitrectomy. RESULTS: In the 25-G, 20-G, and non-core vitrectomy groups, the success rates of IOL implantation were 91.7% (11 of 12 eyes), 88.9% (8 of 9 eyes), and 66.7% (6 of 9 eyes), respectively; the average operation times were 69 minutes, 82 minutes, and 90 minutes, respectively. The 25-G group showed significantly shorter operation time than the other 2 groups. Although not statistically significant, a higher rate of capsular rupture was seen in the non-core vitrectomy group. CONCLUSIONS: A PKP triple procedure with the 25-G system can be a safe treatment that offers a better success rate of IOL implantation and a significantly shorter operation time.