Effect of acyclovir on thermal stress-induced herpesvirus reactivation

PURPOSE: Acyclovir has been shown to be effective in preventing recurrent herpes simplex virus lesions of the genitalia and oral labia. The purpose of the current study was to determine the effect of acyclovir on the appearance of infectious virus in the peripheral nervous system and in an end organ, the eye. MATERIALS AND METHODS: Mice latent for the McKrae strain of herpes simplex virus type 1 were given 3.5 mg/ml acyclovir in their drinking water. Control animals received water without drug. Acyclovir treatment was continued for 4 successive days. On the third day, the mice were subjected to a brief period of hyperthermic stress to induce viral reactivation. Twenty-four hours after stress induction, swabs of the ocular surface and homogenates of the cornea and trigeminal ganglia were analyzed for the presence of infectious herpes simplex virus type 1 and viral DNA. RESULTS: Acyclovir treatment significantly decreased the frequency of infectious virus in the ocular tear film and the cornea but not in the trigeminal ganglion. The corneal homogenates of acyclovir-treated animals contained smaller amounts of viral DNA compared with untreated controls, whereas the amounts of viral DNA in the trigeminal ganglia of acyclovir-treated and untreated animals were similar. CONCLUSIONS: These results suggest that oral administration of acyclovir, at least at the dose used in this study, is effective in modestly reducing viral replication in peripheral tissues such as the eye but is not effective in inhibiting viral reactivation and viral DNA synthesis in the peripheral nervous system in mice subjected to induction of reactivation by hyperthermic stress.     

单纯疱疹病毒Ⅰ型潜伏感染的研究——兔三叉神经节致敏T淋巴细胞检测研究

报告应用抗兔致敏T淋巴细胞的单克隆抗体和ABC免疫组织化学技术对22只实验性单纯疱疹病毒性角膜炎的家兔三叉神经节致敏T淋巴细胞检测的结果，首次揭示在角膜原发感染HSV-I过程中三叉神经节内致敏T淋巴细胞的出现。致敏T淋巴细胞在角膜接种后第13天出现。第85天消失，结果表明：T淋巴细胞在TG内HSV-I潜伏感染的建立过程中起重要作用。本研究对指导临床治疗提供了理论依据。     

Acetylsalicylic acid reduces viral shedding induced by thermal stress

PURPOSE: To investigate the effect of acetylsalicylic acid on ocular shedding of herpes simplex virus type 1 (HSV-1). MATERIALS AND METHODS: Mice that were latent for the McKrae strain of HSV-1 were treated with acetylsalicylic acid, a nonspecific inhibitor of cyclooxygenases, either prophylactically or at the time of heat stress-induced viral reactivation. The effect of the drug on viral shedding in the tear film, infectious virus in the cornea and trigeminal ganglion, and viral DNA in the cornea and trigeminal ganglion was determined. RESULTS: Acetylsalicylic acid inhibited heat stress-induced shedding of virus in the tears and reduced the numbers of corneal and trigeminal ganglion homogenates containing virus. Intraperitoneal therapeutic and oral prophylactic plus therapeutic treatments were similar in their ability to inhibit reactivation. CONCLUSIONS: The results indicate that a cyclooxygenase inhibitor such as acetylsalicylic acid can reduce recurrent viral infection in mice. These findings may implicate prostaglandins as agents in the viral reactivation process and suggest that therapy to suppress viral reactivation using nontoxic inhibitors of prostaglandin synthesis may be effective in humans.     

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.     

Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.

The authors characterized a murine model of herpes simplex virus (HSV) reactivation in which recurrent herpetic keratitis was obtained in up to 80% of animals. Five weeks after ganglionic latency was established in National Institutes of Health inbred mice after corneal inoculation, HSV type 1 (HSV-1) was reactivated by irradiating the previously inoculated eye with ultraviolet (UV) light. Comparison of different UV wavelengths showed UVB to be optimal for reactivation, with peak viral recurrence being induced by a total exposure of approximately 250 mJ/cm2. Reactivated infectious virus generally began to appear in trigeminal ganglia 2 days postirradiation and was subsequently detectable in the cornea by both corneal swabbing and immunostaining for viral antigens. Two consecutive outbreaks of viral recurrence at the ocular surface were induced in selected animals by serial exposure to UVB. Advantages of this model over other models of recurrent keratitis are discussed.     

Effect of immunization on herpes simplex virus type 1 latent infection in the trigeminal ganglion

PURPOSE: To quantify and characterize immune protection from herpes simplex virus (HSV) latent infection in mice following corneal challenge. METHODS: Mice immunized or mock-immunized and boosted in the flank with an HSV replication-deficient mutant were challenged by corneal inoculation with wild type (wt) or thymidine kinase-negative (TK(-)) HSV. At specified times post challenge, trigeminal ganglia were assayed for in vitro reactivation, latent and acute viral load (using quantitative PCR), acute infection, and cellular infiltration (hematoxylin and eosin stained sections). RESULTS: With wt HSV challenge infection, immunization led to reduced reactivation, significantly less latent and acute viral DNA, and no acute viral replication in ganglia, and rapid infiltration of inflammatory cells. Immunization had little effect on viral load following challenge with replication-conditional TK(-) mutant virus. CONCLUSION: These results indicate that immune protection from latent HSV infection in mouse trigeminal ganglia following ocular infection can act under these experimental conditions to block acute viral replication in ganglia and is directed to antigenic targets within the ganglia.     

Inhibition of cyclooxygenase 2 synthesis suppresses Herpes simplex virus type 1 reactivation.

Recurrent herpes virus infection, in which the virus reactivates from the nervous system and causes painful lesions in peripheral tissues, is a significant clinical problem. Our recent studies showing that the amount of cyclooxygenase 2 (COX-2) in the trigeminal ganglia of heat-stressed untreated mice is higher than the amount in heat-stressed mice treated with the COX-2 inhibitor, celecoxib, have indicated that the prostaglandin synthesis pathway--and in particular COX-2--may be an intermediate in the pathway to herpes viral reactivation. To further study this process, we infected the corneas of mice using topical application to a lightly scratched epithelium and waited 30 days for Herpes simplex virus type 1 (HSV-1) latency to be established in the trigeminal ganglia. Prior to the induction of viral reactivation, the mice were treated orally with celecoxib. Treated and untreated mice were induced to undergo reactivation by immersion in 43 degrees C water for 10 min. The shedding of virus at the ocular surface was determined by culturing ocular swabs with indicator cells. The presence of infectious virus in the trigeminal ganglion was evaluated by incubating ganglion homogenates with indicator cells and observing for cytopathic effect. Celecoxib treatment significantly suppressed viral reactivation when given prophylactically by the gastrointestinal route. The numbers of corneas and ganglia containing infectious virus were significantly lower in the celecoxib-treated animals, compared to the placebo-treated mice. These experiments demonstrate that a selective COX-2 inhibitor can suppress hyperthermic stress-induced herpes viral reactivation in the nervous system. It may be possible to use COX-2 inhibitors to prevent viral reactivation in high-risk patients by drug prophylaxis.     

Reactivation of murine latent HSV infection by epinephrine iontophoresis

Iontophoresis of epinephrine into the cornea of previously infected mice was used in an attempt to induce reactivation of latent herpes simplex virus (HSV) infection of the trigeminal ganglia. BALB/c mice infected with HSV-1 strain McKrae following corneal scarification developed a latent infection of the trigeminal ganglia within 15 days. At 28 days postinfection, mice were subjected to a 3-day cycle of iontophoresis of epinephrine (0.01%) into the cornea. Ocular shedding of HSV occurred in 16/23 (70%) of stimulated mice; these animals did not shed HSV in the 3-day period prior to iontophoresis. Spontaneous shedding of HSV, however, was noted in 3/97 (3%) mice not subjected to epinephrine iontophoresis. "Infectious" virus was isolated only from the trigeminal ganglia of stimulated mice, whereas "latent" virus was isolated from the trigeminal ganglia of both stimulated and nonstimulated mice. All virus isolates were verified to be HSV by neutralization with a known HSV-1 antiserum. This ocular system thus allows for the study of the full spectrum of latent HSV infections, including latency, ganglionic reactivation, and peripheral virus shedding.     

单纯疱疹病毒性角膜炎的发病机制

单纯疱疹病毒性角膜炎(herpes simplex keratitis,HSK)是一种常见的眼部疾病,由单纯疱疹病毒(herpes simplex virus,HSV)感染引起。人群中超过90%的人曾经感染过HSV。HSV可以在神经组织及角膜组织长期潜伏。在适宜的刺激下,如紫外线照射、发热、精神压力、高温、低温、手术等,病毒活化增殖导致HSK。HSV感染引起的免疫反应是造成角膜组织损害的主要机制。HSK的免疫反应主要是由CD4+细胞介导的,而CD8+细胞对病毒感染具有保护作用。     

无环鸟苷抑制小鼠单纯疱疹性角膜炎潜伏病毒激活作用的研究

目的 证实无环鸟苷能够作用于潜伏单郊病毒的激活阶段,抑制病毒活化。方法 以Ｂａｌｂ／ｃ小鼠作为潜伏感染模型,以紫外线Ｂ照射为激活条件,治疗组给予无环鸟苷口服,停药后１天处死动物检测活化病毒。结果 治疗组３０例中无１例,而对照组３０例中有１２例检测出活化病毒（Ｐ〈０．０１）;治疗至照射后２天的１０只鼠无１例,而相应对照组１０只中６例检出活化病毒（Ｐ〈０．０１）。结论 预防性应用ＡＶＣ特异作用于病毒激活期,有效的抑制病毒活化。     

实验性单纯疱疹病毒Ⅰ型感染兔眼部扫描电镜观察研究

应用ＤＭＳＯ（Ｄｉｍｅｔｈｙｌｓｕｌｆｏｘｉｄｅ二甲基亚砜）冷冻割断制样扫描电镜技术首次动态观察了不同感染阶段（ｐｏｓｔＩｎｆｅｃｔｉｏｎ，ＰＩ）实验性单纯疱疹病毒性角膜炎（ＨＳＫ）兔的角膜、三叉神经节（ＴＧ）泪腺等组织的病变情况。初步报告并分析了各组织病变的扫描图像结果，并对冷冻割断法观察扫描电镜的原理及优缺点予以讨论，认为该技术应用于眼部单疱病毒感染的研究图像清晰、直观、简便可行，是一种有实用价值的方法。     

Efficacy of a helicase-primase inhibitor in animal models of ocular herpes simplex virus type 1 infection

PURPOSE: The aim of this study was to evaluate the effect of BAY 57-1293, a helicase-primase inhibitor, on herpes simplex virus type 1 (HSV-1) reactivation in mice and its efficacy on established disease in rabbits. METHODS: BALB/c mice latent for McKrae-strain HSV-1 were reactivated via heat stress, treated with BAY 57-1293, and their corneas were swabbed for virus or the trigeminal ganglia (TG) obtained for quantification of viral DNA. New Zealand white rabbits were infected and treated topically or orally in comparison with trifluridine or valacyclovir. RESULTS: Oral BAY 57-1293 suppressed reactivation in HSV-1-infected mice and reduced the viral load in TG up to four orders of magnitude. In the rabbits, the therapeutic efficacies of topical BAY 57-1293 and trifluridine were similar. Once-daily oral BAY 57-1293 was significantly more effective than valacyclovir and as effective as twice a day topical trifluridine. CONCLUSIONS: BAY 57-1293 may be more effective than valacyclovir, without the cytotoxicity or potential healing retardation seen with trifluridine. Oral BAY 57-1293 may be a substitute for eye drops as an effective treatment for herpetic keratitis and might be useful in treating stromal keratitis and iritis, as well as preventing recurrences of ocular herpes.     

T lymphocytes in the trigeminal ganglia of rabbits during corneal HSV infection

The results of this investigation reveal, for the first time, the presence of thymus-derived (T) lymphocytes in the trigeminal ganglia of rabbits undergoing primary corneal infection with herpes simplex virus type 1. Infiltration of T cells into the trigeminal ganglion was evident at 15 days after primary ocular infection but these cells were no longer present by 45 days after infection. Corneas and trigeminal ganglia of rabbits sacrificed at 3, 7, 12, 15, 20, 25, 30, 45, 70 and 90 days after infection were assayed for infectious virus and stained for viral antigen and immunoreactive T cells. Infectious virus and cells expressing viral antigens were present in the corneas and trigeminal ganglia during the acute phase (day 0-day 14) of the infection. T cell infiltration of the trigeminal ganglion was present as a perivascular infiltrate along with a sparse scattering of these cells among the nerve fibers. The perivascular infiltration is characteristic of viral infection of a tissue and was not seen in the sections of trigeminal ganglia obtained earlier than 15 days or in ganglia obtained 45 days or more after primary corneal infection. This investigation demonstrates conclusively that the neural ganglia are not completely shielded from the host immune response, as evidenced by the observation that immunocompetent T lymphocytes infiltrate the ganglia subsequent to the infection of a peripheral tissue such as the cornea of the eye.     

Detection of herpes simplex virus type 1 in human ciliary ganglia

PURPOSE: To determine whether herpes simplex virus type 1 (HSV-1) DNA is present in the ciliary ganglion (CG). METHODS: Fifty CG and 47 trigeminal ganglia (TG) were resected from 63 formalin-fixed cadavers between 56 and 98 years of age that had been embalmed within 12 hours of death. The donors had no known active HSV infection at the time of death. DNA was extracted from all ganglia by proteinase-K digestion (TG) or digestion by a mild lysis buffer (CG). DNA was amplified by polymerase chain reaction for sequences from human chromosome 18, D18S1259 (positive control), and from the HSV-1 DNA polymerase gene, U(L)30. The amplified DNA was separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with the appropriate digoxigenin-labeled probe that was detected by alkaline phosphatase-conjugated monoclonal antibody. RESULTS: The D18S1259 sequence was amplified from 47 TG and 30 CG samples. Of these samples, 32 (68.0%) of the 47 TG samples and 20 (66.6%) of the 30 CG samples were positive for the UL(30) HSV-1 sequence. CONCLUSIONS: Using amplification of HSV-1 DNA as a surrogate marker of latency, the finding that the frequency of HSV-1 in the CG was approximately the same as that of the TG suggests that the CG may be an additional site of HSV-1 latency in humans. Active infection in or reactivation of HSV-1 from non-TG sites may explain why this virus is able to infect sites, such as the retina, that have no direct connections to the trigeminal nerve.     

人三叉神经节细胞体外培养及对HSV—1感染敏感性的实验研究

首次报告了人三叉神经节Trigeminal Ganglian(TG)三种原代培养细胞在体外对单纯疱疹病毒1型(HSV-1)Mckrae株感染后的不同时相的敏感性实验研究。结果表明人TG中的神经节细胞对HSV-1感染最敏感,HSV—1抗原可以在其胞浆及胞核中迅速出现。成纤维细胞和胶质细胞也有部分感染现象。这种结果提示TG中的神经节细胞很可能是HSV-1主要的潜伏感染细胞。     

Ocular reactivation phenotype of HSV-1 strain F(MP)E,a corticosteroid-sensitive strain

PURPOSE: Elucidate the ocular reactivation of HSV-1 strain F(MP)E. METHODS: Rabbit corneas were infected with HSV-1 strains McKrae and F(MP)E. Latency was established and rabbits were treated with epinephrine iontophoresis or corticosteroid injection (immunosuppression). Cultured tear films were used to determine the presence of infectious virus. Eyes of immunosuppressed rabbits were also examined by slit lamp biomicroscopy. Trigeminal ganglia were co-cultured on indicator cells at time of sacrifice for detection of virus. Quantitative real-time PCR was used to detect the number of viral genome copies in the trigeminal ganglia. RESULTS: Acute infections by strains McKrae and F(MP)E were the same. Ocular reactivation of strain F(MP)E by epinephrine iontophoresis was significantly reduced compared to McKrae (P 0.29). Slit lamp examination following corticosteroid injection showed a correlation of recurrent herpetic lesions with infectious virus in tear film swabs for both McKrae and F(MP)E. Trigeminal ganglia from rabbits latently infected with each strain and each method of induced reactivation resulted in infectious virus (P >/= 0.69). Genome copies for both strains were present and were highly variable. CONCLUSIONS: HSV-1 strain F(MP)E has low reactivation following epinephrine iontophoresis compared to McKrae, but has high reactivation like McKrae in response to corticosteroids. The difference in reactivation following epinephrine iontophoresis is not due to a difference in the establishment of latency.