Cytodiagnosis of herpes simplex keratitis by means of an immunoperoxidase technique. A case report

Typical herpes simplex keratitis that developed in a 5-year-old boy was initially diagnosed cytologically in Papanicolaou-stained samples. Subsequently, an immunoperoxidase staining technique was used to identify the specific type of herpes simplex virus (HSV) in the destained cellular samples. The positive staining helped to establish the diagnosis of a type 1 HSV infection, permitting early treatment with acyclovir and subsequent complete recovery from the ocular herpetic infection. Emphasis is placed on the value of the immunoperoxidase technique for the rapid and specific diagnosis of cases of suspected HSV infection.     

Latent Herpes Simplex Virus from Trigeminal Ganglia of Rabbits with Recurrent Eye Infection

LATENTLY infected sensory ganglia have been thought to be the source of virus for various clinical manifestations of recurrent herpetic disease in man^1,2. In direct support of this concept, we recently showed that herpes simplex virus can induce a latent infection in the spinal ganglia of mice³. This murine infection has not, however, been shown to be accompanied by recurrent disease. Recurrent herpetic eye infection can be produced in the rabbit⁴. If sensory ganglia are involved in recurrent disease, then trigeminal ganglia from rabbits undergoing such recurrent infection would be expected to harbour latent virus. We now report that herpes simplex virus does indeed induce latent infection in trigeminal ganglia of rabbits presenting recurrent eye infection. As in the experiments with mice, infectious virus could not be recovered directly; it was only found when ganglia were established as organ cultures in vitro.     

Intensity of the humoral immune response to herpesviruses as an indicator the body immune deficiency

The state of the local immunity system of the oral cavity was studied by the level of saliva immunoglobulins in patients with different processes on their mucous membranes: herpetic infection, respiratory allergosis and malignant tumors of the mouth cavity and the laryngopharynx. The suppression of the production of sIgA, was accompanied by the enhanced production of antibodies to the most widespread herpesviruses (herpes simplex virus, cytomegalovirus and Epstein-Barr virus). The maximum levels of serum IgG to herpesviruses were determined in patients with malignant tumors. The role of herpesviruses in the pathogenesis of immunodeficient states is discussed.     

Lipid metabolism in experimental virus infections]

The effect of herpes simplex virus (HSV) and influenza virus (IV) on lipid metabolism was studied. In conditions of acute herpetic infection of rabbits we detected typical dyslipidemia, characterized by increased contents of total cholesterol, beta-cholesterol and triglycerides in the absence of trustworthy differences in concentrations of alpha-cholesterol. The use of antiherpetic preparation furavir, on the background of infection, corrected lipid spectrum of the infected animals. Blood lipid disturbances in acute influenza virus infection of mice were not detected. HSV infection of cell culture of human aorta was accompanied by increased accumulation of free lipids in cells. IV infection, in the same conditions of experiment, did not change the contents of intracellular lipids. The obtained data deepen the existing notions of herpetic and influenza infections pathogenesis and may be useful in understanding etiopathogenesis of certain somatic metabolism diseases.     

Cytodiagnosis of herpes simplex virus infection in the newborn infant: report of a case

Typical herpetic stomatitis that developed in a premature 6-day-old infant was initially diagnosed cytologically. The cytomorphologic characteristics of herpes simplex virus (HSV) infection in smears of scrapings from the oral mucosa helped to establish the diagnosis of a neonatal HSV infection, permitting early treatment with cytosine arabinoside (ara-C) and subsequent complete recovery from the generalized infection. The diagnosis was later confirmed by rises in the serologic titer of complement-fixing antibodies for HSV type 2.     

Diagnosis of herpes simplex virus in routine smears by an immunoperoxidase technique

Routine Papanicolaou-stained cervicovaginal smears from 59 patients were cytologically screened for herpetic infection. Forty-one of the smears were positive for herpes, 2 were suspicious and 16 were negative. All 59 slides were then destained and restained by a commercial immunoperoxidase kit for the detection of herpes simplex virus (HSV). The immunoperoxidase stain was positive in 23 of the 41 cytologically positive slides. One of the 2 cytologically suspicious slides was also immunoperoxidase positive, as was 1 of the 16 cytologically negative slides. This study indicates that immunoperoxidase staining is very specific but not quite as sensitive as routine Papanicolaou-stained smears in the detection of HSV. The immunoperoxidase method is thus recommended for the confirmation of HSV cases rather than for the routine diagnosis of HSV infection.     

Varicella zoster virus vaccines: potential complications and possible improvements

Varicella zoster virus(VZV) is the causative agent of varicella(chicken pox) and herpes zoster(shingles). After primary infection, the virus remains latent in sensory ganglia, and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine(v-Oka) is highly attenuated in the skin, yet retains its neurovirulence and may reactivate and damage sensory neurons. The reactivation is sometimes associated with postherpetic neuralgia(PHN), a severe pain along the affected sensory nerves that can linger for years, even after the herpetic rash resolves. In addition to the older population that develops a secondary infection resulting in herpes zoster, childhood breakthrough herpes zoster affects a small population of vaccinated children. There is a great need for a neuro-attenuated vaccine that would prevent not only the varicella manifestation, but, more importantly, any establishment of latency, and therefore herpes zoster. The development of a genetically-defined live-attenuated VZV vaccine that prevents neuronal and latent infection, in addition to primary varicella, is imperative for eventual eradication of VZV, and, if fully understood, has vast implications for many related herpesviruses and other viruses with similar pathogenic mechanisms.     

Occurrence of Acute Upper Respiratory Diseases among Children as a Result of Infection by Herpes Simplex Virus

One hundred and twenty-one children showing symptoms of acute upper respiratory disease were bled during a period of nine months, from winter to summer, for detection of complement-requiring neutralizing (CRN) antibody against herpes simplex virus. Six of the cases, all from children under the age of 13 years, were unequivocally related to herpetic infection as evidenced by the presence of anti-herpes CRN antibody. During the same period, 144 other children who were normal healthy or suffering from unrelated non-febrile diseases were tested as controls; anti-herpes CRN antibody was not detected in any of them. Further, the age distribution of individuals with antibodies was compared between patients in the acute upper respiratory disease group and the control group. This analysis showed that approximately 5 to 7% of the acute upper respiratory diseases in the young children in this study would be attributed to herpetic infection.     

Mechanism of virus-induced immunopathology. I. The reactivity of immunocompetent cells in acute herpetic infection in an experiment

The state of general and specific responsiveness of thymocytes and splenocytes in non-inbred white mice has been studied in the reaction of lymphocyte blast transformation under the influence of phytohemagglutinin (PHA) and herpes simplex virus. Experimental herpetic encephalitis has been shown to give rise to the development of pronounced immunosuppression, which is confirmed by a considerable decrease (P less than 0.05) in the levels of the PHA- and virus-induced transformation of thymocytes and splenocytes in infected mice in comparison with the similar transformation characteristics of intact lymphocytes. The inhibition of the general and specific responsiveness of T-lymphocytes has been found to be one of the possible mechanisms of immunosuppression in acute herpes infection.     

Confirmation of genital herpes simplex viral infection by an immunoperoxidase technique

Over the 12-month period from April 1984 to April 1985, 512,000 gynecologic (Papanicolaou) smears were examined in the Provincial Screening Program in British Columbia. During this time, 307 patients were found to have smears that contained cells consistent with, or suggestive of, a herpes simplex viral (HSV) infection. The Papanicolaou-stained smears from these 307 cases were subsequently restained, without prior destaining, using an immunoperoxidase technique specific for type 2 HSV (HSV-2) and cross reactive with HSV-1. Of the 205 smears containing cells considered to be consistent with a herpes infection, 187 were positive using the immunoperoxidase technique. Of the 102 smears showing reactive cell changes though unlikely to be causes by an HSV infection, only 5 were positive using the immunoperoxidase technique. The results show that the immunoperoxidase technique is a rapid and reliable method of confirming a suspected diagnosis of herpetic infection and that it is particularly useful in those patients in whom the Papanicolaou smear findings are equivocal.     

Effect of anti-herpes specific transfer factor

Using a blood cell separator, lymphocytes were collected from otherwise healthy convalescents suffering from herpetic infections. A specific anti-herpes dialysate (AH-DLE) was prepared from the lymphocytes, using standard procedures. Patients with recurrent herpetic infections were treated with a single dose of the dialysate, at the initial signs of herpetic infection (group A), with two doses (group B) or with three doses (group C). A total number of 37 patients (29 women, 8 men, age range 15–73 years) were treated. No improvement was observed in 7 patients (18.9%), whilst 7 patients did not manifest any exacerbation of their herpetic infection in the course of the one-year follow-up. The remaining 62.2% of the patients showed a marked improvement: decrease of the frequency and/or duration or relapses. Before AH-DLE administration, the mean number of herpes relapses in this group of patients was 12 p.a.. After therapy, the number of relapses decreased to 3.5 p.a.. No statistically significant difference was observed between groups A and B. The least favourable results were registered in group C. However, this group included 6 female patients extremely resistant to the previously therapeutic attempts, including inosiplex, non-specific DLE or acyclovir. Thus, even in this group, the therapy was successful in 50% of the patients.     

THE SURGICAL TREATMENT OF HERPETIC KERATITIS

At present, corneal transplantation is the only definitive means of controlling or terminating recurrent or chronic herpetic keratitis. Of 48 keratoplastic operations for various forms of corneal herpes, 16 in quiescent cases and 32 in cases of active keratitis, all but three brought about improvement.Recurrence of keratitis in the graft is particularly likely if the visible lesion is not excised completely and a portion of the graft border lies in contact with diseased tissue.The mode of action of corneal transplantation in improving herpetic keratitis is not clear but several possibilities have been suggested. At least in chronic stromal herpes the removal of diseased and necrotic tissue appears to be a very important factor.     

FTIR spectroscopic method for detection of cells infected with herpes viruses

Microscopic FTIR spectroscopy was used to investigate the spectral differences between normal cells in culture and cells infected with various members of the herpes family of viruses [Herpes simplex (HSV) and Varicella zoster (VZV)]. The main objective of this study is to evaluate the possibility of developing microscopic FTIR spectroscopy as a sensitive assay for the detection of herpetic infections at their early stages. The advantage of this method over conventional FTIR spectroscopy is that it facilitates inspection of restricted regions of tissue. Our results showed significant and consistent differences between all normal and HSV or VZV infected cells that were tested. Detectable and significant spectral differences between normal and infected cells are seen as early as 24 h postinfection, but the damage of the cells (cytopathic effect), caused by the infecting virus, can be seen by optical microscope observations at only 3 days postinfection. An impressive increase in the levels of vital cellular metabolites was seen in the herpes virus infected cells compared to normal cells. It seems that this spectral behavior is unique for infection with herpes viruses, because when these cells were infected with other viruses from different families like retroviruses, a considerable decrease in the levels of vital cellular metabolites was seen in infected cells compared to normal cells. Cluster analysis performed on FTIR mass chromatography yielded 100% accuracy in classifying control uninfected and VZV or HSV infected cells. Our data strongly support the possibility of developing FTIR microscopy as a diagnostic method for early detection of herpetic infections.     

Effects of the suppression of acute herpetic pain by gabapentin and amitriptyline on the incidence of delayed postherpetic pain in mice

The inoculation of mice with herpes simplex virus type-1 (HSV-1) causes herpes zoster-like skin lesions and pain-related responses (tactile allodynia and mechanical hyperalgesia) from day 5 after inoculation. Skin lesions completely heal by day 15 after inoculation, but about half of mice with acute herpetic pain show pain-related responses long after the lesions heal. Using this mouse model, we examined the effects of repeated administration of gabapentin and amitriptyline on the acute herpetic pain and the incidence of postherpetic pain. Gabapentin and amitriptyline were administered three times daily from day 5 to 11 after inoculation. Postherpetic pain-related responses were assessed on day 30 after inoculation. Gabapentin (10-100 mg/kg) produced the dose-dependent inhibition of acute herpetic pain-related responses. This medication produced marked reduction in the incidence of delayed postherpetic pain and the dose of 100 mg/kg produced the complete inhibition. Amitriptyline (10 mg/kg) did not affect the acute pain-related responses in the initial 3- and 2-day periods and then gradually inhibited them. This dosage produced a substantial but non-significant decrease in the incidence of postherpetic pain-related responses. Amitriptyline (1 and 3 mg/kg) was without effects on acute herpetic and postherpetic pain-related responses. The results strongly support the idea that the severity of the acute herpetic pain is a risk factor of postherpetic neuralgia. It may be worth testing the effects of gabapentin on acute herpetic pain and the incidence of postherpetic neuralgia in human subjects.     

Evaluation of Antiherpetic Compounds Using a Gastric Cancer Cell Line: Pronounced Activity of BVDU against Herpes Simplex Virus Replication

We developed a rapid and simple method for the screening of antiviral agents against herpes simplex virus (HSV) in a model of gastrointestinal herpetic infection in vitro. This method was based on inhibition of HSV-induced cytopathogenicity in gastric adenocarcinoma MKN-28 cells, as monitored by an MTT colorimetric assay. From the various compounds that were evaluated for their activity against HSV-1 and HSV-2, brivudine (BVDU) emerged as the most effective. When the 50% effective concentration (EC₅₀) values of the antiherpes agents in MKN-28 cells were compared with those in human embryo lung MRC-5 cells, all compounds, except for BVDU, showed higher EC₅₀ values in MKN-28 cells. For BVDU the EC₅₀ values in MKN-28 cells were 0.8 (HSV-1) and 0.036 (HSV-2) times the EC₅₀ values in MRC-5 cells. Thus BVDU was 27.5 times more active against HSV-2 in MKN-28 cells than in MRC-5 cells. The MKN-28 gastric cancer cells may be useful for the rapid screening of anti-HSV agents and, in particular, those that may be useful in therapy of gastrointestinal HSV infections in gastrointestinal herpetic infection.     

Ocular herpes: the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.     

Ocular herpes:the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus(HSV), varicella zoster virus(VZV), and cytomegalovirus(CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.     

Cytokinin inducing and antiviral activity of cycloferon on experimental herpetic infection

Experimental data on the protective activity and the capacity for inducing the biosynthesis of some cytokins, the low molecular inductors of cycloferon, endogenic interferon of the acridanon group, in herpetic infection are presented. The herpes infection was modelled by intraperitoneal injection of herpes simplex virus, type 1 into BALB/c mice. In the animals with normal immune status cycloferon induced the formation of serum interferon (INF) in high titers (up to 1:20,000) with the peak achieved 4-8 hours after the injection of the preparation. In addition, cycloferon stimulated the synthesis of IL-2 and gamma INF, but decreased the concentration of IL-1b. Following immunosuppression caused by gamma-radiation or cyclophosphamide the titers of serum interferon decreased 4-8 times. In generalized herpes infection in non-inbred white mice with undamaged immune status cycloferon increased survival rate by 30-100% in comparison with the controls (untreated mice), while in case of immunosuppression the protective effect of this preparation was considerably lower. In infected mice the concentrations of gamma INF, IL-2, IL-1b were found to be elevated in comparison with their concentrations in healthy animals. In the course of the infectious process cycloferon suppressed the production of IL-2 and IL-1b, but did not influence the synthesis of gamma INF.     

Contribution of vascular endothelial growth factor in the neovascularization process during the pathogenesis of herpetic stromal keratitis

This report analyzes the role of vascular endothelial growth factor (VEGF)-induced angiogenesis in the immunoinflammatory lesion stromal keratitis induced by ocular infection with herpes simplex virus (HSV). Our results show that infection with replication-competent, but not mutant, viruses results in the expression of VEGF mRNA and protein in the cornea. This a rapid event, with VEGF mRNA detectable by 12 h postinfection (p.i.) and proteins detectable by 24 h p.i. VEGF production occurred both in the virus-infected corneal epithelium and in the underlying stroma, in which viral antigens were undetectable. In the stroma, VEGF was produced by inflammatory cells; these initially were predominantly polymorphonuclear leukocytes (PMN), but at later time points both PMN and macrophage-like cells were VEGF producers. In the epithelium, the major site of VEGF-expressing cells in early infection, the infected cells themselves were usually negative for VEGF. Similarly, in vitro infection studies indicated that the cells which produced VEGF were not those which expressed virus. Attesting to the possible role of VEGF-induced angiogenesis in the pathogenesis of herpetic stromal keratitis were experiments showing that VEGF inhibition with mFlt(1-3)-immunoglobulin G diminished angiogenesis and the severity of lesions after HSV infection. These observations are the first to evaluate VEGF-induced angiogenesis in the pathogenesis of stromal keratitis. Our results indicate that the control of angiogenesis represents a useful adjunct to therapy of herpetic ocular disease, an important cause of human blindness.