Latent herpes simplex virus reactivation in the guinea pig. An animal model for recurrent disease.

The purpose of this investigation was to establish an animal model by which latent herpetic disease could be mechanically reactivated, yielding an adequate number of recurrent clinical lesions in the guinea pig. To determine strain virulence, hairless guinea pigs were inoculated with three different strains of herpes simplex virus (HSV) using a spring-loaded multiple puncture apparatus. HSV-1 strains SC-16 and McKrae produced an average of 21 and 8 lesions per infected area, respectively. The HSV-2 strain (333) produced the lowest number at an average of 3 lesions per site. Following the determination of strain virulence, a larger number of guinea pigs, Hartley and hairless, were inoculated with the same HSV strains in a similar fashion as previously described. The primary infection was evident from 6 to 12 hours postinfection (PI) by the initial appearance of small pustules, which peaked by day 2, seen as dome-shaped fluid-filled sacs. These initial lesions burst, crusted (day 6 PI), and had resolved and flattened between days 9 and 12 PI. At 4-6 weeks PI the inoculated areas were stripped 6 times per area with cellophane tape. Recurrent lesions were seen in the majority of the stripped areas (89-100%). The best results were achieved with the HSV-1 (SC-16) strain in hairless guinea pigs, which peaked on day 3 poststripping, producing an average of 12.25 lesions per area. The hairless guinea pig is ideal for this type of experiment because its use eliminates the trauma associated with denudation, a procedure necessary when using haired (Hartley) animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Herpes simplex virus-specific antibodies present in tears during herpes keratitis.

We examined the specificity and levels of antibodies present in rabbit tears after induced infection of the rabbit cornea. Two strains of herpes simplex virus-1 (HSV) with different patterns of ocular disease were used: RE which produces stromal disease, and F which produces epithelial disease. We found that (i) IgG, IgA, and IgM antibodies were produced, (ii) the number of specific HSV antigens recognized by these antibodies was no significantly different, and (iii) postinfection (PI) timing and concentration of antibodies varied according to the disease pattern of the virus strain. The animals infected with strain F produced high levels of IgG antibodies early PI which remained constant, while IgA and IgM antibodies also increased early PI but declined after Day 16 PI. Animals infected with strain RE showed low levels of IgA and IgM antibodies which remained low. IgG antibodies increased early PI but declined at Day 16 PI. These differences in times of appearance and in amounts of antibodies in tears may be related to the clinical course of the disease. It has been shown that stromal disease has an immunopathologic basis. Inflammation, cellular infiltration of lymphocytes, and plasma cells are seen in the stroma of RE-infected animals, but these are not present in the stroma of F-infected animals. Infectious virus was not isolated from corneal explants taken from animals during the quiescent stage of the disease. The difference in pathogenicity cannot be explained in terms of specificity of tear antibodies. Even though the disease patterns were different, the number and types of HSV polypeptides recognized by both sets of tears was similar. Consequently, we believe that the immunopathology seen in the stromal disease may be due to the anatomical site of HSV antigens, rather than to differences in specificity of tear antibodies.

Heterogeneity within an HSV-1 wild-type strain and its importance in pathogenesis.

A herpes simplex virus-type 1 low passage, clinical eye isolate, E-43 at P2, was compared with its variant progeny, SLi-43 at P8, in terms of ocular disease, cytopathic effects, and genomic variation. In New Zealand White (NZW) rabbits, E-43 produced mild epithelial defects and punctate lesions with full recovery by Day 10 postinfection (pi). SLi-43 caused dendritic lesions, progressing to geographic ulceration and death from herpes simplex virus encephalitis in 10 days postinfection. In RK, Hep-2, and Vero cells, E-43 displayed the syn+ phenotype (aggregation and cell rounding); SLi-43 showed the syn phenotype (syncytium formation). DNA digestion profiles of E-43, SLi-43, and isolates from the brains of infected animals showed that the genomic differences map within the terminal repeat of the unique long segment and the internal joint region, specifically in bands B, E, N, and S (Bam HI) and bands M and N (Hind III). Analysis of the DNA of virus recovered from the brain stem of SLi-43-infected, encephalitic rabbits demonstrated that an in vivo selection for neurotropic virions had taken place. Plaque purification of 20 clones from the original E-43 strain showed that one of 20 was the syn phenotype, indicating that the SLi-43 variant was present in the original E-43 isolate and did not develop de novo by rapid mutation. The parent-progeny relationship between E-43 and SLi-43 forms an ideal model in which to compare differences in pathogenicity at the genomic level, and underscores the importance of heterogeneity within a single herpes simplex virus-type 1 wild-type population in terms of variations in ocular disease.

The effect of modulating the synthesis of arachidonic acid cascade products on HSV lesion recurrence.

Induction of HSV lesion recurrence may be achieved by a variety of stimuli. Trauma of almost any kind (physical, chemical, electromagnetic and thermal) to the healed primary lesion site has been successful for induction of recurrence. In common with each of these mechanisms is the release of inflammatory mediators (arachidonic acid (AA), complement, kinins, etc.) following trauma. Because blockade of the AA cascade with steroids has been noted to abort HSV skin lesions, and because steroids have numerous side effects making them a poor therapeutic choice in ocular lesions, we decided to test several relatively different types of AA cascade inhibitory drugs in mouse ear HSV recurrence models. In this series of experiments, it was found that topical steroids gave the greatest initial decrease in lesion number (80% fewer than control on day 3 post recurrence induction (PRI), while meclofenamate resulted in the greatest reduction of lesions by day 5 PRI (85% fewer lesions than control and 60% fewer than the steroid treated group). The NDGA treated group exhibited the least reduction in recurrence severity (27% fewer lesions than control on day 5 PRI and 200% more lesions than the steroid group. Chlorpromazine (thorazine) acted roughly equivalent to the steroid treated group by day 5 PRI (70% fewer lesions than the untreated control group). Relative efficacy in lesion reduction between groups by day 5 PRI is: meclofenamate greater than steroid = chlorpromazine greater than NDGA greater than control. Meclofenamate, steroid and chlorpromazine significantly reduced lesions (p less than .05) when compared with the saline treated control mice. NDGA did not significantly reduce lesions by day 5 PRI.

Effect of the herpes simplex virus genome on the response of infection to corticosteroids.

The type and severity of ocular herpetic disease, as well as the pattern of recurrence, have been shown to be determined by the virus genome. We infected rabbit eyes with two closely related recombinant strains of herpes simplex virus type 1 and treated one half of the eyes in each group with corticosteroids before or immediately after virus inoculation. The severity of disease in the first week was similar in the treated and untreated eyes infected with the F(MP)F strain; however, with F(MP)E infection, the disease in the treated eyes was significantly worse than the disease in the untreated eyes. Cultures of corneal virus showed similar titers in all of the groups, but cultures of trigeminal ganglia indicated that increased severity of disease did not result in an increased tendency toward ganglionic colonization. The results suggest that the response to corticosteroids is another factor that is determined by the genetics of the infecting virus, but that there is no correlation between worsening of disease with corticosteroid treatment and the establishment of virus latency.

Epidermal growth factor receptors on corneal endothelium.

If a growth factor could bind to and stimulate human endothelial healing, corneal disease could be minimized. To this end, primary cultures of feline and human corneal endothelium were tested in receptor binding assays for radiolabeled epidermal growth factor (EGF). Both of these cells bound ten times as much 125I-EGF as did the negative control cell lines. The time course of association of 125I-EGF to cat corneal endothelium was found to be complete after approximately 120 minutes at 22 degrees C. The 125I-EGF was shown not to dissociate greatly when fresh binding buffer was added to endothelial cultures that had bound the radiolabeled peptide. The pH optimum for binding was determined to be approximately 6.4. The receptor number per cell and the affinity constant for binding were determined to be 40,000 receptors per cell and 1.1 x 10(9) L/mole, respectively, using a Scatchard plot. Parallel cultures of human fetal corneal endothelium grew in vitro only when the growth medium was supplemented with low concentrations of EGF. These studies provide evidence that EGF is specifically bound to the corneal endothelium.

Strain specificity of clinical isolates of herpes simplex virus.

Herpes simplex virus (HSV) produces a wide variety of ocular disease in man. Although host factors are important in determining this variation, it is possible that the different clinical patterns of herpetic ocular disease may be attributed at least partially to the differing biological behavior of specific strains of HSV. To test this theory, we compared the anterior segment disease produced by infecting rabbit corneas with seven different strains of HSV. We found that these seven different strains produced different patterns of ocular disease in the rabbit eye. This also may occur in humans, and we hope to define the biological differences that cause one strain to produce disease more severe than that produced by another strain.

9-(2-hydroxyethoxymethyl)guanine as an inhibitor of herpes simplex virus replication.

The drug 9-(2-hydroxyethoxymethyl)guanine effectively inhibits herpes simplex virus replication. It is selectively phosphorylated by the virus-induced thymidine kinase but not by normal cellular thymidine kinase.

Effect of 9-(2-hydroxyethoxymethyl)guanine on herpesvirus-induced keratitis and iritis in rabbits.

Drugs used for the inhibition of DNA viruses, such as iododeoxyuridine, adenine arabinoside, or trifluorothymidine, are not biochemically selective in their action and also interfere with normal cellular functions. The recently reported 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) is selectively phosphorylated by viral thymidine kinase but not by normal cellular thymidine kinase. Our present studies show that the acycloguanosine is as effective in treating herpetic keratitis in the rabbit as iododeoxyuridine and trifluorothymidine when given topically as an ointment. It is also effective when given intravenously for the treatment of herpetic iritis and is effective in preventing death from encephalitis in rabbits.

Prevention of surface bacterial contamination of donor corneas.

A simple method has been developed to reduce contamination in postmortem donor human eyes in anticipation of corneal transplantation. In vivo investigation of albino rabbits demonstrates that vigorous saline solution irrigation is extremely effective in decreasing the surface bacterial counts of the postmortem eye. In vitro and in vivo studies show that Neosporin kills bacteria at room temperature and further show that a tenfold increase in the thimerosal concentration of the Neosporin will kill fungus. Postmortem eyes contaminated by pathogenic organisms can be effectively cleaned by a combination of saline solution irrigation and the new Neosporin-thimerosal solution. No substantial damage of the donor tissue was noted by scanning electron microscopy. Human eyes cultured before this procedure were all contaminated, but after cleansing and immersion, no bacterial or fungal growth occurred.

A comparison of serum immunoglobulins from patients with non-neoplastic prostates and prostatic carcinoma.

The major immunoglobulin classes were surveyed among 23 patients with carcinoma of the prostate, 14 patients with clinically manifest benign prostatic hyperplasia and 23 healthy, elderly men without evidence of prostatic disease to determine if differences in immunoglobulin levels existed. Levels of IgG,IgA and IgM were determined by single radial immunodiffusion. Serum IgM levels were depressed in patients with all stages of carcinoma of the prostate as compared to levels in controls. These depressions were significant statistically for the tumor group considered as a whole and for patients with stages A and B tumors; the depression of IgM levels in patients with stages C and D tumors bordered on statistical significance. Serum IgG levels were depressed significantly in patients with stages A and B carcinoma of the prostate as compared to controls. Levels in patients with stages C and D lesions exceeded control levels but the difference was not statistically significant. Serum IgA levels in patients with stages A and B tumors were comparable to control levels but levels in patients with stages C and D lesions were significantly higher than controls.

Herpesvirus type2: study of semen in male subjects with recurrent infections.

Semen from 30 healthy male subjects with recurrent infections with herpesvirus type 2 was obtained when subjects were free of lesions and surveyed by tissue culture for an infectious virus in an attempt to elucidate the transmission of this disease. Inclusion bodies compatible with herpesvirus were found in tissue cultures of semen from 2 participants but an infectious virus could not be cultured directly from any sample. The data suggest that herpevirus type 2 is not ubiquitous in semen of male subjects with recurrent genital infections. The possible role of seminal inhibitors and a defective virus in causing the observed results is discussed, as are the current theories of herpesvirus type 2 transmission.

Leukocyte migration inhibitory factor in HSV infections.

The cell-mediated immune (CMI) response as measured by a direct assay of leukocyte migration inhibition factor (LMIF) was determined in a population of patients with recurrent herpes simplex virus (HSV) infections in the quiescent stage as well as in healthy volunteers. The migration of leukocytes incubated in the presence of HSV antigens was compared to that without viral antigens for the calculation of the migration index (MI). Eleven of 41 control subjects (16.8%) had a MI below 0.8, indicating a positive CMI response. In contrast, all the herpes patients tested had a MI above 0.8, suggesting an impairment in the production of LMIF at this stage of their disease. This difference was statistically significant (t = 4.296; p less than 0.001) and was not dependent on the age of the population. This study indicates that individuals with recurrent HSV infections have impaired CMI response betweeen attacks which may be associated with the stage of the disease.

Dextran flux in M-K medium-stored human corneas.

Dextran, with a minimal molecular weight of 40,000, can pass in and out of the corneal endothelium during storage in M-K medium. Results suggest that the degree of penetration of dextran depends on the length of storage and the condition of the endothelium.

In vitro studies on the mechanism of herpesvirus plaque growth inhibition by sensitized lymphocytes.

Inhibition of herpesvirus plaque growth was observed when herpes simplex virus (HSV)-sensitized rabbit lymphocytes were placed in contact with an HSV-infected human foreskin monolayer. This inhibition was obtained as early as 3 h when a ratio of 6 viable lymphocytes to target cells was used, and the supernatants of these cultures also demonstrated plaque size reduction when put onto newly infected cell monolayers. Interferon, which is present in this system, had no effect on HSV when tested on human foreskin monolayers, indicating that interferon was not the mechanism for plaque size reduction. Plaque growth inhibition was attributed to the T lymphocyte, because purified T cells reduced plaque growth and anti-rabbit thymocyte serum eliminated the effect of T cells. The specificity of this assay was determined by the facts that nonsensitized lymphocytes did not reduce the size of a plaque and the recognition of an infected cell by the sensitized lymphocyte was necessary for the release of a soluble mediator into the supernatant fluid. This cytotoxic lymphocyte was detected in the peripheral blood of rabbits as early as 4 days after initial corneal infection, with a maximum response at 7 to 10 days.

Herpesvirus type 2: isolation from seminal vesicle and testes.

Reproductive tissues from 10 recent male cadavers were examined. Herpesvirus type 2 was isolated from testes, seminal vesicle, or both in 4 cases. This is the first report of the isolation of herpesvirus type 2 from human seminal vesicular tissue. The data support previous evidence that herpesvirus type 2 can be isolated from the reproductive tissues of males without active or prior infection and suggest that these tissues may serve as a reservoir for transmission of this virus. The study also documents, for the first time, the ability to culture herpesvirus type 2 in organ explants from cadaveric reproductive tissues with the subsequent release of infectious virus.

Failure of systemically administered adenine arabinoside to affect humoral and cell-mediated immunity.

The effect of a high dosage (250 mg/kg of body weight) of adenine arabinoside or ara-A (9-beta-D-arabinofuranosyladenine) on humoral immunity was studied in New Zealand white rabbits infected with the McKrae strain of herpes simplex virus. The rabbits were treated daily for 14 days with subcutaneous injections of ara-A. The primary and secondary humoral responses, as measured by neutralizing antibody titers, developed similarly in control and treated groups. Similar drug treatment was used on guinea pigs before or after sensitization with BCG vaccine. Subsequent challenge of the sensitized animals with Old tuberculin solution indicated that ara-A treatment had no effect on the induction or previously established cell-mediated immunity. The lack of immunosuppressive activity of ara-A at dosage levels higher than those used in primates makes this drug a potentially effective agent in the systemic treatment of herpetic infections.

Anti-herpes activity of adenine arabinoside monophosphate.

Adenine arabinoside monophosphate (Ara-AMP) demonstrated antiviral activity equivalent to adenine arabinoside (Ara-A) against herpesvirus Types 1 and 2 in tissue culture. Against established herpes virus Type 1 epithelial keratitis in rabbits, Ara-AMP 2 per cent was comparable in efficacy to Ara-A 3 per cent, and Ara-AMP 20 per cent was superior to Ara-A 3 per cent. These results are especially significant in that Ara-A's high solubility will permit (1) more concentrated formulations to be presented topically and (2) adequate absorption by parenteral routes with smaller fluid loads than required for Ara-A.

Immune host response to corneal grafts sensitized to herpes simplex virus.

Experiments were performed to study the fate of corneal grafts sensitized to herpes antigens when grafted to HSV-sensitized hosts. Nonsensitized grafts in systemically sensitized hosts remained clear, whereas all locally sensitized grafts in systemically sensitized hosts became opaque within ten days. The stroma showed severe lymphocytic and plasma cell infiltration and neovascularization. One-half of the corneas from systemically sensitized hosts grafted to eyes previously infected opacified in an accelerated manner. Round cells were found in the stroma and endothelium. The histological reaction in both groups was similar to that found in the immune graft reaction even though the clinical picture was not typical (peripheral and progressive edema with a rejection line). The accelerated reaction was probably related to the presence of virus antigens in graft stroma and subepithelial areas of the graft. The significance of HSV-sensitized donor tissue grafted onto HSV-sensitized hosts is discussed because of its possible implication in tissue transplantation.