Dru E. Willey

Detection of herpes simplex virus DNA sequences in corneal transplant recipients by polymerase chain reaction assays

Polymerase chain reaction (PCR) assays were used to amplify herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) sequences in DNA extracted from formalin-fixed, paraffin embedded corneas of patients undergoing corneal transplantation. PCR reamplification with an internal (nested) set of primers was required for detection in 10 of the 12 positive corneas indicating very low abundance of viral sequences. Three of the positive corneal samples were from failed corneal grafts. Overall, TK sequences were detected in 8 of 11 corneas from subjects with a past history of herpes keratitis and in 4 of 11 corneas from subjects with no past history of herpetic eye disease.

Effect of Intensive Acyclovir Therapy during Artificial Reactivation of Latent Herpes Simplex Virus

Abstract The chemotherapeutic effect of intensive acyclovir (ACV) treatment was evaluated in New Zealand white rabbits with confirmed latent herpes simplex virus type 1 (HSV-1). Acyclovir was administered orally (1 mg/ml of drinking water), topically (three times per day), and intramuscularly (16.5 mg/kg body wt twice per day) beginning 36 days after ocular inoculation and continued for 4 consecutive weeks, during which time the rabbits underwent concurrent stimulation by bilateral iontophoresis of 0.01% epinephrine into the cornea to induce reactivation of latent virus. None of the eight rabbits receiving intensive ACV shed virus in their tear film sample during 4 weeks of concurrent epinephrine iontophoresis and antiviral therapy. All five rabbits receiving placebo treatment shed HSV in tear film samples. The total number of positive ocular samples was 14 during the first week of dual treatment (5/5 animals positive) and decreased to 2 positive samples (2/5 animals) during the fourth week of dual treatment. Upon sacrifice, HSV was isolated from 8 of 16 trigeminal ganglia of ACV-treated animals and 6 of 10 placebo-treated animals. No increased resistance to ACV was detectable in any of the virus isolated from trigeminal ganglia. Antibody and cellular immune responses were not significantly different between the ACV-treated and placebo-treated groups.

A recombinant vaccinia virus expressing herpes simplex virus type 1 glycoprotein B induces cytotoxic T lymphocytes in mice.

Spleen cells from BALB/c (H-2d) mice vaccinated with vgB11, a recombinant vaccinia virus which expresses glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1), lysed EMT6 (H-2d) target cells infected with vgB11 or with HSV-1 but did not lyse uninfected EMT6 cells or infected L-929 (H-2k) target cells. Unlabelled target cell competition of lysis showed that only syngeneic cells infected with vgB11 or HSV-1 inhibited lysis of radiolabelled HSV-1-infected targets. These results demonstrate that vgB11 induces H-2-restricted anti-HSV-1 cytotoxic T lymphocytes and that gB is the target antigen.

Efficacy of BW759 (9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine) against herpes simplex virus type 1 keratitis in rabbits

A promising new nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy] methyl]-guanine (BW759), which is structurally similar to acyclovir, was tested against acute herpetic keratitis in the rabbit ocular model. Topical 1-0.1% BW759 given 3-5x per day gave beneficial results in that corneal epithelial involvement, conjunctivitis, iritis, and corneal clouding were reduced even when chemotherapy was initiated at 3 days postinoculation. Under the same conditions, topical BW759 therapy gave slightly better results than acyclovir, and both were better than idoxuridine therapy. Mortality rate and colonization of the trigeminal ganglia by HSV-1 were unaffected by BW759 therapy. Duration of virus, shed into the tear film was reduced by BW759.

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

Sequential analysis of antibody responses in serum, aqueous humor and tear film during latent and induced recurrent HSV infections.

To study HSV specific antibody responses during latent HSV infection and induced HSV ocular shedding, rabbits were infected binocularly with McKrae strain HSV-1. The titer and class of anti-HSV antibody response in serum, aqueous humor and eye washes were determined sequentially during latent HSV infection and following intentional reactivation. In all instances the only antibody class detected was IgG. The highest anti-HSV titers were in the serum and aqueous humor, whereas a significantly lower level of anti-HSV IgG was found in eye washes. Anti-HSV IgG antibody titers were consistently 100-fold higher in serum than in the aqueous humor, which suggests an absence of local ocular antibody synthesis. Neither anti-HSV IgM nor secretory IgA antibodies were detected in any samples.

Ocular acyclovir delivery by collagen discs: A mouse model to screen anti-viral agents

Discs 1.6 mm in diameter trephined from corneal collagen shields were used to deliver acyclovir (ACV) to the cornea of mice inoculated with herpes simplex virus type 1 (HSV-1). In the first minute after application to the cornea, there was a 23% decrease of ACV in the discs. After the first minute, ACV clearance from the discs appeared to be exponential with a half-life of 21 minutes. Treatment given 3 times a day reduced HSV-1 titer in tear film, corneal tissue, and trigeminal ganglia. This animal model should be useful to conserve novel potential anti-viral drugs undergoing initial screening.

Intentional reactivation of latent ocular herpes infection during BVDU therapy.

Sixteen adult New Zealand white rabbits with previously confirmed herpes simplex virus type 1 (HSV-1) infections were stimulated by iontophoresis of 6-hydroxydopamine into the cornea and were followed-up by topical epinephrine treatment to confirm latency. A total of 224 ocular cultures were obtained, of which 73 were positive for HSV during the seven day cycle. Twenty-seven of the 32 eyes (84%) had at least one positive culture. Animals were randomly divided into two treatment groups. Upon repeat stimulation (Cycle 2), concurrent with oral and topical bromovinyl-deoxyuridine (BVDU) therapy, only 3/104 ocular cultures were HSV positive, while 24/112 ocular cultures from placebo-treated animals were positive. Anti-HSV serum titers were comparable before and after BVDU therapy and the HSV isolates from BVDU treated animals did not develop drug resistance (i.e., ED-50 values were approximately 0.1-0.2 ug/ml both before and after therapy). It was concluded that BVDU had a demonstrable therapeutic effect on the recovery of HSV-1 from ocular cultures during intentional reactivation, but the latent ganglionic infection was not eliminated.

Antibody response following implantation of xenogenic lenticules

Rabbits underwent implantation of either lyophilized or fresh porcine lenticules into the central or peripheral cornea. All animals were followed for up to four months by slit-lamp examination and macrophotography to determine implant rejection. Serum antibody levels to soluble porcine cornea extract were determined by an enzyme-linked immunosorbant assay. Only those animals receiving lenticules into the peripheral cornea experienced a rejection and developed an antibody response to the porcine cornea extract. The production of antibody preceded the appearance of vascularization of the implanted lenticules. Thus, the site of lenticule implantation, not the type of tissue preparation, determined the outcome of the graft.

A Specular Microscopic Viewing System for Donor Corneas

A system for routine specular microscopic assessment of donor corneal endothelium including a chamber that allows both storage and viewing of the donor tissue was evaluated. A specular microscope modified to enable noncontact scanning of donor cornea from the endothelial side was used. Forty-five human corneas were examined upon arrival at the eyebank. Storage in the new chamber was compared with that in standard vials for 13 paired corneas. This study demonstrates the feasibility and simplicity of routine specular microscopy on donor corneal tissue, and discusses its advantages.