Keith A. Laycock

Reactivation of Latent Herpes Simplex Virus by Excimer Laser Photokeratectomy

We tested whether excimer laser photorefractive and phototherapeutic keratectomy may reactivate latent herpes simplex and cause recurrent keratitis in mice. Two of ten latently infected mice that were treated with ten excimer laser pulses to the corneal epithelium shed herpes simplex virus type 1, as did four of ten mice that were treated with 50 excimer laser pulses. Ocular shedding of herpes simplex virus was detected in four of ten mice that were treated with ethylenediamine-tetraacetic acid (EDTA) scraping of the corneal epithelium without laser keratectomy, and in six of ten mice on which combined EDTA-facilitated epithelial removal was performed followed by the application of ten excimer laser pulses. In both EDTA-treated groups, viral shedding was prolonged and 18 of 20 mice developed marked corneal opacification or neovascularization, or both. Corneal photoablation with the excimer laser may induce reactivation of latent herpes simplex virus, even in mice with clear and smooth-appearing corneas, and should be considered in the differential diagnosis of humans with persistent corneal epithelial defects after refractive or therapeutic excimer procedures.

AIDS-related optic neuropathy: a histological, virological and ultrastructural study

Abstract• Background: Clinical and histopathological evidence of optic nerve axonal loss has been reported in AIDS patients without retinitis. The study was carried out to investigate the possible involvement of HIV-infected cells in the development of optic nerve degeneration. • Methods: Optic nerves were obtained from eight AIDS patients and four normal controls. These nerves were morphologically and immunohistochemically analyzed. Additionally, using PCR amplification techniques, the retina and optic nerve samples obtained from three HIV-seropositive patients and one control were examined for the presence of HIV and cytomegalovirus antigens. • Results: We noted various stages of axonal degeneration in the optic nerves obtained from AIDS patients in whom there was an absence of retinal findings. Characteristic glial changes involving hypertrophic astrocytes, vacuolated oligodendrocytes, and mononuclear phagocyte series cells were also seen in the AIDS optic nerves. HIV DNA was present in at least four of five optic nerves but in only one of five retinas. Control specimens were each negative for all cytomegalovirus and HIV antigens. • Conclusions: Degeneration in the optic nerve may be mediated by HIV-infected macrophages rather than by direct viral infection of neurons. Axonal degeneration due to AIDS at the level of the optic nerve can occur independently of retinal infection.

Detection of herpes simplex virus type 1 gene expression in latently and productively infected mouse ganglia using the polymerase chain reaction

The polymerase chain reaction (PCR) was employed to detect herpes simplex virus (HSV) sequences in the DNA, and HSV gene expression in total cell RNA, extracted from cervical and trigeminal ganglia of mice during productive and latent infection with HSV-1, strain SC16. Such gene expression was detected in 1 microgram or less of RNA, the quantity anticipated to be present in one or two cervical ganglia. Within the limits of the primers available, gene expression during latency appeared to be restricted to the latency-associated transcript (LAT). The 195 base portion of the LAT amplified by the PCR was sequenced and found to contain several base changes and deletions with respect to published sequences for different HSV strains. These mutations, within the putative open reading frame 2 of the LAT, formed stop or terminator signals, which suggests that the LAT does not act to establish or maintain latency through translation to a protein. The primers for the LAT also amplified a 300 bp fragment from any murine and some other mammalian RNAs. Apart from the oligonucleotide primers, this fragment did not show any homology with HSV.

Postexposure vaccination with a virion host shutoff defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice

A herpes simplex virus type 1 (HSV-1) recombinant (UL41NHB) deficient in the virion host shutoff (vhs) function was tested as a therapeutic vaccine in an ultraviolet (UV) light-induced mouse ocular reactivation model. Mice were infected with HSV-1 via the cornea. Following the establishment of latency by HSV-1 the mice were subsequently vaccinated intraperitoneally with one dose of UL41NHB or with uninfected cell extract. Mice were subsequently UV-irradiated to induce viral reactivation and during the 7 days post-UV irradiation, numbers of mice shedding virus were reduced from 13/23 (57%) to 3/25 (12%), and numbers of virus-positive eye swabs were reduced from 40/161 (25%) to 6/175 (3%) by the vaccine (P < 0.001). These data suggest that deletion of vhs may be a useful strategy in the development of attenuated therapeutic HSV vaccines.

Therapeutic vaccination with vhs− herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice

Virion host shutoff (vhs)-deficient herpes simplex virus (HSV) was tested as a therapeutic vaccine in a mouse model of UV light-induced recurrent herpetic stromal keratitis. Four weeks after primary corneal infection, mice were vaccinated intraperitoneally with vhs(-) vaccine or control. Four weeks after vaccination, the eyes of latently infected mice were UV-B irradiated to induce recurrent virus shedding and disease. Post-irradiation corneal opacity in latently infected, vhs(-)-vaccinated mice was significantly reduced compared to control-vaccinated mice (P=0.007 to 0.035). The incidence and duration of recurrent virus shedding were the same in both groups. Antibody titres were increased (P=0.05) and delayed type hypersensitive responses were unaffected by vhs(-) vaccination. Combined with studies using different vaccination timing and vhs(-) genotypes, these data suggest that deletion of vhs is a useful strategy in the development of a therapeutic HSV vaccine, and that temporal and genetic factors influence vaccination outcome.

An In Vivo Model of Human Cytomegalovirus Retinal Infection

PURPOSE To develop an animal model system in which human retina implanted in the anterior chamber of the eyes of rats would support human cytomegalovirus replication. Cytomegalovirus retinitis currently represents the most common cause of posterior uveitis in many urban areas in North America. Despite the tremendous interest in cytomegalovirus retinitis as a result of the acquired immunodeficiency syndrome (AIDS) epidemic, human cytomegalovirus infection has been difficult to model in vivo because of its extreme species-specificity. METHODS Human retina was introduced into the anterior chamber of athymic rats and allowed to attach to the rat iris. A human cytomegalovirus mutant carrying a beta-galactosidase indicator gene was then injected into the anterior chamber to infect the implanted tissue. After 4 weeks, the eyes were removed, sectioned, and developed with a chromogenic substrate to demonstrate the presence and location of beta-galactosidase expression. RESULTS Multiple spreading foci of beta-galactosidase expression were found in the retinal implants, indicating that human cytomegalovirus replication had occurred within the human tissue. There was no infection of rat tissue. CONCLUSIONS This model allows human cytomegalovirus infection of human retina to be established in vivo and sustained long enough to permit multiple cycles of viral replication to occur. The model thus has potential for evaluating antiviral therapies directed against human cytomegalovirus retinal disease.

Comparative Diagnosis of Neonatal Chlamydial Conjunctivitis by Polymerase Chain Reaction and McCoy Cell Culture

Cervical and ocular swabs from 100 mother/newborn pairs delivering on the clinic service were assayed for Chlamydia trachomatis with standard McCoy cell culture and with standard and biotinylated polymerase chain reaction techniques, using primers directed against the major outer membrane protein gene and C. trachomatis-specific cryptic plasmid, respectively. Using the polymerase chain reaction, 20 (20%) mothers and seven (7%) neonates were positive for Chlamydia. All neonates positive by polymerase chain reaction were from mothers positive by polymerase chain reaction, yielding a 35% transmission rate. Only five of 20 (25%) mothers and two of seven (28%) neonates positive by polymerase chain reaction were positive by cell culture. All cell culture samples were positive by polymerase chain reaction testing. Culture and polymerase chain reaction analysis two weeks after treatment with oral erythromycin were negative. The polymerase chain reaction assay appears to be equally specific and more sensitive than McCoy cell culture for the detection of C. trachomatis from ocular specimens.

The role of nerve growth factor in modulating herpes simplex virus reactivation in vivo

The role of nerve growth factor (NGF) in the modulation of herpes simplex virus (HSV) latency and reactivation was investigated in a mouse model. To determine whether NGF depletion would reactivate latent virus, concentrated anti-NGF serum antibodies were administered intraperitoneally to latently infected mice for 9 consecutive days. To determine whether NGF given prophylactically could suppress UV-B-induced viral reactivation, mice were irradiated with UV-B while being treated with NGF using diverse regimes over a 4-day period. Following intraperitoneal administration of anti-NGF antibodies, viral shedding was detected in a small number (10%) of mice, but it was not possible to pharmacologically suppress UV-B-induced viral reactivation with NGF. It would appear, therefore, that HSV latency in neurons innervating the cornea can be sustained and disrupted by physiological factors independent of NGF levels.

Screening Corneas for Human Immunodeficiency Virus Type 1 Proviral DNA by Polymerase Chain Reaction

PURPOSE We evaluated the sensitivity of the polymerase chain reaction as a technique to directly screen potential donor corneas for human immunodeficiency virus type 1 (HIV-1) proviral DNA. METHODS DNA from the central 8.0-mm cornea, limbal cornea, aqueous humor, and retina from 22 eyes of 11 cadavers seropositive for HIV was extracted and amplified by polymerase chain reaction using primers specific for the gag and env regions of the HIV-1 genome. The identity of amplification products was confirmed by Southern blot hybridization. RESULTS Viral DNA was detected in four (18.2%) of 22 central corneas, one (4.5%) of 22 limbal corneas, one (6.3%) of 16 aqueous humor samples, and seven (31.8%) of 22 retinas. No correlation was noted between the presence of HIV-1 proviral DNA in samples from the central cornea and from the other tissues tested from the same eye. CONCLUSIONS Within the limits of our assay, processing and analysis of limbal cornea, aqueous humor, and retina by polymerase chain reaction may not reliably ascertain the presence of HIV-1 in the central, transplantable cornea.

Screening Potential Corneal Donors for HIV-1 by Polymerase Chain Reaction and a Colorimetric Microwell Hybridization Assay

PURPOSE Current screening of potential corneal donors for human immunodeficiency virus type 1 (HIV-1) involves serologic detection of antibodies to the virus. However, this approach cannot detect infection during the seronegative window period of the disease. We therefore evaluated the polymerase chain reaction (PCR) assay for viral nucleic acid as a possible alternative to screening cadaveric blood for HIV-1. METHODS Blood specimens from cadavers diagnosed at autopsy with acquired immunodeficiency syndrome (AIDS) (n = 21), at high risk for HIV-1 infection (n = 47), and at no known risk (n = 350) were screened by PCR for HIV-1 proviral DNA and human leukocyte antigen (HLA)-DQ alpha sequences, and for HIV antibodies. RESULTS All AIDS group samples were seropositive; of these, 18 (86%) and 20 (95%) of 21 were positive for HIV by PCR of proteinase K- and Chelex-extracted pellets, respectively. The seropositive samples negative by PCR testing were shown to inhibit PCR amplification. Nine (19%) of 47 high-risk specimens were HIV-positive. The no-known-risk group yielded negative results. The overall sensitivities for PCR in the proteinase K- and Chelex-treated groups were 90% and 97%, respectively, compared with Western blot reactivity. If PCR-inhibitory samples and HLA-DQ alpha-negative samples had been eliminated, sensitivity would have been 100%. Specificity was 100% for each group. CONCLUSIONS Screening cadaveric blood by PCR may be feasible, but further refinement of the assay and blood specimen collection practices will be necessary for it to become routine. Future studies should focus on optimizing specimen procurement and preparation to reduce or eliminate specimens that inhibit PCR.