Detection of herpes simplex-1 and -2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis / Detecção de vírus herpes simplex tipo 1 e 2 e varicella zoster por reação em cadeia de polimerase em tempo real em córneas de pacientes com ceratites bacterianas

ABSTRACT Objective: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. Methods: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. Results: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. Conclusions: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.

RESUMO Objetivo: Ceratites bacterianas ocorrem mundialmente e apesar dos novos desenvolvimentos permanece como uma condição que pode levar à cegueira. Avaliar a presença de herpes simples (-1 e -2) e vírus varicella zoster (VZV) por reação em cadeia quantitativa de polimerase em tempo real (qPCR) em raspados corneanos de pacientes com ceratite bacteriana. Métodos: Sessenta e cinco pacientes com ceratite infecciosa foram submetidos a raspados corneanos estudados para gram, Giemsa, cultura e qPCR (grupo de estudo). Foram avaliados fatores de risco e epidemiológicos. O grupo controle foi composto por 25 casos de úlcera dendrítica típica por herpes analisados por qPCR. Resultados: Do grupo de estudo (n=65), nove pacientes (13,8%) apresentaram cultura, qPCR e raspado negativos. Cinquenta e seis (86,2%) pacientes apresentaram cultura positiva, 51 para bacteria, 4 para fungo e 1 para ameba. A qPCR identificou 10 pacientes do grupo de cultura positiva para bactéria que também foram positivos para vírus, um VZV e 9 para HSV-1. Dos 25 pacientes que compunham o grupo controle, 21 apresentaram qPCR positivo para HSV-1. Conclusão: Herpes pode estar presente em pacientes com úlceras de córnea bacterianas e a qPCR pode ser útil na sua detecção.

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.

Induction of cellular transcription factors in trigeminal ganglia of mice by corneal scarification, herpes simplex virus type 1 infection, and explantation of trigeminal ganglia.

In a mouse model for herpes simplex virus type 1 (HSV-1) latency in which the virus was inoculated via the eye after corneal scarification, HSV-1 replicated in corneal epithelial cells and infected the nerve cell endings. HSV-1 reached the trigeminal ganglia by fast axonal transport between 2 and 10 days postinfection (p.i.) and established a latent infection in neuronal cells or replicated and spread to nonneuronal cells. By using in situ hybridization, we showed that cellular transcription factors are stimulated by HSV-1 infection in trigeminal ganglia. This stimulation is biphasic, peaking at 1 and 3 to 4 days p.i. The first peak involves c-jun and oct-1 expression in neurons, and the second involves c-jun, c-fos, and oct-1 expression in neurons and nonneuronal cells. Corneal scarification, alone or followed by infection with UV-inactivated HSV-1, induced monophasic c-jun and oct-1 expression in some neurons of the trigeminal ganglia, with a peak at 1 day p.i. Corneal infection without prior scarification induced c-jun, c-fos, and oct-1 expression in some neuronal and nonneuronal cells of the trigeminal ganglia 2 to 9 days p.i. Explanation of ganglia from latently infected animals resulted in reactivation of the latent virus. Independently of the presence of latent HSV-1 in explanted ganglia, expression of c-fos, c-jun, and oct-1 was induced first in nonneuronal cells, peaking 6 to 10 h postexplantation, and then in neuronal cells, with a peak at 24 h after explantation when expression of viral replicative genes was first detectable. Since ocular HSV-1 infection, corneal scarification, and explantation of trigeminal ganglia all resulted in induction of expression of cellular transcription factors in ganglia, these factors may play a critical role in the permissiveness of cells for HSV-1 replication during acute infection, latency, and reactivation.

Detection of herpes viral genomes in normal and diseased corneal epithelium.

Herpetic ocular disease is one of the major causes of corneal blindness. Clinical diagnosis of corneal disease is based principally on corneal appearance. However, abnormal morphology of the corneal epithelium (CE) is not an indicator for the presence of a herpes virus. Further, it has not been established if herpes viruses are present in normal corneal epithelial tissue. In these studies, the polymerase chain reaction was used to evaluate normal and diseased corneal epithelium for the presence of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) genomic sequences. Thirty-two normal corneal epithelium specimens obtained from cadavers shortly after death were analyzed for HSV-1, EBV and CMV genomic sequences. Three of the 32 normal CE specimens were positive for amplified EBV DNA, 1 was positive for HSV-1 DNA, and none was positive for CMV DNA. We also tested eight herpetic dendritic lesions of which 3 were HSV-1 culture and PCR positive. The remaining five dendritic lesions were HSV-1 culture and PCR negative. Since these lesions were not evaluated for other herpesviruses, the etiology of these dendritic lesions is unknown. Six corneal epithelium samples from HIV-infected donors were negative for EBV, CMV and HSV-1 amplified sequences. Positive EBV, CMV and HSV-1 serology on all normal donors and on donors with clinically apparent disease did not correlate with positive PCR results. The results of these studies suggest that EBV and HSV-1 DNA can be amplified from a small percentage of apparently normal corneal epithelium.

[Retinal lymphoma, retinal necrosis: identification of retinal viral particles and anti-herpetic antibodies in the vitreous body]. / Lymphome rétinien, nécrose rétinienne: identification de particules virales rétiniennes et d'anticorps anti-herpétiques dans le vitré.

Clinical data and histopathologic report of the association of a retinal malignant lymphoma and acute retinal necrosis. Demonstration of virus particle and vitreous herpetic antibodies.

Characterization of glycoprotein C-negative mutants of herpes simplex virus type 1 isolated from a patient with keratitis.

Recently three strains of herpes simplex virus type 1 (HSV-1), which did not react with Micro Trak Herpes (Syva Co.), were isolated by us from a patient with recurrent herpetic keratitis. In this study we characterized these strains of HSV-1 and found them to be HSV-1 gC- mutants which are very rare isolates from humans. The properties of the HSV-1 strains regarding plaque morphology on Vero cells and chick embryo fibroblasts and viral DNA analysis were the same as those of the usual HSV-1 strains. An immunofluorescence study using anti-gC-1 monoclonal antibody and SDS-PAGE analysis of radiolabeled viral glycoproteins showed that these strains are deficient in gC-1. They were virulent for mice and sensitive to acyclovir and bromovinyldeoxyuridine. Furthermore the infectivity of the strains was inactivated by complement though the phenomenon was not observed in the usual HSV-1 strains. This finding suggests that protection from damages by complement is an important function of gC. In keratitis the effects of complement are thought to be minimal because of the scanty blood supply and this may be the reason why these strains were isolated from the cornea.

Cryogenic induced ocular HSV-1 reactivation is enhanced by an inhibitor of the lipoxygenase pathway.

Reactivation of latent herpes simplex virus type 1 (HSV-1) has been shown to occur in response to localized inflammation. Prostaglandins and lipoxygenase products [eg. hydroxyeicosatetraenoic acids, (HETEs)] are associated with inflammation and, therefore, may play a role in HSV-1 infection and reactivation. In the rabbit cornea, alkali injury, cryogenic injury, and acute HSV-1 infection promote the synthesis of HETEs. Recently, a platelet activating factor antagonist, ginkgolide B (BN 52021) has been found to specifically inhibit the corneal synthesis of HETEs after alkali injury. If the induction of HETEs after injury is related to HSV reactivation and severity of infection, BN 52021 may alter HSV reactivation and the severity of infection by reducing the production of HETEs. To study the effect of BN 52021 on HSV-1 reactivation, cryogenic corneal lesions were produced in ten HSV-1 latently infected rabbits. Five rabbits were treated with topical and intravenous BN 52021 while the remaining five rabbits received topical artificial tears and intravenous saline. In the BN 52021 treated group, 90% (9/10) of the eyes and 53% (35/66) of the total ocular cultures were positive for HSV-1. In the control group, 60% (6/10) of the eyes, and 27% (18/66) of the ocular swabs were positive for HSV-1. The total number of positive cultures was significantly greater (p less than .05) in the BN 52021 treated rabbits. By increasing the number of positive HSV ocular cultures, BN 52021 appeared to act similarly to other inhibitors of arachidonic acid metabolism such as steroidal and nonsteroidal anti-inflammatory agents.

The relationship of corneal Langerhans cells to herpes simplex antigens during dendritic keratitis.

The corneal migration and topographic distribution of Langerhans cells (LC) in relation to herpes simplex virus antigens was studied during the course of dendritic keratitis in inbred mice. Corneal epithelial sheets from infected mice at selected time points were "double stained" for Ia-positive Langerhans cells and HSV antigens, using a sequential avidin biotin immunoperoxidase and glucose oxidase technique. The amount of HSV antigen was maximum at day 2 paralleling the clinical time course, with most corneal epithelium HSV antigen negative by day 8. LC were seen in peripheral corneas by day 2 and in paracentral and central cornea by day 8, with peak numbers detected between days 8 and 11 post-infection. Although HSV antigens and LC were simultaneously detected within corneal epithelium, LC were not observed in anatomic juxtaposition to HSV antigens, even after reinoculation of infected corneas with HSV on day 14 following the primary infection. These data suggest that local factors in the corneal epithelium other than HSV antigens per se may be chemotactic for LC during the course of dendritic keratitis.

Susceptibility to acyclovir of herpes simplex virus: emergence of resistance in patients with lymphoid and myeloid neoplasia.

We have examined the susceptibility to acyclovir (ACV) of herpes simplex virus isolated from immunocompetent and immunocompromised patients. Susceptibility to ACV was determined in a dye-uptake assay with vero cells grown in 96-well microtitre plates. Resistant strains were found in two out of 35 (5.7%) patients and partially resistant strains in four out of 35 (11.4%) patients immunodeficient as a result of treatment for lymphoid or myeloid malignancy. Strains resistant to ACV were not found in organ transplant recipients or in immunocompetent patients.

Alterations in the antigenic structure of two major HSV-1 glycoproteins, gC and gB, influence immune regulation and susceptibility to murine herpes keratitis.

We previously demonstrated that anterior chamber (AC) injection of HSV-1 before or simultaneous with topical corneal HSV-1 infection resulted in cellular immune tolerance of HSV-1 Ag and a reduced frequency of corneal stromal lesions. In the present study, we have investigated the role of the HSV-1 cell-surface glycoproteins gC and gB in the induction of tolerance, and the resulting reduced susceptibility to HSV-1 corneal stromal disease. These studies utilized mutant strains of HSV-1 with deletion or point mutations in the gene coding for gC or gB. Groups of mice received topical corneal infections with wild-type HSV-1, followed by AC injection of the same eye with wild-type HSV-1 or a mutant strain. Varying the antigenic composition of the virus injected into the AC resulted in three distinct patterns of immune responsiveness. In agreement with our previous findings, AC injection of wild-type HSV-1 induced a state of HSV-1 specific tolerance that extended to both the delayed type hypersensitivity (DTH) and CTL responses. A mutant strain lacking gC (gC-) induced partial tolerance characterized by undetectable CTL activity but a normal DTH response. A mutant strain lacking gB (gB-) caused partial suppression of the CTL response and no reduction of the DTH response. Thus, whereas gB may be involved in CTL tolerance induction in this model, gC clearly is not involved. In contrast, both gC and gB must be present in the AC to induce detectable DTH tolerance. The latter interpretation was strengthened by the observation that AC injection of a mixture of gC- (expressing normal gB) and gB- (expressing normal gC) effectively suppressed the DTH response to wild-type HSV-1. A panel of mar mutants with individual point mutations affecting gC and gB was used to identify the epitopes responsible for induction of DTH tolerance. Two of the gC mutants failed to induce DTH tolerance to wild-type HSV-1 when injected into the AC, suggesting that the sites on the gC molecule that are altered by these mutations are important for the induction of DTH tolerance. Similarly, one of the mar mutants for gB uniformly failed to suppress the DTH response, while another had a variable effect. The unique pattern of cellular immune reactivity exhibited by the mice receiving simultaneous topical corneal infection with wild-type HSV-1 and AC injection of gC- (no CTL but normal DTH) was associated with significantly reduced susceptibility to HSV-1 corneal stromal lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid detection of herpes simplex virus (HSV) antigen in human ocular infections.

The new HERPCHEK (Dupont, No. Billerica, MA) enzyme immunosorbent assay (EIA) was used in a double-blind clinical study for rapid and specific detection of ocular herpes simplex virus (HSV) infection. This 4-hour assay can be used to demonstrate conclusively the presence of HSV antigen without culture and thereby rapidly differentiate between HSV and other clinically similar ocular infectious diseases. Ocular samples were collected from 180 individuals including 30 patients with acute HSV, 90 with latent HSV (ie, currently asymptomatic but with a positive history), 11 with acute or latent varicella zoster virus, 30 with nonherpetic infections (due to adenovirus, Acanthamoeba or bacteria), and 19 normal controls. A clinical diagnosis was made by one of us (DPL) and duplicate tear-film samples obtained by swabbing the conjunctival cul-de-sac and cornea. Coded samples were tested by routine viral culture on Vero cell monolayers and also were run independently in the HERPCHEK test. During active HSV infection, the HERPCHEK correlated 100% with clinical diagnosis, and virus culture correlated 90% with clinical diagnosis. In all latent HSV ocular infections, other nonherpetic ocular infections and normal samples, both the HERPCHEK and culture assays were negative.

Evidence that the gene for herpes simplex virus type 1 DNA polymerase accounts for the capacity of an intertypic recombinant to spread from eye to central nervous system.

HSV-1(17) replicates 100-fold more efficiently than HSV-2(186) within trigeminal ganglia following ocular infection. In order to identify the nucleotide sequences responsible for the differences in the capacity of the two HSV strains to grow within the peripheral nervous system, an intertypic recombinant was generated by infecting neuroblastoma cells with HSV-2(186) and a HSV strain possessing nucleotide sequences from HSV-1(17). The genome of the intertypic recombinant was composed entirely of HSV-2(186) DNA except for 2.0 kb of HSV-1(17) DNA positioned between m.u. 0.413 and 0.426. Following corneal infection of mice, the intertypic recombinant grew to higher titers in both ocular tissues and trigeminal ganglia than did the HSV-2 parent. Most significantly, the intertypic recombinant could spread into the brain from the trigeminal ganglion and kill the host whereas mice inoculated with the HSV-2(186) parent survived infection. The 2.0 kb of HSV-1(17) DNA inserted into the genome of the intertypic recombinant encodes the 5' terminus of the HSV-1 gene for DNA polymerase. Thus, the results suggest that the difference in the capacity of two HSV strains to replicate within the trigeminal ganglion of its host and to spread into the brain is determined by nucleotide sequences within the gene for DNA polymerase.

Immunologic modulation of virus-induced pathology in a murine model of acute herpetic retinal necrosis.

Unilateral inoculation of herpes simplex virus Type 1 (KOS strain) into the anterior chamber of BALB/c eyes produces an ocular disease with a distinctive differential pattern of retinal pathology. Specifically, the retina of the inoculated eye remains histologically intact, whereas the contralateral retina becomes necrotic. We demonstrate that retinal necrosis in opposite uninjected eyes directly correlates with the presence of herpes simplex viral antigens, whereas the intact retinas of virus-injected eyes are devoid of immunocytochemically detectable viral antigens. Immunosuppression or lack of a thymus results in bilateral retinal necrosis, with positive immunoperoxidase staining for viral antigens in both eyes. We have shown previously that retinal protection in both eyes can be restored to irradiated recipients by adoptive transfer of spleen cells from mice primed by AC injection of HSV. Our results with reconstituted and normal mice suggest that virus-mediated cytopathic effects underlie contralateral retinal necrosis since HSV antigens are localized to areas of retinal necrosis and their presence precedes the local inflammatory response; immunosuppression does not alter the development of contralateral retinal necrosis. They also indicate that ipsilateral retinal preservation reflects T cell-mediated inhibition of viral spread to retinas of injected eyes. Reconstitution of irradiated recipients with AC primed donor cells prevents immunohistochemically detectable virus and retinal necrosis in both eyes. In all experimental groups we failed to detect viral antigens in the absence of retinal pathology.

The antiherpesvirus activity and cytotoxicity of sangivamycin.

Sangivamycin, 4-amino-5-carboxamido-7-(beta-D-ribofuranosyl)-pyrrolo[2,3-d]-pyrimidine is a structural analog of adenosine belonging to a group of nucleosides classified as pyrrolopyrimidines. Sangivamycin, an adenosine deaminase resistant analog, was found to inhibit the replication of three strains of herpes simplex virus type 1 (HSV-1) by 50% (ED50) at a concentration approximately equal to the concentration which inhibits cell growth by 50% (LD50). Both Vero cells and rabbit corneal stromal cells in exponential growth were about 10-fold more sensitive to the drug than quiescent cells. The selectivity indices of sangivamycin indicated that the drug was not a highly selective antiviral agent and, therefore, would offer no advantage over drugs currently available for the treatment of herpetic keratitis.

Application of immunoperoxidase staining in the cytodiagnosis of herpes simplex keratitis.

In corneal scraping smears from 13 patients with clinically suspected herpes simplex keratitis (HSK), HSK is demonstrated by means of peroxidase-antiperoxidase (PAP) technique with antisera to herpes simplex virus (HSV) in Papanicolaou-destained cellular samples. The staining for HSV antigen was present in seven cases of corneal scraping smears with superficial keratitis (dendritic and geographic ulcers) while six cases of stromal keratitis (deep keratitis) failed to show HSV antigen except in one case. Specific antigen for HSV was predominantly present in the cytoplasm rather than in the nucleus. Immunoreactions were negative with HSV antisera in patients with other infections and in those in a normal control group. Using the PAP technique, detection of HSV antigen in corneal scraping smears was of great value in the diagnosis of HSK, especially in cases of superficial keratitis.

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.