641
British Journal of Ophthalmology, 1991, 75, 641-642
BRITISH JOURNAL OF OPHTHALMOLOGY
Editorial Herpes simplex latency and the eye The herpes simplex virus (HSV) exerts its ocular damage by recurrent corneal infection, having lain dormant in the host tissues for some time. Latency of the virus is an essential part of its natural history, yet the mechanism of latency and recurrence has, for both the clinician and the research worker, remained an enigma. The definition of HSV latency has been described as the capacity of the virus to exist in a host cell for an extended period of time in a virion-free state and, upon stimulation, to reactivate and replicate to produce infectious progeny virions.' As a result of recent advances in molecular biology and genetics, the phenomenon of latency and reactivation is now being explained in terms of specific chemical components found or localised in the chromosomes. After primary infection, usually of ectodermally derived tissues, the viral genetic material travels by fast axonal transport as a nuclear capside to the site of latency, either the trigeminal ganlgion or other extraneuronal sites, where it exists in an extrachromosomal state as a circular episome.2 HSV reactivation can be thought of in terms of the classic Crick model of the HSV DNA molecule acting as a template to use the host cell DNA to copy or express, by DNA replication, the HSV genetic information which is transmitted (by transcription) into RNA nucleic acid, which in turn directs the synthesis in the host cell of the specific HSV protein to manufacture the entire virus. Genetic studies have shown that during the acute productive infection many genes are expressed, including the immediate early, early and late viral genes. During latent infection, when the virus is in a non-replicating but transcriptionally active state, only part of the genome is present, specifying latency associated transcripts (LATs), of which four have been identified from the viral DNA strand opposite that which encodes the infected cell protein ICPO, an immediately early regulatory protein. The LATs overlap at least 30% of the terminus of this protein but the transcription pattern of the LAT encoding region is complex and, as yet, not fully defined. The exact localisation of the genes concerned with the establishment, maintenance and reactivation of latency remain unknown, nor has any protein been found associated with these functions. It has been suggested that reactivation involves expression of a subset of genes, which pass to the surface tissue, such as cornea or skin, initiating a replication cascade and leading ultimately to corneal involvement or skin vesicles. The mechanism which controls the switch between acute and latent phases of infection is poorly understood. Expression of LATs is not essential for the maintenance of latency'
but is concerned in viral reactivation. This has been demonstrated by the finding that viral strains lacking LATs can still establish latency in the experimental animal, but have reduced recurrence rates compared with those strains with a LAT present.45 (See ref 6 for overall review.) It has been suggested that promoters of LATs of herpes simplex come from a local tissue response. It is likely that a number of reactivation inducing stimuli in humans occur, including ultraviolet irradiation, trauma and the experimental process of iontophoresis of adrenaline into the cornea. Prostaglandins have been thought to be implicated in the reactivation of viral latency.7 Indomethazine, a potent inhibitor of prostaglandin synthesis, has been shown to reversibly inhibit the reactivation of HSV from experimental latently infected ganglia.' In mammalian cells such stimuli lead to release of an agent which interacts with a cell surface receptor activating adenylate cyclase which synthesises cyclic adenosine monophosphate, a cyclic nucleotide that regulates a large number of metabolic processes and is derived from ATP. The DNA sequence of HSV Type 1 contains an adenine phosphate monophosphate response element in the enhancer region of the LAT promoter. Cyclic adenosine monophosphate binds to the regulatory subunit of cyclic adenosine monophosphate dependent protein kinases, releasing active catalytic subunits that can phosphorylate specific transcription factors. Such a factor or phosphoroprotein has been identified in HSV9 and is believed to be involved in regulating many cyclic adenosine monophosphate inducible genes.'0 This suggests that tissue responses have a role in triggering viral reactivation. 1 Whilst there is no doubt that trigeminal ganglia are the main source of latent HSV in the eye, with up to 20 copies of viral DNA per latently infected neuron being present,6 the question remains whether the cornea is also a site of herpes simplex latency. In animal work, HSV has been isolated from rabbit cornea after organ culture but with varying frequency, sometimes depending on the strain of virus used. In the rabbit model, after corneal inoculation with the virus, adrenaline corneal iontophoresis resulted in the virus reappearing only in those animals that had been inoculated with virus strains in which LATs were expressed, and not in those where it was deficient,'2 Electron microscopic studies of human cornea removed by keratoplasty after previous HSV infection have shown incomplete extracellular viral particles in deep stromal levels"; (for review see ref 14), which could signify low grade infection still being present. Organ culture of keratoplasty specimens has resulted in the isolation of viral particles from
McGill
642
the cornea, but whilst none of previously infected human corneas showed any clinical signs of active herpetic infection immediately prior to keratoplasty, it is possible that the HSV isolated after these corneas were cultured in tissue culture (organ culture) was either a contaminant or from low grade corneal infection. Although electron microscopic studies only found HSV particles in such corneas after but not before organ culture,'56 it is possible that low grade infection persisted with relatively few viral particles present and were missed by the relatively limited numbers of slides examined with the electron microscope. In situ hybridisation studies using gene probes for the whole HSV genome have found HSV DNA material in 50% of corneas examined 60 days after experimental corneal inoculation.'7 This contrasts with HSV detection rates of between 17 and 24% after experimental or human corneal organ culture,'8 but this does not mean that the gene is either functional, or that it is all present. In situ hybridisation techniques will not pick up small amounts of viral nucleic acids present, so the polymerase chain reaction has been used to amplify and detect any viral DNA and RNA present (for summary of techniques see ref
20).
Latency associated transcripts (LATs RNA) have been found in corneas removed at the time of keratoplasty from patients seropositive to HSV, either with recurrent but clinically quiescent herpes simplex keratitis, or non HSV corneal disease.2' In this issue Cook et al, after experimental corneal HSV infection, during the latent period found HSV DNA from the ICPO/LAT and thymidine kinase (TK) genes in all of the trigeminal ganglia and three of five corneas investigated, signifying that infection had taken place. HSV LAT RNA was found in all of the trigeminal ganglia and 9% of corneas, signifying latency had been established in these areas. The absence of TK RNA in the corneas rules out active low grade corneal infection, but this potential certainly existed as TK DNA was found in both trigeminal ganglia and corneas. In order to definitely establish that active infection is not taking place, and that the corneal latency has been established after HSV infection, such a study should incorporate both the
operational definition of latency and molecular studies involving LATs. This implies that the same corneas be assessed for the absence of infectious HSV protein, the presence of only LATs or other latency associated transcripts, virus only being produced after organ culture, and no virus seen on electron microscopy prior to corneal organ culture. If such LATs are definitely found in the cornea, and the protein that they express can be identified, it should be possible, either by drugs or monoclonal antibodies directed against those herpes proteins, to block either the establishment of latency or reactivation of the HSV. This would lead to the long term goal of prevention of recurrence of HSV from the human cornea. The cause of herpes simplex induced stromal disease is controversial. Incomplete herpes simplex viral particles have been found in deep stromal levels in quiescent stromal keratitis,'3 complete particles within stromal keratocytes after graft rejection22 and the cornea has now been shown in this issue to be a potential source of viral latency. This would suggest that local viral replication plays a major part in the pathogenesis of herpes simplex stromal disease, but it is
uncertain whether either just viral replication or a local immune response or both are responsible for all the disease process. It does mean though that antiviral chemotherapy is essential in the treatment of this condition and it raises the question whether topical steroids are also required. For the future, genetic manipulation of the whole HSV genome could result in the manufacture of a non-virulent variant, not expressing latency, that could be used as a vector to transport genes responsible for enzymes deleted or deficient in the target organ as a result of acquired or congenital disease. For instance, the herpes virus could be used to transport genes to those corneas with congenital dystrophies such as classic lattice where amyloid material accumulates, or in macular dystrophy where glycosoaminoglycans accumulates as a result of a defect in the conversion of a glycoprotein precursor. JAMES McGILL 1 Gordon YJ, Romanowski E, Araullo-Cruz T, McKnight JLC. HSC-l corneal latency. Invest Ophthalmol Vis Sci 1991; 32: 663-5. 2 Mellerick DM, Fraser RW. Physical state of the latent herpes simplex virus genome in a mouse model nodal system: evidence suggesting an episomal state. Virology 1987; 158: 265-75. 3 Sedarati F, Izumi KM, Wagner EK, Stevens JG. Herpes simplex virus type 1 latency-associated transcription plays no role in establishment or maintenance of a latent infection in murine sensory neurons. J Virol 1989; 63: 4455-8. 4 Lieb DA, Bogard CL, Kosz-Vnenchak M, Hicks KA, Coen DM, Knipe DM, et al. A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency. J Virol 1989; 63: 2893-900. 5 Hioll JM, Sedarati F, Javier RT, Wagner EK, Stevens JG. Herpes simplex virus latent phase transcription facilitates in vivo reactivation. Virology 1990; 174: 117-25. 6 Stevens JG. Human herpes viruses: a consideration of the latent state. Microbzol Rev 1989; 53: 318-32. 7 Blyth WA, Hill TJ, Field HJ, Harbour DA. Reactivation of herpes simplex virus infection by ultraviolet light and possible involvement of prostaglandins. J Gen Virol 1976; 33: 547-50. 8 Kurane I, Tsuchiya Y, Sekizawa T, Kumagi K. Inhibition by indomethacin of in vitro reactivation of latent herpes simplex virus type 1 in murine trigeminal ganglia. J Gen Virol 1984: 65: 1665-74. 9 Leib DA, Nadeau KC, Rundle SA, Schaffer PA. The promoter of the latencyassociated transcripts of herpes simplex virus type 1 contains a functional cAMP-response element: Role of the latency-associated transcripts and cAMP in reactivation of viral latency. Proc Nadl Acad Sci USA Medical Seience 1991: 88: 48-52. 10 Jones RH, Jones NC. Mammalian cAMP-responsive element can activate transcription in yeast and binds a yeast factor(s) that resembles the mammalian transcription factor ATF. Proc Natl Acad Sci USA Biochemistrn 1989; 86: 2176-80. 11 Sears AE, Roizman B. Amplification by host cell factors of a sequence contained within the herpes simplex virus 1 genome. Proc Natl Acad Sci USA Microbiology 1990; 87: 9441-4. 12 Hill JM, Sedarati F, Javier RT, Wagner EK, Stevens JG. Herpes simplex virus latent phase transcription facilitates in vivo reactivation. Virology 1990; 174: 117-25. 13 Davson C, Togni E, Moore TE. Structural changes in chronic herpetic keratitis. Arch Ophthalmol 1968; 79: 740-7. 14 Cook SD, Hill JM. HSV: Molecular biology and the possibility of latent corneal infections. Surv Ophthalmol 1991 (in press). 15 Cook SD, Aitken DA, Loeftler WU, Brown SM. Herpes simplex virus in the cornea; An ultrastructural study on viral reactivation. Trans Ophthalmol Soc UK 1986; 105: 634-41. 16 Easty DL, Shimeld C, Claoue CMP, Menage M. Herpes simplex virus isolation in chronic stromal keratitis: human and laboratory studies. Czirr Eye Res
1987;6:68-74.
17 Sabbaga EMH, Pavan-Langston D, Bean KM, Dunkel EC. Detection of HSV nucleic acid sequences in the cornea during acute and latent ocular disease. Exp Eye Res 1988; 47: 545-53. 18 Shield C, Tullo AB, Easty DL, Thomsitt J. Isolation of herpes simplex virus from the cornea in chronic stromal keratitis. BrJt Ophthlamol 1982; 66: 6437. 19 Tullo AB, Easty DL, Shimeld PE, Stirling PE, Darville JM. Isolation of herpes simplex virus from corneal discs of patients with chronic stromal keratitis. Trans ofOphthal Soc UK 1985; 104: 159-65. 20 Easty DL. Molecular biology for the ophthalmologist [Editorial]. r j
Ophthalmol 1991; 75: 193-4. 21 Kaye SB, Lynas C, Patterson A, Risk JM, McCarthy K, Hart CA. Evidence for herpes simplex viral latency in the human cornea. BrJ' Ophthlamiol 1991; 75: 195-200. 22 Holbach LM, Font RL, Wilhelmus KR. Recurrent herpes simplex keratitis with concurrent epithelial and stromal involvement. Arch Ophthalmtol 1991: 109: 692-5.
British Journal of Ophthalmology, 1991, 75, 641-642
BRITISH JOURNAL OF OPHTHALMOLOGY
Editorial Herpes simplex latency and the eye The herpes simplex virus (HSV) exerts its ocular damage by recurrent corneal infection, having lain dormant in the host tissues for some time. Latency of the virus is an essential part of its natural history, yet the mechanism of latency and recurrence has, for both the clinician and the research worker, remained an enigma. The definition of HSV latency has been described as the capacity of the virus to exist in a host cell for an extended period of time in a virion-free state and, upon stimulation, to reactivate and replicate to produce infectious progeny virions.' As a result of recent advances in molecular biology and genetics, the phenomenon of latency and reactivation is now being explained in terms of specific chemical components found or localised in the chromosomes. After primary infection, usually of ectodermally derived tissues, the viral genetic material travels by fast axonal transport as a nuclear capside to the site of latency, either the trigeminal ganlgion or other extraneuronal sites, where it exists in an extrachromosomal state as a circular episome.2 HSV reactivation can be thought of in terms of the classic Crick model of the HSV DNA molecule acting as a template to use the host cell DNA to copy or express, by DNA replication, the HSV genetic information which is transmitted (by transcription) into RNA nucleic acid, which in turn directs the synthesis in the host cell of the specific HSV protein to manufacture the entire virus. Genetic studies have shown that during the acute productive infection many genes are expressed, including the immediate early, early and late viral genes. During latent infection, when the virus is in a non-replicating but transcriptionally active state, only part of the genome is present, specifying latency associated transcripts (LATs), of which four have been identified from the viral DNA strand opposite that which encodes the infected cell protein ICPO, an immediately early regulatory protein. The LATs overlap at least 30% of the terminus of this protein but the transcription pattern of the LAT encoding region is complex and, as yet, not fully defined. The exact localisation of the genes concerned with the establishment, maintenance and reactivation of latency remain unknown, nor has any protein been found associated with these functions. It has been suggested that reactivation involves expression of a subset of genes, which pass to the surface tissue, such as cornea or skin, initiating a replication cascade and leading ultimately to corneal involvement or skin vesicles. The mechanism which controls the switch between acute and latent phases of infection is poorly understood. Expression of LATs is not essential for the maintenance of latency'
but is concerned in viral reactivation. This has been demonstrated by the finding that viral strains lacking LATs can still establish latency in the experimental animal, but have reduced recurrence rates compared with those strains with a LAT present.45 (See ref 6 for overall review.) It has been suggested that promoters of LATs of herpes simplex come from a local tissue response. It is likely that a number of reactivation inducing stimuli in humans occur, including ultraviolet irradiation, trauma and the experimental process of iontophoresis of adrenaline into the cornea. Prostaglandins have been thought to be implicated in the reactivation of viral latency.7 Indomethazine, a potent inhibitor of prostaglandin synthesis, has been shown to reversibly inhibit the reactivation of HSV from experimental latently infected ganglia.' In mammalian cells such stimuli lead to release of an agent which interacts with a cell surface receptor activating adenylate cyclase which synthesises cyclic adenosine monophosphate, a cyclic nucleotide that regulates a large number of metabolic processes and is derived from ATP. The DNA sequence of HSV Type 1 contains an adenine phosphate monophosphate response element in the enhancer region of the LAT promoter. Cyclic adenosine monophosphate binds to the regulatory subunit of cyclic adenosine monophosphate dependent protein kinases, releasing active catalytic subunits that can phosphorylate specific transcription factors. Such a factor or phosphoroprotein has been identified in HSV9 and is believed to be involved in regulating many cyclic adenosine monophosphate inducible genes.'0 This suggests that tissue responses have a role in triggering viral reactivation. 1 Whilst there is no doubt that trigeminal ganglia are the main source of latent HSV in the eye, with up to 20 copies of viral DNA per latently infected neuron being present,6 the question remains whether the cornea is also a site of herpes simplex latency. In animal work, HSV has been isolated from rabbit cornea after organ culture but with varying frequency, sometimes depending on the strain of virus used. In the rabbit model, after corneal inoculation with the virus, adrenaline corneal iontophoresis resulted in the virus reappearing only in those animals that had been inoculated with virus strains in which LATs were expressed, and not in those where it was deficient,'2 Electron microscopic studies of human cornea removed by keratoplasty after previous HSV infection have shown incomplete extracellular viral particles in deep stromal levels"; (for review see ref 14), which could signify low grade infection still being present. Organ culture of keratoplasty specimens has resulted in the isolation of viral particles from
McGill
642
the cornea, but whilst none of previously infected human corneas showed any clinical signs of active herpetic infection immediately prior to keratoplasty, it is possible that the HSV isolated after these corneas were cultured in tissue culture (organ culture) was either a contaminant or from low grade corneal infection. Although electron microscopic studies only found HSV particles in such corneas after but not before organ culture,'56 it is possible that low grade infection persisted with relatively few viral particles present and were missed by the relatively limited numbers of slides examined with the electron microscope. In situ hybridisation studies using gene probes for the whole HSV genome have found HSV DNA material in 50% of corneas examined 60 days after experimental corneal inoculation.'7 This contrasts with HSV detection rates of between 17 and 24% after experimental or human corneal organ culture,'8 but this does not mean that the gene is either functional, or that it is all present. In situ hybridisation techniques will not pick up small amounts of viral nucleic acids present, so the polymerase chain reaction has been used to amplify and detect any viral DNA and RNA present (for summary of techniques see ref
20).
Latency associated transcripts (LATs RNA) have been found in corneas removed at the time of keratoplasty from patients seropositive to HSV, either with recurrent but clinically quiescent herpes simplex keratitis, or non HSV corneal disease.2' In this issue Cook et al, after experimental corneal HSV infection, during the latent period found HSV DNA from the ICPO/LAT and thymidine kinase (TK) genes in all of the trigeminal ganglia and three of five corneas investigated, signifying that infection had taken place. HSV LAT RNA was found in all of the trigeminal ganglia and 9% of corneas, signifying latency had been established in these areas. The absence of TK RNA in the corneas rules out active low grade corneal infection, but this potential certainly existed as TK DNA was found in both trigeminal ganglia and corneas. In order to definitely establish that active infection is not taking place, and that the corneal latency has been established after HSV infection, such a study should incorporate both the
operational definition of latency and molecular studies involving LATs. This implies that the same corneas be assessed for the absence of infectious HSV protein, the presence of only LATs or other latency associated transcripts, virus only being produced after organ culture, and no virus seen on electron microscopy prior to corneal organ culture. If such LATs are definitely found in the cornea, and the protein that they express can be identified, it should be possible, either by drugs or monoclonal antibodies directed against those herpes proteins, to block either the establishment of latency or reactivation of the HSV. This would lead to the long term goal of prevention of recurrence of HSV from the human cornea. The cause of herpes simplex induced stromal disease is controversial. Incomplete herpes simplex viral particles have been found in deep stromal levels in quiescent stromal keratitis,'3 complete particles within stromal keratocytes after graft rejection22 and the cornea has now been shown in this issue to be a potential source of viral latency. This would suggest that local viral replication plays a major part in the pathogenesis of herpes simplex stromal disease, but it is
uncertain whether either just viral replication or a local immune response or both are responsible for all the disease process. It does mean though that antiviral chemotherapy is essential in the treatment of this condition and it raises the question whether topical steroids are also required. For the future, genetic manipulation of the whole HSV genome could result in the manufacture of a non-virulent variant, not expressing latency, that could be used as a vector to transport genes responsible for enzymes deleted or deficient in the target organ as a result of acquired or congenital disease. For instance, the herpes virus could be used to transport genes to those corneas with congenital dystrophies such as classic lattice where amyloid material accumulates, or in macular dystrophy where glycosoaminoglycans accumulates as a result of a defect in the conversion of a glycoprotein precursor. JAMES McGILL 1 Gordon YJ, Romanowski E, Araullo-Cruz T, McKnight JLC. HSC-l corneal latency. Invest Ophthalmol Vis Sci 1991; 32: 663-5. 2 Mellerick DM, Fraser RW. Physical state of the latent herpes simplex virus genome in a mouse model nodal system: evidence suggesting an episomal state. Virology 1987; 158: 265-75. 3 Sedarati F, Izumi KM, Wagner EK, Stevens JG. Herpes simplex virus type 1 latency-associated transcription plays no role in establishment or maintenance of a latent infection in murine sensory neurons. J Virol 1989; 63: 4455-8. 4 Lieb DA, Bogard CL, Kosz-Vnenchak M, Hicks KA, Coen DM, Knipe DM, et al. A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency. J Virol 1989; 63: 2893-900. 5 Hioll JM, Sedarati F, Javier RT, Wagner EK, Stevens JG. Herpes simplex virus latent phase transcription facilitates in vivo reactivation. Virology 1990; 174: 117-25. 6 Stevens JG. Human herpes viruses: a consideration of the latent state. Microbzol Rev 1989; 53: 318-32. 7 Blyth WA, Hill TJ, Field HJ, Harbour DA. Reactivation of herpes simplex virus infection by ultraviolet light and possible involvement of prostaglandins. J Gen Virol 1976; 33: 547-50. 8 Kurane I, Tsuchiya Y, Sekizawa T, Kumagi K. Inhibition by indomethacin of in vitro reactivation of latent herpes simplex virus type 1 in murine trigeminal ganglia. J Gen Virol 1984: 65: 1665-74. 9 Leib DA, Nadeau KC, Rundle SA, Schaffer PA. The promoter of the latencyassociated transcripts of herpes simplex virus type 1 contains a functional cAMP-response element: Role of the latency-associated transcripts and cAMP in reactivation of viral latency. Proc Nadl Acad Sci USA Medical Seience 1991: 88: 48-52. 10 Jones RH, Jones NC. Mammalian cAMP-responsive element can activate transcription in yeast and binds a yeast factor(s) that resembles the mammalian transcription factor ATF. Proc Natl Acad Sci USA Biochemistrn 1989; 86: 2176-80. 11 Sears AE, Roizman B. Amplification by host cell factors of a sequence contained within the herpes simplex virus 1 genome. Proc Natl Acad Sci USA Microbiology 1990; 87: 9441-4. 12 Hill JM, Sedarati F, Javier RT, Wagner EK, Stevens JG. Herpes simplex virus latent phase transcription facilitates in vivo reactivation. Virology 1990; 174: 117-25. 13 Davson C, Togni E, Moore TE. Structural changes in chronic herpetic keratitis. Arch Ophthalmol 1968; 79: 740-7. 14 Cook SD, Hill JM. HSV: Molecular biology and the possibility of latent corneal infections. Surv Ophthalmol 1991 (in press). 15 Cook SD, Aitken DA, Loeftler WU, Brown SM. Herpes simplex virus in the cornea; An ultrastructural study on viral reactivation. Trans Ophthalmol Soc UK 1986; 105: 634-41. 16 Easty DL, Shimeld C, Claoue CMP, Menage M. Herpes simplex virus isolation in chronic stromal keratitis: human and laboratory studies. Czirr Eye Res
1987;6:68-74.
17 Sabbaga EMH, Pavan-Langston D, Bean KM, Dunkel EC. Detection of HSV nucleic acid sequences in the cornea during acute and latent ocular disease. Exp Eye Res 1988; 47: 545-53. 18 Shield C, Tullo AB, Easty DL, Thomsitt J. Isolation of herpes simplex virus from the cornea in chronic stromal keratitis. BrJt Ophthlamol 1982; 66: 6437. 19 Tullo AB, Easty DL, Shimeld PE, Stirling PE, Darville JM. Isolation of herpes simplex virus from corneal discs of patients with chronic stromal keratitis. Trans ofOphthal Soc UK 1985; 104: 159-65. 20 Easty DL. Molecular biology for the ophthalmologist [Editorial]. r j
Ophthalmol 1991; 75: 193-4. 21 Kaye SB, Lynas C, Patterson A, Risk JM, McCarthy K, Hart CA. Evidence for herpes simplex viral latency in the human cornea. BrJ' Ophthlamiol 1991; 75: 195-200. 22 Holbach LM, Font RL, Wilhelmus KR. Recurrent herpes simplex keratitis with concurrent epithelial and stromal involvement. Arch Ophthalmtol 1991: 109: 692-5.
