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Accumulation of amyloid-β (Aβ) in the brain is a central component of pathology in Alzheimer's disease. A growing volume of evidence demonstrates close associations between periodontal pathogens including Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola) and AD. However, the effect and mechanisms of T. denticola on accumulation of Aβ remain to be unclear. In this study, we demonstrated that T. denticola was able to enter the brain and act directly on nerve cells resulting in intra- and extracellular Aβ 1-40 and Aβ 1-42 accumulation in the hippocampus of C57BL/6 mice by selectively activating both β-secretase and γ-secretase. Furthermore, both KMI1303, an inhibitor of β-secretase, as well as DAPT, an inhibitor of γ- secretase, were found to be able to inhibit the effect of T. denticola on Aβ accumulation in N2a neuronal cells. Overall, it is concluded that T. denticola increases the expression of Aβ 1-42 and Aβ 1-40 by its regulation on beta-site amyloid precursor protein cleaving enzyme-1 and presenilin 1.

Herpes simplex encephalitis (HSE) is characterized by severe focal brain inflammation leading to substantial loss of nervous tissue. The authors established a model of Herpes simplex virus type 1 (HSV)-1-induced acute encephalitis in the rat by injecting into the whiskers' area a virus strain isolated from a fatal human HSE case. The model might resemble natural propagation of HSV-1 in humans; spreading from the mouth and lips via the trigeminal nerve to trigeminal ganglia and subsequently entering the central nervous system (CNS). HSV-1 infected Dark Agouti (DA) rats developed a well-synchronized disease and died 5 days after inoculation. HSV-1 detection by quantitative polymerase chain reaction (qPCR), virus isolation and immunohistochemistry, magnetic resonance imaging, and histopathological examination verified dramatic encephalitis mainly in the brainstem, but also in the olfactory bulb and other segments of the brain of diseased rats. In contrast, Piebald Virol Glaxo (PVG) rats were completely resistant to disease, displaying a more rapid clearance of peripheral infection and no evidence of virus entering into neither the trigeminal ganglia nor the CNS. These results suggest a regulation of susceptibility to HSV-1-induced encephalitis at the level of peripheral infection and subsequent neuronal uptake/transport of the virus. This provides a basis for future positioning of genetic polymorphisms regulating HSE and for dissection of important pathogenetic mechanisms of this severe human disease.

Streptococcus pneumoniae cause considerable morbidity and mortality, with persistent neurological sequelae, particularly in young children and the elderly. It is widely assumed that carriage occurs through direct mucosal colonization from the environment whereas meningitis results from invasion from the blood. However, the results of published studies can be interpreted that pneumococci may enter the brain directly from the nasal cavity by axonal transport through olfactory nerves. This hypothesis is based on findings that (i) teichoic acid of the pneumococcal cell wall interact with gangliosides (GLS), (ii) the interaction of GLS with cholera toxin leads to axonal transport through the olfactory nerves into the brain, and (iii) viruses enter the brain through axonal transport into olfactory nerves. After nasal inoculation, we observe high numbers of pneumococci in nasal washes and the olfactory nerves and epithelium. Significant numbers of pneumococci also infected the olfactory bulbs, brain, and the trigeminal ganglia. The absence of bacteremia in this model makes it unlikely that the bacteria entered the brain from the blood stream. Recovery of colony-forming units from the brain, lungs, olfactory nerves, and epithelium and nasal washes was inhibited by incubating pneumococci with GLS before nasal inoculation. These findings, confirmed by PCR and immunohistochemistry, support a GLS-mediated process of infection and are consistent with pneumococci reaching the brain through retrograde axonal transport.

By using mice genomically lacking the mononuclear phagocytic growth factor colony-stimulating factor 1 and thereby deficient in macrophage and dendritic cell populations, we show that these cells play a dual role: they constitute a major defense against systemic infection but also facilitate cerebral bacterial invasion by Listeria monocytogenes.

The purpose of this investigation was to use molecular and immunological techniques to determine whether oral Treponema infected the human brain. Pieces of frontal lobe cortex from 34 subjects were analyzed with species-specific PCR and monoclonal antibodies. PCR detected Treponema in 14/16 Alzheimer's disease (AD) and 4/18 non-AD donors (P < 0.001), and AD specimens had more Treponema species than controls (P < 0.001). PCR also detected Treponema in trigeminal ganglia from three AD and two control donors. Cortex from 15/16 AD subjects and 6/18 controls contained Treponema pectinovorum and/or Treponema socranskii species-specific antigens (P < 0.01). T. pectinovorum and/or T. socranskii antigens were also found in trigeminal ganglia and pons from four embalmed cadavers, and 2/4 cadavers also had Treponema in the hippocampus. These findings suggest that oral Treponema may infect the brain via branches of the trigeminal nerve.

The pathologic features of cerebral Listeria monocytogenes infection strongly suggest that besides hematogenous spread, bacteria might also spread via a neural route. We propose that after snout infection of recombination activating gene 1 (RAG-1)-deficient mice, L. monocytogenes spreads to the brain via a neural route. The neural route of invasion is suggested by (i) the immunostaining of L. monocytogenes in the trigeminal ganglia (TG) and brain stem but not in other areas of the brain; (ii) the kinetics of bacterial loads in snout, TG, and brain; and (iii) the increased resistance of mice infected with a plcB bacterial mutant (unable to spread from cell to cell). Gamma interferon (IFN-gamma) plays a protective role in neuroinvasion; inducible nitric oxide synthase (iNOS) accounts only partially for the protection, as shown by a comparison of the susceptibilities of IFN-gamma receptor (IFN-gamma R)-deficient, iNOS-deficient, and wild-type mice to snout infection with L. monocytogenes. The dramatically enhanced susceptibility of RAG-1-deficient, IFN-gamma R gene-deficient mice indicated the overall importance of innate immune cells in the release of protective levels of IFN-gamma. The source of IFN-gamma appeared to be NK cells, as shown by use of RAG-1-deficient, gamma-chain receptor gene-deficient mice; NK cells played a relevant protective role in neuroinvasion through a perforin-independent mechanism. In vitro evidence indicated that IFN-gamma can directly induce bacteriostatic mechanisms in neural tissue.

The ganglionic cell type in which varicella-zoster virus (VZV) is latent in humans was analyzed by using antibodies raised against in vitro-expressed VZV open reading frame 63 protein. VZV open reading frame 63 protein was detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is, to our knowledge, the first identification of a herpesvirus protein expressed during latency in the human nervous system.

Infection by standard thymidine kinase-positive (TK+) and TK- mutant herpes simplex virus (HSV) was performed in order to evaluate the role of HSV TK expression in neurovirulence and in HSV latency. In newborn mice, mortality and trigeminal ganglion (TG) HSV titer correlated (both were high) for TK+ and TK- HSV. In adult mice after TK- HSV infection they also correlated (both were low). After TK+ infection of adult mice, correlation was not present; mortality was low while HSV titer was moderately high. During the period of HSV latent infection (> 28 days after HSV infection), the number of neurons expressing HSV latency-associated transcript (LAT) was much greater for TK- HSV newborn-inoculated mice (average of 943/ganglion) than adult-inoculated mice (average of 138/ganglion). In addition, total amount of TG LAT was greater in the former than the latter. Reactivation from latency was restricted, however, for both groups. This result supported the important role of HSV TK expression in HSV reactivation, even when the number of LAT-positive neurons was greatly increased. The following conclusions were drawn from the study of TK- HSV in newborn mice: (i) HSV TK expression was of limited importance for neurovirulence and in vivo HSV TG infection (but was of importance in adult mice); (ii) increased in vivo HSV TG infection correlated with increased number of LAT-positive neurons, so that HSV replication and establishment of latency were not completely separable; and (iii) even with greatly increased numbers of latently infected neurons, HSV TK expression was important for reactivation from latency. Results in newborn mice suggested that the role of HSV TK expression in reactivation from latency and in neurovirulence were separable. To further investigate HSV replication in newborn and adult mice, ganglia were infected with HSV in vitro and either maintained in vitro or transplanted beneath the renal capsule of adult recipients. In both of these studies, HSV titers in ganglia were much higher in newborn than adult ganglia. This suggested that in addition to the well-know role of the immune system in HSV neurovirulence in newborn mice, it is likely that HSV replication per se in neural tissue is greater in newborn than adult mice. This may be related to the high level of HSV neurovirulence in newborn mice.

Herpes simplex virus establishes latency in nervous tissue in which it is maintained for the life of the mammalian host, with occasional reactivation leading to subsequent spread. Latency-associated transcripts are abundant during latency, but viral proteins and productive cycle RNAs have not been detected. Using sensitive, quantitative PCR assays, we have quantified certain viral RNAs specific to productive-cycle genes in mouse ganglia latently infected with herpes simplex virus type 1. Sense-strand RNA specific to the essential immediate-early gene, ICP4, was present in most ganglia in variable amounts relative to the amount of viral DNA, with one to seven molecules of RNA per viral genome in about 20% of ganglia. In contrast, the amount of latency-associated transcripts was much less variable, at an average of 4 x 10(4) molecules per viral genome. The amounts of ICP4-specific RNA were similar at 30 and 60 days postinfection, and at least some of these transcripts initiated within a region consistent with utilization of the ICP4 promoter. RNA specific to the thymidine kinase gene, whose transcription in productive infection is dependent on ICP4, was present in latently infected ganglia at a maximum level of 3.2 x 10(6) molecules per ganglion (500 molecules per viral genome). ICP4-specific and tk-specific RNAs measured from the same samples showed a positive correlation extending over 2 orders of magnitude. We conclude that ICP4-specific RNA is expressed in the absence of detectable reactivation and discuss possible implications of our findings for latent gene expression.

The number of TNF-alpha and IL-1 beta producing cells was investigated during the acute replication phase of herpes simplex virus (HSV) in trigeminal ganglia after intranasal infection with strains of different virulence. The highly virulent strain WAL replicated strongly and induced many cytokine producing cells early in the ganglia. The low virulent strain HFEM replicated less, only few cytokine producing cells were detected late. The thymidine-kinase negative (TK-) virus 1301 did not replicate but produced some lymphocytic inflammation. The higher the virulence of strains of HSV-1 or -2 was, the stronger was the extent of histopathological lesions; moreover, a dissociation in time between replication and cellular reaction (granulocytic and lymphocytic) could be observed after infection with strains HFEM and TK- virus 1301. CD4 and CD8 positive cells could be detected mainly at the rim of necrotic areas, TNF-alpha and IL-1 beta producing cells, however, were scattered throughout the ganglia.

Varicella zoster virus (VZV) establishes latency in sensory ganglia following primary infection (chickenpox) and may reactivate decades later to produce zoster (shingles). The presence of VZV DNA in latently infected ganglia has been demonstrated by Southern blot hybridization as well as by polymerase chain reaction of DNA extracted from latently infected ganglia. Conflicting results have been obtained by in situ hybridization studies to determine the cell type in the ganglia harboring the latent VZV. To address this controversy we have utilized a more sensitive method than the previous studies. We have applied the technique of polymerase chain reaction to sections of ganglia from latently infected individuals and combined this with in situ hybridization to detect the amplified product. Primers specific for VZV were used to amplify VZV DNA in latently infected human trigeminal ganglia and demonstrated the presence of VZV DNA in neurons only. Sections from human kidney and ganglia from neonates as well as monkey ganglia served as controls and did not show amplification of VZV sequences. Amplification using primers for human genes, alpha tubulin and the oncogene Bcl-2, demonstrated the presence of these sequences in nearly all cells in the human tissues while only weak signals were seen in the monkey tissue. This is the first report where in situ amplification has been utilized to detect latent VZV in human ganglia.

Four specific pathogen-free ponies were infected intranasally with equine herpesvirus 1 (EHV-1) and two were similarly infected with an EHV-1 thymidine kinase deletion mutant. The primary infections were characterized by a transient fever accompanied by virus shedding into nasal mucus and viraemia. No virus was detected in clinical specimens after 15 days post-infection. Two months later a reactivation stimulus was administered to all six ponies and only the four that had been previously inoculated with wild-type EHV-1 shed virus into nasal mucus (for 10 days), proving the presence of a latent infection. No recurrence of viraemia was observed. The animals were monitored for a further 6 weeks and were consistently shown to be free from infectious virus. Tissues were then obtained postmortem. Co-cultivation of explanted trigeminal ganglia from two out of the four ponies that carried the wild-type virus yielded cultures positive for infectious virus. Apart from nasal epithelium, no infectious virus was recovered from any other tissue. PCR confirmed the presence of virus DNA in the ganglia from all six ponies. Lymphoid tissues also yielded positive signals using this technique. The relevance of virus detection by PCR in lymphoid and neural tissues is discussed in relation to the potential for reactivation of latent virus in the host. However, evidence is presented to show that EHV-1 is neurotropic and, in common with other members of the alpha-herpesvirus subfamily, establishes latency in sensory ganglia from which virus can be reactivated.

Glycoprotein D (gD) is an essential component of the herpes simplex virus (HSV) envelope. It is essential for viral penetration and for cell to cell spread of virus in vitro, and is also important for neuroinvasiveness. We investigated the contribution of N-linked oligosaccharides (N-CHO) on gD to viral pathogenesis. We used F-gD(QAA), a mutant virus derived from strain F of HSV-1. This virus contains three mutations in the gD gene which eliminate all signals for addition of N-CHO. These mutations affect the antigenic structure of gD and also lead to a small plaque phenotype. Otherwise the virus appears normal in in vitro assays. We used the mouse eye model of HSV latency to examine whether the mutations alter the phenotype of the virus in vivo. At 4 days postinfection similar amounts of F-gD(QAA) and F-gD(WT), its wild-type parent, were found in either eyes or trigeminal ganglia (TG) of infected mice. Moreover, both mutant and wild-type viruses exhibited the same ability to establish, maintain, and be reactivated from latency. We conclude that N-CHO on gD are not essential for HSV-1 pathogenesis in this model.

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.

Clinical resistance to antiviral drugs requires that a virus evade drug therapy yet retain pathogenicity. Thymidine kinase (TK)-negative mutants of herpes simplex virus are resistant to the drug, acyclovir, but are attenuated for pathogenicity in animal models. However, numerous cases of clinical resistance to acyclovir have been associated with viruses that were reported to express no TK activity. We studied an acyclovir-resistant clinical mutant that contains a single-base insertion in its tk gene, predicting the synthesis of a truncated TK polypeptide with no TK activity. Nevertheless, the mutant retained some TK activity and the ability to reactivate from latent infections of mouse trigeminal ganglia. The mutant expressed both the predicted truncated polypeptide and a low level of a polypeptide that comigrated with full-length TK on polyacrylamide gels and reacted with anti-TK antiserum, providing evidence for a frameshifting mechanism. In vitro transcription and translation of mutant tk genes, including constructs in which reporter epitopes could be expressed only if frameshifting occurred, also gave rise to truncated and full-length polypeptides. Reverse transcriptase-polymerase chain reaction analysis coupled with open reading frame cloning failed to detect alterations in tk transcripts that could account for the synthesis of full-length polypeptide. Thus, synthesis of full-length TK was due to an unusual net +1 frameshift during translation, a phenomenon hitherto confined in eukaryotic cells to certain RNA viruses and retrotransposons. Utilization of cellular frameshifting mechanisms may permit an otherwise TK-negative virus to exhibit clinical acyclovir resistance.

Restricted gene expression takes place during latent infection of herpes simplex virus type 1 (HSV-1) in the human peripheral nervous system and has been linked with viral reactivation. The state of HSV-1 gene expression in the central nervous system (CNS) during latency is unclear and we, therefore, examined gene expression in the brainstem of experimental mice and normal humans. Only part of the transcription pattern present during latent infection in peripheral sensory ganglia (PSG) was identified in the human brainstem by in situ hybridization and Northern blot analysis for HSV-1-specific transcripts. Instead of three HSV-1 latency-associated transcripts (LATs) present in PSG and demonstrated by Northern blot analysis, only one was identified in mouse brainstem and none was detected in human brainstem. These findings might be attributed to the relatively low amounts of HSV-1-specific latency-associated RNAs in brainstem tissue. Combined with our inability to reactivate HSV-1 from explanted mouse brainstem, these findings suggest that tissue levels of latency-associated gene expression play a role in HSV-1 reactivation and have relevance to the very low incidence of HSV-1-induced CNS disease compared with peripheral mucocutaneous disease.

Herpes simplex virus type I (HSV-1) establishes a latent infection in sensory ganglion neurons. During latency no viral-specific proteins are detected and virus gene expression is restricted to the latency-associated transcripts. We report here that trigeminal ganglia of mice with severe combined immunodeficiency contain individual sensory neurons exhibiting restricted viral gene expression characteristic of latency; this occurred during acute (4-6 days) infection with the wild-type HSV-1 strain 17+ and after prolonged (4 weeks) infection with the replication impaired HSV-1 mutant in 1814. These results indicate that T and B lymphocytes, while important for the recovery from viral infections, are not required for the establishment or maintenance of latency in neurons.

Herpes simplex virus type 1 (HSV-1) expresses a unique series of RNA molecules, the latency-associated transcripts or LATs, during latent infection of neuronal tissues. Previous studies by others have described a TATA box-containing latency-active promoter, referred to here as LAP1, located approximately 700 bp upstream of the 5' end of the major 2.0-kb LAT. In this report, transient gene expression assays were employed to identify a second, novel latency-active promoter (LAP2) present within a region downstream of LAP1 and 5' proximal to the major 2.0-kb LAT. In contrast to LAP1, this promoter lacks a TATA box but possesses cis-acting regulatory elements and other features frequently observed within eukaryotic housekeeping gene promoters. Unlike most other HSV promoters, LAP2 was down-regulated by the viral transcriptional activators ICP4 and ICP0. The majority of LAP2-positive regulatory elements were located within sequences from -257 to -58 relative to the 5' end of the 2.0-kb LAT, and the basal promoter mapped within sequences from -14 to +28. RNase protection experiments demonstrated that chimeric LAT-chloramphenicol acetyltransferase transcripts produced in the transient assays initiated at or near the 5' end of the major 2-kb LAT. Tn5 insertional mutagenesis of the ICP4 regulatory gene determined that down-regulation of LAP2 required the ICP4 transactivating domain and targeted the minimal promoter region as the site of action by ICP4. Replicating recombinant viruses containing a LAP2-lacZ reporter gene cassette in an ectopic site (glycoprotein C locus) were shown to be active in mouse trigeminal ganglia. Taken together, these experiments suggest that the LAT region of the HSV-1 genome contains at least two latency-active promoters which may play different roles in expressing the various LATs. Alternatively, these promoters may comprise a larger promoter-regulatory complex which may influence transcription during latency.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus DNA replication and gene expression in two murine in vitro models for virus reactivation. We examined latent infections with wild-type (wt), precisely defined latency-associated transcript-negative (LAT-) mutants, and LAT+ rescuants of these mutants of the 17syn+ strain of virus in both murine trigeminal and lumbosacral ganglia and of the KOS(M) strain in the latter. In explants of ganglia latently infected with the LAT- mutant of strain 17syn+ virus, a reduction in number of cultures exhibiting cytopathic effects due to virus reactivation and measurable delays in virus recovery were observed compared with wt or the LAT+ rescuant. This LAT-specific effect was not seen in explants of lumbosacral ganglia latently infected with mutants derived from the KOS(M) strain of virus. Although there was appreciable variation between individual animals, no significant difference between LAT+ and LAT- virus in time of onset of viral DNA replication in explanted ganglia was seen with use of either virus strain. There was a slight decrease in the relative amount of viral DNA recovered compared with internal cellular controls in latently infected ganglia harboring the LAT- mutant of 17syn+ compared with the wt virus or the LAT+ rescuant. This reduced relative amount ranged from 0 to as much as 50% but averaged 20%. Such differences were not seen in infections with KOS(M)-derived mutants. In contrast, although expression of productive-cycle transcripts could be detected within 4 h following explant cultivation of latently infected ganglia, no differences between LAT+ and LAT- viruses could be seen. As discussed, these data place specific constraints on possible models for the role of LAT expression in in vitro reactivation systems.

Herpes simplex virus type 1 (HSV-1) establishes latent infection in the sensory ganglia. To investigate the process of reactivation from latency, we used the RNA polymerase chain reaction (RNA-PCR) to detect the expression of several HSV genes. BALB/c mice were inoculated in the anterior ocular chamber with HSV-1 strain KOS and the trigeminal ganglia were examined at least 8 weeks after inoculation. Latency-associated transcripts (LATs) were found in the latently infected ganglia and remained detectable 120 h after explantation. Besides LATs, we detected transcripts for infected cell protein 0 (ICP0) (Vmw110) 24 h after explantation, but RNAs encoding ICP4 (Vmw175), ICP27, thymidine kinase and VP16 (ICP25; Vmw65) remained undetectable for 120 h after explantation. Following in vivo reactivation of HSV-1 by administration of cyclophosphamide and dexamethasone, all viral transcripts including ICP0 RNA became detectable. The RNA-PCR enabled us to detect ICP0 RNA much earlier than has been previously reported in studies using the Northern blot technique and has laid a foundation for further study of viral and cellular transcripts during reactivation. Our results suggest that the process of reactivation of HSV-1 from trigeminal ganglia may be divided into at least two steps: (i) initiation of ICP0 gene transcription and (ii) detectable transcription of the other genes. The second step may be regulated in part by the host immune system, since cyclophosphamide and dexamethasone administration enabled the detection of several viral transcripts.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus (HSV) DNA replication and gene expression in the rabbit corneal model for virus reactivation in vivo. We used carefully defined latency-associated transcript-negative (LAT-) and LAT+ promoter mutants of the 17syn+ strain of HSV type 1. In agreement with earlier studies using a more extensive LAT- deletion mutant, the 17 delta Pst(LAT-) virus reactivated with extremely low frequency upon epinephrine induction. In contrast to our findings with murine latency models, amounts of viral DNA recovered from rabbit ganglia latently infected with either LAT+ or LAT- virus were equivalent. Also in contrast with the murine models, no net increase in viral DNA was seen in latently infected rabbit trigeminal ganglia induced to reactivate in vivo by iontophoresis of epinephrine. Despite this, transcription of lytic-phase genes could be detected within 4 h following induction of rabbits latently infected with either LAT+ or LAT- virus; this transcription diminished by 16 h following induction. These results are discussed in relation to models for the mechanism of action of HSV LAT.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) were used as a new means to immunize mice against HSV-1-mediated ocular infection and disease. The effects of the induced immune responses on pathogenesis of acute and latent infection by challenge virus were investigated after corneal inoculation of immunized mice with virulent HSV-1. A single subcutaneous injection of replication-defective mutant virus protected mice against development of encephalitis and keratitis. Replication of the challenge virus at the initial site of infection was lower in mice immunized with attenuated, wild-type parental virus (KOS1.1) or replication-defective mutant virus than in mice immunized with uninfected cell extract or UV-inactivated wild-type virus. Significantly, latent infection in the trigeminal ganglia was reduced in mice given one immunization with replication-defective mutant virus and was completely prevented by two immunizations. Acute replication in the trigeminal ganglia was also prevented in mice immunized twice with wild-type or mutant virus. The level of protection against infection and disease generated by immunization with replication-defective mutant viruses was comparable to that of infectious wild-type virus in all cases. In addition, T-cell proliferative and neutralizing antibody responses following immunization and corneal challenge were of similar strength in mice immunized with replication-defective mutant viruses or with wild-type virus. Thus, protein expression by forms of HSV-1 capable of only partially completing the replication cycle can induce an immune response in mice that efficiently decreases primary replication of virulent challenge virus, interferes with acute and latent infection of the nervous system, and inhibits the development of both keratitis and systemic neurologic disease.

Glycoprotein C (gC)-negative clinical isolates of herpes simplex virus type 1 (HSV-1) are very rare. An HSV-1 strain (TN-1), isolated from a patient with herpetic keratitis, exhibited a gC-negative phenotype. While a gC-negative mutant showed reduced pathogenicity and failed to induce herpetic stromal keratitis (HSK) in a previously reported mouse model, TN-1 induced HSK in mice comparable to RTN-1-20-3, a gC-positive recombinant virus derived from TN-1. Virus growth in eyes and brains and the mortality of TN-1-inoculated mice were equal to or higher than those of RTN-1-20-3-inoculated mice.

The directional movement of many cellular organelles in neurons is dependent on polarized microtubules and direction-specific motor molecules. Microtubules are also thought to mediate the retrograde transport of herpes simplex virus (HSV) in sensory neurons. To define the cellular machinery responsible for retrograde axonal transport of HSV, we have investigated the polarity of microtubules in the peripheral axons of trigeminal ganglion neurons. The long ciliary nerves of rabbits were prepared for a standard "hook assay" of microtubule polarity. Axons in cross-sectioned nerves contained microtubules with almost uniform orientation. The fast-growing, plus ends of these axonal microtubules are located distal to the cell body and the slow-growing, minus ends are directed centrally. To determine the role played by microtubules in the retrograde transport of HSV in these axons, we injected the retrobulbar space of mice with the microtubule-inhibiting drugs colchicine, vinblastine, or nocodazole or with the microfilament inhibitor cytochalasin D and 1 d later inoculated the cornea with HSV. We found that colchicine, vinblastine, or nocodazole reduced by 52-87% the amount of virus recovered from the ganglion 3 d postinoculation, compared to vehicle-treated animals. In contrast, cytochalasin D or beta-lumicolchicine did not significantly reduce the amount of HSV recovered from the ganglion. We conclude that the retrograde axonal transport of HSV from axon endings in the cornea to the trigeminal ganglion cell bodies requires intact microtubules and occurs in a plus-to-minus direction on the microtubules. Our data are consistent with the hypothesis that the retrograde axonal transport of HSV is mediated by a minus end-directed motor molecule, for example, cytoplasmic dynein.

To detect and localize a herpes simplex virus type 2 (HSV-2) thymidine kinase gene sequence in paraffin sections of brains and trigeminal ganglia of infected mice, an in situ polymerase chain reaction (ISPCR) protocol was developed. Using a single pair of primers, a 110 base pair DNA target sequence, and incorporation of a digoxigenin-labelled nucleotide during amplification, this procedure permitted rapid, specific, reproducible detection of infected cells. During acute brain infection, cells labelled by ISPCR were in the same infected foci that, in adjacent sections, contained viral antigen. This, together with controls, gave evidence of method specificity. In mice surviving acute infection, latently infected cells were labelled by ISPCR. In brains, focal areas contained labelled cell nuclei, and in trigeminal ganglia, neuronal nuclei were likewise labelled. Latent infection was confirmed by several methods, including identification of an HSV-specific sequence in DNA extracts of brains and ganglia, virus isolation from explanted ganglia, and HSV-2 latency-associated transcript (LAT) RNA localization in ganglionic neurons by in situ hybridization. Evidence in brains of ISPCR-labelled cells in regions where HSV-2 LAT-positive cells were not detected, and in ganglia of more ISPCR-labelled neurons than were LAT-positive, indicated that ISPCR is more sensitive in detecting latently infected cells than previous methods.

Poxvirus recombinants, based on the highly attenuated NYVAC strain of vaccinia virus (Tartaglia et al., 1992), containing single gene inserts encoding the pseudorabies virus (PRV) gII, gIII, or gp50 glycoproteins were tested for their immunogenicity in pigs. Twenty-four pigs were randomly divided into six groups of four. Groups 1-3 were inoculated with 10(7) CCID50 of NYVAC/PRV gII, NYVAC/PRV gIII, or NYVAC/PRV gp50, respectively, while groups 4 and 5 received the NYVAC parent virus or an inactivated PRV vaccine control, respectively. Group 6 represented the sham vaccinated control group. All inoculations were given by the intramuscular route on weeks 0 and 4. The candidate vaccines were shown to be safe with no local or systemic reactions. At 4 weeks following the second inoculation, all pigs were challenged by an oronasal administration of a virulent PRV strain. Pigs were monitored before and after challenge for clinical manifestations resulting from vaccination and challenge exposure, respectively. Sera were analyzed for PRV neutralizing activity. Virological analyses after challenge included assessment of virus shedding and the development of latent PRV infections. All but one animal developed latent PRV infection following challenge exposure; however, significant protection against PRV-induced signs was afforded by vaccination with either the NYVAC/PRV gp50 or NYVAC/PRV gII recombinant viruses, as well as with the inactivated PRV vaccine. The NYVAC/PRV gp50 also reduced overall virus shedding after challenge. The extent of protection against PRV-induced clinical signs, in general, was associated with the level of pre-challenge virus neutralizing activity.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.

Intracerebral inoculation of the MS strain of herpes simplex virus type 2 (HSV-2) into mice causes an acute encephalitis associated with multifocal demyelination and necrotizing retinitis. We have studied the distribution of latent virus in mice that had recovered from the acute encephalitis. Four weeks or longer after inoculation, HSV-2 could be recovered from the trigeminal ganglia of all mice examined by co-culture of explants in roller tubes. The virus could not be recovered from explants of retina or brain stem. HSV-2 latency associated transcript (LAT) was readily detected in the trigeminal ganglia by reverse transcriptase-PCR more than 4 months after inoculation. LAT was also demonstrated in the brain but this required nested PCR for consistent detection. Both LAT and ICP0 mRNA were detected in brain tissue during the acute encephalitis but, unlike LAT, ICP0 mRNA could not be amplified from the trigeminal ganglia or brain beyond 4 weeks after inoculation of the virus. In situ hybridisation with a double-stranded DNA probe to the ICP0/LAT overlap region of HSV-2 revealed signal in trigeminal ganglion neurons and occasional cells in the brain stem. These findings indicate that HSV-2 introduced by intracerebral inoculation becomes latent in the trigeminal ganglia and that transcription of LAT also persists within the brain.

Herpes simplex viruses maintained in a latent state in sensory neurons in mice do not reactivate spontaneously, and therefore the factors or procedures which cause the virus to reactivate serve as a clue to the mechanisms by which the virus is maintained in a latent state. We report that cadmium sulfate induces latent virus to reactivate in 75 to 100% of mice tested. The following specific findings are reported. (i) The highest frequency of induction was observed after two to four daily administrations of 100 micrograms of cadmium sulfate. (ii) Zinc, copper, manganese, or nickel sulfate administered in equimolar amounts under the same regimen did not induce viral reactivation; however, zinc sulfate in molar ratios 25-fold greater than those of cadmium induced viral replication in 2 of 16 ganglia tested. (iii) Administration of zinc, nickel, or manganese prior to the cadmium sulfate reduced the incidence of ganglia containing infectious virus. (iv) Administration of cadmium daily during the first week after infection and at 2-day intervals to 13 days after infection resulted in the recovery from ganglia of infectious virus in titers 10- to 100-fold higher than those obtained from animals given saline. Moreover, infectious virus was recovered as late as 11 days after infection compared with 6 days in mice administered saline. (v) Administration of cadmium immediately after infection or repeatedly after establishment of latency did not exhaust the latent virus harbored by sensory neurons, inasmuch as the fraction of ganglia of mice administered cadmium and yielding infectious virus was similar to that observed in mice treated with saline. We conclude that induction of cadmium tolerance precludes reactivation of latent virus. If the induction of metallothionein genes was the sole factor required to cause reactivation of latent virus, it would have been expected that all metals which induce metallothioneins would also induce reactivation, which was not observed. The results therefore raise the possibility that in addition to inducing the metallothionein genes, cadmium inactivates the factors which maintain the virus in latent state.

The results of studies in several laboratories suggest that a TATA box-containing promoter located in the herpes simplex virus type 1 internal long repeat and long terminal repeat elements drives expression of the latency-associated transcripts (LATs). In the present study, we show that expression of a 2-kb LAT-related transcript can occur in the absence of this LAT TATA promoter, indicating the existence of a cryptic promoter. By Northern (RNA) blot analysis, we have examined LAT expression by herpes simplex virus type 1 variant strains KOS/29 and 1704, which contain deletions of the LAT promoter region. Our data indicate that KOS/29, despite lacking the 203-bp fragment which contains the LAT TATA box, can express a 2-kb LAT-related transcript during productive infection in tissue culture and in mouse trigeminal ganglia during acute infection and reactivation. Similarly, strain 1704, which contains a larger deletion in this promoter region, also expresses a 2-kb LAT-related transcript during tissue culture infection and reactivation of latently infected trigeminal ganglia. However, LATs are not expressed with either virus during latency. Northern blot analysis with a single-stranded, oligonucleotide probe demonstrates that the 2-kb LAT and LAT-related transcript are colinear and share a large area of sequence similarity. These findings suggest the existence of a second promoter in the LAT gene which can function during lytic infection and reactivation, at least in the absence of the LAT TATA promoter. We propose that this cryptic promoter is located either in a proximal region approximately 300 bp upstream of the start site of the 2-kb LAT or in a distal region starting over 1,226 bp upstream of this site.

Mechanisms of postinfectious pruritus and peracute death in mice by pseudorabies virus (PRV) were investigated by inoculating the Yamagata-S81 strain of PRV peripherally or intracerebrally into 4-week-old ICR and BALB/c mice. Clinical signs developed most rapidly in mice inoculated intracerebrally, with intermediate speed in mice inoculated intraocularly, and slowly in mice inoculated subcutaneously. Since intraocularly inoculated mice showed an acute reaction and this is considered a peripheral route, the distribution of viral antigens in the nervous system of intraocularly inoculated mice was examined immunohistologically. Viral antigens were mainly detected along the trigeminal and the oculomotor nerves, but neither necrosis nor an inflammatory response was observed in these areas. The infectious virus was efficiently recovered from the viral antigen-positive tissues. In the pruritic skin lesions, viral antigens were not observed. These findings indicate that the main route of viral spread in intraocularly inoculated mice is the trigeminal and oculomotor nerves and that the virus in the trigeminal nerve may trigger pruritus.

From marker rescue, sequencing, transcript, and latency analyses of the thymidine kinase-negative herpes simplex virus mutant dlsactk and studies using the thymidine kinase inhibitor Ro 31-5140, we infer that the virus-encoded thymidine kinase is required in murine trigeminal ganglia for acute replication and lytic gene expression, for increasing the numbers of cells expressing latency-associated transcripts, and for reactivation from latent infection.

Herpes simplex virus type 1 (HSV-1) is transcriptionally active during latent infection in human peripheral sensory ganglia. Viral gene expression includes the latency-associated transcripts (LATs) which have been linked to the ability of the virus to resume replication and reactivate. However, the molecular basis of reactivation and the mechanisms of action of these transcripts are unknown. In order to study these parameters, an in vivo reactivation model is needed. We investigated use of the mouse as the experimental animal, modifying the route of infection, the viral strain and the reactivation protocol. Following administration of human immunoglobulin 1 day prior to corneal infection, no infectious virus was detected in trigeminal ganglia (TG). However, latency was established in all infected animals as indicated by explant reactivation of TG, and in vivo reactivation was achieved in 30 to 40% of them. DNA quantification revealed that TG of immunized mice contained more HSV-1 DNA than did those of non-immunized mice. By in situ hybridization twice as many neuronal cells in TG of immunized mice were positive for LATs, compared with infected but non-immunized, mice. These findings suggest that suppression of primary infection facilitates reactivation by increasing HSV-1 copy number in latently infected nervous tissue.

In order to examine if mutations within the LAT promoter region of HSV-1 are sufficient to change the reactivation phenotype, a mutant (KOS/29) containing a deletion of the LAT TATA element was used to establish latent infections in mouse ganglia by corneal inoculation. During the acute phase of infection, KOS/29 replicated as efficiently as its wild-type parent. As previously noted, latent KOS/29 infections were totally devoid of LAT gene transcripts (Dobson A. T., Sederati F., Devi-Rao G., Flanagan J., Farrell M. J., Stevens J. G., Wagner E. K., and Feldman L. T., J. Virol. 63, 3844-3851 (1989))). However, unlike other null mutants, KOS/29 reactivated from explanted ganglia with a kinetics similar to that of the LAT competent parent. These data show that the deletion created in KOS/29, removing the LAT TATA promoter element and small upstream and downstream flanking sequences, is not enough to alter the reactivation phenotype and that efficient reactivation can occur in the absence of any detectable LAT expression during latency.

Thymidine kinase (TK)-negative (TK-) mutant strains of herpes simplex virus type 1 (HSV-1) show reduced expression of alpha and beta viral genes during acute infection of trigeminal ganglion neurons following corneal infection (M. Kosz-Vnenchak, D. M. Coen, and D. M. Knipe, J. Virol. 64:5396-5402, 1990). It was surprising that a defect in a beta gene product would lead to decreased alpha and beta gene expression, given the regulatory pathways demonstrated for HSV infection of cultured cells. In this study, we have examined viral gene expression during reactivation from latent infection in explanted trigeminal ganglion tissue. In explant reactivation studies with wild-type virus, we observed viral productive gene expression over the first 48 h of explant incubation occurring in a temporal order (alpha, beta, gamma) similar to that in cultured cells. This occurred predominantly in latency-associated transcript-positive neurons but was limited to a fraction of these cells. In contrast, TK- mutant viruses showed greatly reduced alpha and beta gene expression upon explant of latently infected trigeminal ganglion tissue. An inhibitor of viral TK or an inhibitor of viral DNA polymerase greatly decreased viral lytic gene expression in trigeminal ganglion tissue latently infected with wild-type virus and explanted in culture. These results indicate that the regulatory mechanisms governing HSV gene expression are different in trigeminal ganglion neurons and cultured cells. We present a new model for viral gene expression in trigeminal ganglion neurons with implications for the nature of the decision process between latent infection and productive infection by HSV.

Following primary infection with varicella-zoster virus (VZV), the virus establishes a latent infection in humans. The molecular pathogenesis of VZV latency is not well understood, mainly due to the lack of an adequate animal model. We report here that we have developed a mouse model for VZV infection that involves corneal inoculation of mice. Although infected animals showed no signs of disease, most of the animals could not eliminate the virus early after infection. By PCR, we demonstrated that at 33 days post-infection (p.i.), viral DNA was still present in more than 60% of the animals (14/21). VZV DNA was most frequently detected in the trigeminal ganglia (7/14) followed by the brain stem (10/21), kidneys (4/21), spleen (3/20), liver (2/21) and brain (1/21). By in situ hybridization, a few cells positive for VZV mRNA were detected in the trigeminal ganglia, brain stem, cerebellum and spleen of a small number of the infected animals as late as 33 days p.i. No viral proteins were detected at the site of inoculation or in any other tissue by immunostaining. Our results suggest that VZV spreads in mice by both viraemia and axonal transport and establishes a non-productive (latent) infection.

During latent infection of neurons with herpes simplex virus type 1 (HSV-1), several RNA transcripts of varying abundance arise from a single locus within the virus repeats. The functions of latency-associated transcripts (LATs) are unknown and the relationship between the various RNA species requires further clarification. Reported here is a novel approach to the study of HSV transcripts during latency, based on the increasing realization that cellular and viral RNAs are synthesized and processed by macromolecular complexes that occupy discrete compartments within the nucleoplasm of a cell. High resolution non-isotopic in situ hybridization was used to study the intranuclear topology of HSV-1 LATs in primary sensory neurons of latently infected mice and humans. Low abundance (minor) LATs were localized to sharply defined intranuclear foci of 1 to 3 microns in diameter. On average, there were 2.6 to 2.8 foci/LAT+ neuronal profile (5 microns), representing 13 to 14 foci/cell. In contrast to the focal deployment of minor LATs, the more abundant latency-associated RNAs were distributed diffusely throughout the nucleoplasms of latency infected neurons, with prominent sparing of nucleolar regions. These data establish a foundation for studying the synthesis, processing and transport of LATs in vivo. It should now be possible to investigate the nature of those cellular products which associate with HSV-1 encoded LATs in vivo and thereby determine whether minor LATs are associated with previously characterized macromolecular complexes, such as those responsible for processing of pre-messenger RNA.

Latency represents a challenge for any herpesvirus vaccine. Vaccines could differ in their ability to minimize latency of pseudorabies virus (PRV). To study this possibility, a quantitative and differential PRV-specific polymerase chain reaction (PCR) was developed by the authors. Efficiency of amplification in different tubes is measured by co-amplification with the viral targets (either gI or gp50 genes) of the porcine gene nuclear factor 1. The criteria used to select this approach to a quantitative PCR, as well as for the selection of the standard, are discussed. The amplified products are measured by Southern blot or, alternatively, high performance liquid chromatography. Sensitivity and reproducibility of the technique are evaluated. The ability of this technique to discriminate between the level of latency established by two different PRV strains in trigeminal ganglia is also evaluated. Using this technique, the authors are currently studying whether different vaccines could possess differing levels of ability to minimize, by interference, establishment of wild-type latency.

Herpes simplex virus type 1 (HSV-1) is the cause of a serious and often fatal encephalitis. Patients who survive herpes simplex encephalitis (HSE) experience behavioral abnormalities including profound cognitive dysfunctions. We have developed a rat model of acute HSE to investigate the cognitive impairments caused by HSV-1 central nervous system (CNS) infection. Following intranasal inoculation of Lewis rats with a neurovirulent strain of HSV-1, animals shed virus in both ocular and nasal secretions and developed clinical signs of infection, including partial complex motor seizures that eventually generalized. Homogenization assays demonstrated infectious virus in the trigeminal ganglia, olfactory bulbs, and the piriform and entorhinal cortices. Histopathological assessment revealed inflammatory and hemorrhagic lesions in the trigeminal ganglia, olfactory bulbs, amygdala, hippocampus, the piriform and entorhinal cortices, and the spinal trigeminal nuclei. Viral antigens and nucleic acids were also detected within these structures by immunofluorescence microscopy and in situ hybridization, respectively. Viral-induced astrocytic hypertrophy in the CNS was demonstrated by glial fibrillary acidic protein immunoreactivity. Together, these results indicate that HSV-1 has the ability to invade, replicate, and induce site-specific CNS damage in the Lewis rat.

Latency in nervous tissue caused by herpes simplex virus 1 (HSV-1) and by varicella-zoster virus (VZV) is an intriguing feature of herpes-virus' neurotropism. HSV-1 and VZV latency are the causes of ophthalmic zoster and recurrent HSV infections in the distributions of the trigeminal branches. HSV-1 neuronal latency may play a role in the etiopathogenesis of HSV encephalitis. We attempted to determine the prevalence and age distribution of VZV and HSV latency. We applied nested polymerase chain reaction (PCR) assays to detect HSV-1 and VZV genome in trigeminal ganglia and olfactory bulbs which were obtained from 109 human corpses at forensic postmortems. HSV-1 latency was found in 72.5% of trigeminal ganglia and in 15.5% of olfactory bulbs. VZV latency was 63.3% in trigeminal ganglia and 1% in olfactory bulbs. Simultaneous latency of VZV and HSV genome occurs in 48.8% of trigeminal ganglia. The age-group specific prevalence of HSV neuronal latency increases from 18.2% in 0-20 years to reach finally 100% in persons older than 60 years. Age specific prevalences of VZV peaked for a first time with 82% between 21-30 years, fell to 50% for 40-50 years, and rose to 89% for 71-80 years. If the latent trigeminal ganglion HSV-1 genome were the source of endogenously acquired encephalitis, the peak incidence of HSV encephalitis in older subjects correlates with our findings. Increased VZV latency prevalence in nervous tissue of younger people without subsequent disease indicates sufficient immune surveillance.

Competitive polymerase chain reaction was used to quantitate latent varicella-zoster virus (VZV) DNA in human trigeminal ganglia. Ganglionic DNA from five subjects was amplified with oligonucleotide primers specific for VZV gene 28. Two of the samples were also analyzed with primers specific for VZV gene 62. Our results indicated that there are 6 to 31 copies of the VZV genome in every 100,000 ganglionic cells.

Our laboratory previously reported in situ hybridization analyses showing varicella-zoster virus (VZV)-specific RNA transcripts in latently infected human trigeminal ganglia. These transcripts were found exclusively in non-neuronal cells and emanated from at least three separate regions of the VZV genome, those encompassing restriction fragments BamHI-EY, EcoRI-B, and BamHI-J. Extending these in situ studies, we now report that VZV RNAs colinear with open reading frames (ORFs) 29 and 62 are demonstrable in non-neuronal cells; however, neither VZV RNA colinear with ORF 28 nor antisense RNAs of ORFs 29 and 62 are detected. Northern hybridization analyses of poly(A)+ RNA from human trigeminal ganglion pools also reveal VZV-specific transcripts corresponding to ORFs 29 and 62, but not those of ORFs 28 or 61. The ORF 29- and 62-specific transcripts are similar to those expressed in productive infection, with regard to size and polyadenylation. The cumulative findings suggest that VZV latency is characterized by selected expression of multiple genes, which appear to include some but not all putative immediate-early and early genes.

Purpose: The purpose of this study was to determine whether herpes simplex virus (HSV) transcripts are present in the corneas of patients with chronic herpetic stromal keratitis., Methods: Corneal buttons from patients with a history of stromal keratitis, but no ongoing active disease, together with positive and negative control tissues, were analyzed by in situ hybridization using single-stranded RNA probes for all three classes of viral lytic cycle transcripts as well as for the latency-associated transcripts (LATs). Tissues also were screened for presence of HSV genomic DNA using the polymerase chain reaction (PCR)., Results: HSV DNA was detected in 7 of 13 quiescent corneas by PCR, but no viral transcripts were detected in any of these corneas by in situ hybridization., Conclusions: At the level of detection afforded by in situ hybridization, HSV persistent in scarred human corneas after stromal keratitis appears to be transcriptionally dormant. This contrasts with the situation in neurons of latently infected sensory ganglia, in which LATs are present at high levels.

A polymerase chain reaction (PCR) assay was developed to detect the thymidine kinase gene of feline herpesvirus 1 (FHV-1) and to study the active and latent carrier state in a group of naturally FHV-1 infected specific pathogen free (SPF) cats. The detection limit of PCR products on ethidium bromide stained gels was 390 fg or about 3 x 10(3) copies of the FHV-1 genome. The PCR was 25% more sensitive than conventional cell culture based virus isolation techniques in detecting FHV-1 in oral/ocular swabs and 100 times more sensitive in detecting virus in cell culture supernatants. Sites of FHV-1 latency in FHV-1 carriers as determined by PCR were mainly tissues of the head, especially the trigeminal ganglia, optic nerves, olfactory bulbs and corneas. Oral fauces, salivary glands, lacrimal glands, cerebellum and conjunctiva were less consistently positive. The cerebral cortex, thymus, trachea, lung, liver, spleen, kidney, and peripheral blood mononuclear cells were consistently negative for FHV-1 genome. The distribution of FHV-1 DNA in the tissues of the head was similar whether or not corticosteroid-induced virus shedding was occurring at the time the tissues were collected. Infectious virus was never recovered from tissue homogenates regardless of the PCR status of the tissues.

A polymerase chain reaction (PCR) was designed which is specific to Macaca fascicularis (cynomolgus monkey) isolates of B virus. The PCR primers produced the expected 188 basepair product from the Cyno 2 strain and seven other cynomolgus monkey isolates of B virus. Oligomer hybridization with a 31-mer oligonucleotide was used to confirm the origin of this product. The PCR failed to amplify DNA of Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, and other alphaherpesviruses (herpes simplex virus types 1 and 2, four SA 8 isolates and three rhesus isolates of B virus). PCR testing of swabs obtained from four orally-infected cynomolgus monkeys confirmed the presence of B virus DNA in samples previously shown to be positive by culture. In addition, PCR detected B virus in several swabs from infected monkeys that were culture negative. Total DNA extracts from the trigeminal and sacral ganglia of these animals were tested by nested PCR and B virus DNA was detected in the trigeminal ganglia of 3 of the 4 orally-infected cynomolgus monkeys. Nested PCR did not detect B virus DNA in total DNA extracts obtained from the brains of the four monkeys.

Herpes simplex virus can cause acute retinal necrosis, a blinding retinal disease in man. A unilateral intracameral inoculation of herpes simplex virus type 1 (HSV-1) in mice induces retinal necrosis primarily in the contralateral eye and provides an experimental model for the disease. Previous studies suggested that a major envelope glycoprotein of HSV-1, glycoprotein C (gC), is required for retinal necrosis. We studied HSV-1 strain TN-1, a gC-deficient clinical isolated from a lesion of herpetic keratitis, for its pathogenicity in mice with an intracameral inoculation of the virus and found that TN-1 could induce severe necrotizing retinitis in both inoculated and uninoculated eyes of BALB/c mice. Inoculation with a lower dose of TN-1 resulted in a unilateral necrotizing retinitis in the uninoculated eyes. Tissue virus titration of infected mice killed at various times after inoculation detected an infectious virus in various organs including the eyeballs, trigeminal ganglia, brain and adrenal glands. Anterior chamber-associated immune deviation (ACAID) was observed in TN-1-inoculated mice as well as in mice inoculated with gC-positive laboratory strain KOS 7 days postinoculation. Our findings suggested that gC of HSV-1 is not necessary for either the induction of retinal necrosis, neural spread of the virus, or ACAID.

Inoculation of the scarified cornea with herpes simplex virus (type 1) leads to herpetic infection of trigeminal ganglion cells. A recent study of the susceptibility of ganglion cells revealed that there may be at least four populations of trigeminal ganglion cells that are infectable by herpes. Two classes were identified by their neuropeptide content: Substance P or calcitonin gene-related peptide. One class was identified by its affinity for a monoclonal antibody, SSEA-3. The fourth class was recognized by its common affinity for both the monoclonal antibody LD2 and for the lectin Bandeiraea simplicifolia isolectin. However, there has been no direct evidence of which types are infected directly as a result of retrograde transport from the corneal site and which may be infected by cell-to-cell spread. The aim of this study was to determine which classes of neurons, which are known to become infected with HSV after ocular inoculation, supply corneal innervation. We have identified four classes of trigeminal ganglion neurons that supply axons to the central cornea of the mouse, on the basis of their ability to transport Fluoro-Gold retrograde from axons in the central corneal epithelium and stroma. About 40% of the neurons that innervate the cornea contain Substance P or calcitonin gene-related peptide; about 60% of the neurons that innervate the cornea react with the monoclonal antibody SSEA-3. About 36% of all neurons in the whole ophthalmic division react with the LD2 or Bandeiraea simplicifolia isolectin, and Fluoro-Gold labels only 2% of them.(ABSTRACT TRUNCATED AT 250 WORDS)

Ocular infection of immunocompetent (BALB/c) mice with wild-type herpes simplex virus type 1 (HSV-1) 17+ may lead to acute fatal encephalitis; however, in surviving animals, a latent (nonproductive) infection of the nervous system is established. In contrast, 17+ infection invariably kills mice with severe combined immunodeficiency (SCID mice) within 2 weeks. Ocular infection of immunocompetent mice with a mutant HSV-1 strain, in1814, which does not produce a functional alpha-transinducing protein, results in no detectable viral replication in the nervous system during the time corresponding to the acute phase of infection, no mortality, and the establishment of latency. In SCID mice, however, the in1814 virus establishes a unique, slowly progressing infection. In studying the courses of in1814 infection in SCID and BALB/c mice, we found that although intact B- and/or T-lymphocytic functions were required for the control of viral replication in the nervous system, some of the infected neurons of SCID mice seemed to be able to restrict in1814 replication and harbor the virus in a latent state.