Latent Herpes Simplex Virus Type 1 Infection Research Articles

A dual fluorescent herpes simplex virus type 1 recombinant reveals divergent outcomes of neuronal infection.

Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.

Varicella-zoster virus proteome-wide T-cell screening demonstrates low prevalence of virus-specific CD8 T-cells in latently infected human trigeminal ganglia

BackgroundTrigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized.MethodsPaired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts.ResultsVZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses.ConclusionsThe low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.

T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers

BackgroundTrigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ.MethodsPeripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis.ResultsCollagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical TRM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher TRM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1+), which occasionally showed features of proliferation (KI-67+) and activation (CD137+), but without signs of degranulation (CD107a+) nor damage (TUNEL+) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a+) were not detected.ConclusionsThe human TG represents an immunocompetent environment in which both CD4 and CD8 TRM are established and retained. Based on our study insights, we advocate for TRM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

A Cell Culture Model of Latent and Lytic Herpes Simplex Virus Type 1 Infection in Spiral Ganglion

Purpose of the Study: Reactivation of latent herpes simplex virus type 1 (HSV-1) in spiral ganglion neurons (SGNs) is supposed to be one of the causes of idiopathic sudden sensorineural hearing loss. This study aims to establish a cell culture model of latent and lytic HSV-1 infection in spiral ganglia. Procedures: In the presence of acyclovir, primary cultures of SGNs were latently infected with HSV-1 expressing green fluorescent protein. Four days later, these cells were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1. TCID₅₀ was used to measure the titers of virus in cultures on Vero cells. RNA from cultures was detected for the presence of transcripts of ICP27 and latency-associated transcript (LAT) using reverse transcription polymerase chain reaction. Results: There is no detectable infectious HSV-1 in latently infected cultures, whereas they could be observed in both lytically infected and latently infected/TSA-treated cultures. LAT was the only detectable transcript during latent infection, whereas lytic ICP27 transcript was detected in lytically infected and latently infected/TSA-treated cultures. Conclusion: Cultured SGNs can be both latently and lytically infected with HSV-1. Furthermore, latently infected SGNs can be reactivated using TSA, yielding infectious virus.

The Effects of Dexamethasone and Acyclovir on a Cell Culture Model of Delayed Facial Palsy

Pretreatment with antiherpetic medications and steroids decreases likelihood of development of delayed facial paralysis (DFP) after otologic surgery. Heat-induced reactivation of herpes simplex virus type 1 (HSV1) in geniculate ganglion neurons (GGNs) is thought to cause of DFP after otologic surgery. Antiherpetic medications and dexamethasone are used to treat DFP. Pretreatment with these medications has been proposed to prevent development of DFP. Rat GGN cultures were latently infected with HSV1 expressing a lytic protein-GFP chimera. Cultures were divided into pretreatment groups receiving acyclovir (ACV), acyclovir-plus-dexamethasone (ACV + DEX), dexamethasone alone (DEX), or untreated media (control). After pretreatment, all cultures were heated 43°C for 2 hours. Cultures were monitored daily for reactivation with fluorescent microscopy. Viral titers were determined from culture media. Heating cultures to 43°C for 2 hours leads to HSV1 reactivation and production of infectious virus particles (59 ± 6.8%); heating cultures to 41°C showed a more variable frequency of reactivation (60 ± 40%), compared with baseline rates of 14.4 ± 5%. Cultures pretreated with ACV showed lower reactivation rates (ACV = 3.7%, ACV + DEX = 1.04%) compared with 44% for DEX alone. Viral titers were lowest for cultures treated with ACV or ACV + DEX. GGN cultures harboring latent HSV1 infection reactivate when exposed to increased temperatures that can occur during otologic surgery. Pretreatment with ACV before heat provides prophylaxis against heat-induced HSV reactivation, whereas DEX alone is associated with higher viral reactivation rates. This study provides evidence supporting the use of prophylactic antivirals for otologic surgeries associated with high rates of DFP.

Chapter 38 - Bell's palsy and vestibular neuronitis

Abstract Bell's palsy and vestibular neuritis are uncommon conditions but can be the cause of significant acute neurologic disability. Bell's palsy is the most common cause of facial weakness, whereas vestibular neuritis ranks second or third as the most frequent cause of sudden onset of dizziness and vertigo. The two conditions can occur either singly or in combination to cause facial weakness and debilitating dizziness. The disorders have been shown to be caused by a number of conditions, with the most frequent being idiopathic. The etiology of the disorders is also frequently presumed to be inflammatory and potentially related to reactivation of latent viral infection, most notably infection with herpes simplex virus type 1 (HSV-1). Bell's palsy has an incidence of 15–30 per 100 000, whereas the incidence of vestibular neuritis is less common, at 3.5 per 100 000. Men and women are equally affected, with a peak age of onset at 40 years of age. Individuals may spontaneously recover if left untreated; however, many do not fully recover from either disorder. Anatomically, connections between the facial and vestibular nerve bundles have been demonstrated, which may explain the occasional concurrent involvement of the nerves. Many histopathologic studies done for both conditions have identified the presence of latent HSV-1 infection in the geniculate ganglia as well as the vestibular ganglia, suggesting that the infection may contribute to causality of both entities, although evidence still remains circumstantial. The treatment is similar for the two conditions. Recent randomized double-blinded control studies done in both diseases using corticosteroids, antiviral agents, and the combination of both demonstrated that only the use of steroids as single therapy was effective in improving outcomes for both Bell's palsy and vestibular neuritis. The use of antiviral agents alone and in combination with steroids did not provide additional benefit.

Chronic Progressive Deficits in Neuron Size, Density and Number in the Trigeminal Ganglia of Mice Latently Infected with Herpes Simplex Virus

Numerous epidemiological studies have proposed a link between herpes simplex virus (HSV) infection and several common chronic neuropsychiatric and neurodegenerative diseases. Experimental HSV infection of mice can lead to chronic behavioral and neurological deficits and chronic pain. While neuron injury and loss are well-documented consequences of the acute phase of infection, the pathologic consequences of latent HSV infection are poorly understood. To determine whether latent HSV infection can cause neuronal injury in mice, trigeminal ganglia (TG) derived from adult BALB/c mice 1, 12 and 31 weeks after corneal HSV type 1 (HSV-1) inoculation were analyzed for evidence of productive or latent HSV-1 infection, inflammation and changes in neuron size, density and number. We found that latent HSV-1 infection between 12 and 31 weeks after corneal virus inoculation was associated with inflammation and progressive deficits in mean neuron diameter, neuronal nucleus diameter, neuron density and neuron number in the TG relative to mock-infected controls. The extent of neuronal injury during latent infection correlated with the extent of inflammation. These studies demonstrate that latent HSV infection is associated with progressive neuronal pathology and may lead to a better understanding of the role of HSV infections in chronic neurological diseases.

Analysis of herpes simplex virus type 1 restriction fragment length polymorphism variants associated with herpes gladiatorum and Kaposi's varicelliform eruption in sumo wrestlers

The geographical distribution of herpes simplex virus type 1 (HSV-1) restriction fragment length polymorphism (RFLP) variants BgK(L) and BgO(L) and the high relative frequency (RF) of BgK(L) in orolabial lesions has led to a dispersion-replacement hypothesis for these variants. The pathogenic properties of HSV-1 variants in mice and professional sumo wrestlers were examined here. The wrestlers herpes gladiatorum (HG) was caused by primary and non-primary HSV-1 infections and recurred in many wrestlers. HSV-1 neutralizing antibody titres in sera from wrestlers who did not develop HG were relatively high. HG was caused by distinct HSV-1 variants and strains from wrestlers living in the same sumo stable. The BgK(L) RF was significantly higher in HG cases, particularly in those with Kaposi's varicelliform eruption. These data indicated that reactivation and transmission of latent HSV-1 infections, especially BgK(L), occurred frequently among wrestlers and was caused by severe skin damage. These results support the BgK(L) dispersion hypothesis.

Modulation of HLA-G and HLA-E Expression in Human Neuronal Cells After Rabies Virus or Herpes Virus Simplex Type 1 Infections

Human Leukocyte Antigen (HLA)-G and E are nonclassical human MHC class I molecules. They may promote tolerance leading to virus and tumor immune escape. We recently described that the herpes simplex virus type 1 (HSV-1), a neurotropic virus inducing chronic infection and neuron latency, and rabies virus (RABV), a neuronotropic virus triggering acute neuron infection, up-regulate HLA-G expression in human neurons (NT2-N). Surface expression was only detected after RABV infection. We investigated here whether RABV and HSV-1 up-regulate HLA-E expression in human neuronal precursors (Ntera-2D/1). We found that RABV, not HSV-1, up-regulates HLA-E expression, nevertheless HLA-E could not be detected on the surface of RABV-infected Ntera-2D/1. Altogether these data suggest that HLA-G and not HLA-E could contribute to the immune escape of RABV. In contrast, there was no evidence that these molecules are used by latent HSV-1 infection. Thus, neurotropic viruses that escape the host immune response totally (RABV) or partially (HSV-1) regulate HLA-G expression on human neuronal cells differentially.

Herpes simplex virus type-1 latency inhibits dendritic growth in sympathetic neurons

Herpes simplex virus type-1 (HSV-1) initially infects mucoepithelial tissues of the orofacial region, the eye and to a lesser extent the genitalia. Subsequently, the virus is retrogradely transported through the axons of the sensory and sympathetic neurons to their nuclei, where the virus establishes a life-long latent infection. During this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). LAT has been shown to affect apoptosis, but little else is known regarding its effects on neurons. To understand how HSV-1 latency might affect dendrites in sympathetic neurons, we transfected primary cultures of sympathetic neurons obtained from rat embryos, with LAT expressing plasmids. LAT inhibited initial dendritic growth and induced dendritic retraction in sympathetic neurons. Latent HSV-1 infection of cultured sympathetic neurons inhibited dendritic growth indicating that this is likely also a consequence of natural infection.

Methods for detecting the HSV-1 LAT anti-apoptosis activity in virus infected tissue culture cells

Plasmids expressing the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) reduce apoptosis in transient transfection assays in tissue culture. LAT also reduces apoptosis in the context of the virus in trigeminal ganglia of rabbits and mice at approximately 6–7 days post-infection during the switch from acute to latent HSV-1 infection, a time at which LAT is the only abundantly transcribed viral gene. Analysis of LAT’s anti-apoptosis function is complicated in tissue culture by the expression of at least five additional viral gene products that can block apoptosis, and by the fact that apoptosis usually occurs in only a fraction of the cells. Here, we present two approaches for detecting LAT’s anti-apoptosis activity in the context of the whole virus in tissue culture. Using a combination of serum starvation to both partially synchronize the cells and induce apoptosis, and Hoechst staining to detect chromatin condensation, we found that there was a small window of time post-infection during which Schwann cells infected with the LAT(−) mutant dLAT2903 reproducibly had more apoptotic nuclei than identically treated cells infected with the LAT(+) parental virus HSV-1 strain McKrae. Using serum starvation and/or UV treatment and a method to isolate fragmented DNA away from large chromosomal DNA, we found a similar window of time post-infection during which Neuro2A cells infected with dLAT2903 had increased DNA fragmentation (as judged by a DNA laddering assay) compared to identically treated cells infected with wild type McKrae or the LAT(+) marker rescued dLAT2903R virus. These assays should permit the use of culture assays, rather than labor intensive animal models, to examine LAT’s anti-apoptosis activity in the context of the virus in a large number of existing LAT mutant viruses.

Methods for detecting the HSV-1 LAT anti-apoptosis activity in virus infected tissue culture cells

Plasmids expressing the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) reduce apoptosis in transient transfection assays in tissue culture. LAT also reduces apoptosis in the context of the virus in trigeminal ganglia of rabbits and mice at approximately 6–7 days post-infection during the switch from acute to latent HSV-1 infection, a time at which LAT is the only abundantly transcribed viral gene. Analysis of LAT’s anti-apoptosis function is complicated in tissue culture by the expression of at least five additional viral gene products that can block apoptosis, and by the fact that apoptosis usually occurs in only a fraction of the cells. Here, we present two approaches for detecting LAT’s anti-apoptosis activity in the context of the whole virus in tissue culture. Using a combination of serum starvation to both partially synchronize the cells and induce apoptosis, and Hoechst staining to detect chromatin condensation, we found that there was a small window of time post-infection during which Schwann cells infected with the LAT(−) mutant dLAT2903 reproducibly had more apoptotic nuclei than identically treated cells infected with the LAT(+) parental virus HSV-1 strain McKrae. Using serum starvation and/or UV treatment and a method to isolate fragmented DNA away from large chromosomal DNA, we found a similar window of time post-infection during which Neuro2A cells infected with dLAT2903 had increased DNA fragmentation (as judged by a DNA laddering assay) compared to identically treated cells infected with wild type McKrae or the LAT(+) marker rescued dLAT2903R virus. These assays should permit the use of culture assays, rather than labor intensive animal models, to examine LAT’s anti-apoptosis activity in the context of the virus in a large number of existing LAT mutant viruses.

Herpes simplex virus 1 infected neuronal and skin cells differ in their susceptibility to complement attack.

Herpes simplex virus type 1 (HSV-1) infection in neurons is lifelong and generally asymptomatic. Reactivation of this latent infection results in skin blistering whereas the respective peripheral neurons are rarely affected. Why the neuronal cells are spared while the skin cells are sacrificed is not well understood. In the present study our aim was to study whether neuronal and skin cells differ in their ability to control complement attack during HSV-1 infection. Human embryonal skin (HES) cells and neuronal Paju cells were infected by HSV-1 in vitro. Both types of infected cells activated complement but were initially resistant to membrane attack complex (MAC) deposition. During the first hours of infection the expression of the endogenous complement regulators decay accelerating factor (DAF) and CD59 increased on both HES and Paju cells. By 12 hr the infected HES cells had lost their ability to control complement attack. The expression of DAF and CD59 decreased and the cells became targets for MAC attack. In contrast, complement regulator expression on the Paju cells did not decrease below the initial level and complement C5b-9 deposition was found only on 10% of the Paju cells at 12 hr. The results suggest that HSV-infected neuronal cells are better than skin cells in protecting themselves against complement attack. This may contribute to the persistence of a latent HSV-1 infection in neuronal cells for prolonged periods.

The Immediate-Early Protein, ICP0, Is Essential for the Resistance of Herpes Simplex Virus to Interferon-α/β

Herpes simplex virus type 1 (HSV-1) is resistant to the antiviral effects of interferon (IFN)-α, -β, or -γ. The fact that ICP0− mutants replicate like wild-type virus in IFN-α/β receptor knockout mice (Leib et al., 1999, J. Exp. Med. 189, 663) suggested that ICP0 may serve a direct role in the resistance of HSV-1 to IFN. To test this hypothesis, the effects of IFN-α, -β, and -γ were compared against wild-type HSV-1 and an ICP0− mutant virus, 7134. In Vero cells, 7134 was more sensitive to inhibition by low doses of type I IFN (-α/β) or type II IFN (-γ) than vesicular stomatitis virus, a well-studied IFN-sensitive virus. At a concentration of 100 U/ml, IFN-α, -β, or -γ reduced the efficiency of 7134 plaque formation by 120-, 560-, and 45-fold, respectively. In contrast, none of the IFNs reduced wild-type HSV-1 plaque formation by more than 3-fold. Even when Vero cells were infected with 10 pfu per cell, IFN-α and -β inhibited 7134 replication by over 100-fold, but inhibition by IFN-γ decreased to less than 10-fold. While IFN-β efficiently inhibited 7134 replication in primary mouse kidney and SK-N-SH cells, IFN-γ did not inhibit 7134 to a comparable extent in these cells. ICP0 provided in trans from an adenovirus vector allowed 7134 to replicate efficiently in Vero cells in the presence of IFN-α, -β, or -γ. While IFN-β or -γ efficiently repressed the ICP0 promoter-lacZ reporter gene in 7134 (i.e., ∼60-fold reduction in β-galactosidase activity), ICP0 provided in trans almost completely reversed IFN-mediated repression of the lacZ gene in 7134. The results suggest that the rate of ICP0 expression in infected cells in vivo may be critical in determining whether host IFNs repress the HSV-1 genome. This concept is discussed in light of its potential relevance to the establishment of latent HSV-1 infections.

Herpes simplex type 1 infects and establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice.

The majority of the human population has been infected with herpes simplex virus type 1 (HSV-1). During a typical primary episode, HSV-1 spreads from the oral pharynx to the trigeminal ganglia, where a latent HSV-1 infection is established. Cold sores at the mucocutaneous junction of the lip are the typical manifestation of recurrent HSV-1. We investigated whether HSV-1 also infects the brain during the primary infection. We used HSV-1 infected BALB/c mice and inbred cotton rats as models. While both species were susceptible to HSV-1 infection, the time course of lesion formation and healing in the cotton rat more closely reflected that seen in humans. In both species, HSV-1 replicated in the brainstem and cerebellum, as well as the trigeminal ganglia during a primary infection of the lip. The brain infection was produced by a low inoculation dose, and did not cause observable neurologic signs or mortality. Using PCR and RT-PCR techniques, we demonstrated HSV-1 thymidine kinase in the absence of infectivity in the brains of both species 30-40 days after primary infection.

Mouse model of Bell's palsy induced by reactivation of herpes simplex virus type 1.

In order to investigate the mechanism of Bell's palsy, we developed an animal model of facial nerve paralysis induced by the reactivation of herpes simplex virus type 1 (HSV-1). Eight weeks after recovery from facial nerve paralysis caused by inoculation with HSV-1, the mice were treated with auricular skin scratch at the site of the previous inoculation, or with intraperitoneal injection of anti-CD3 monoclonal antibody (mAb), or combination of both procedures. No mice developed facial nerve paralysis when they were treated with either auricular scratch or mAb injection alone. In contrast, 20% of mice developed facial nerve paralysis with the combined treatment. With one exception, no mouse treated with either auricular scratch or mAb injection showed HSV-I DNA in their facial nerve tissue, whereas 4 out of 6 mice receiving both treatments showed HSV-1 DNA on day 10 after treatment. Histopathological findings showed neuronal degeneration in the geniculate ganglion and demyelination of the facial motor nerve in paralyzed mice. These findings suggest that a combination of stimuli, local skin irritation, and general immunosuppression is essential for successfully inducing facial nerve paralysis in mice with latent HSV-1 infection.

Herpes Simplex Virus Type 1 Latency in the Murine Nervous System Is Associated with Oxidative Damage to Neurons

The pathological consequences of herpes simplex virus type 1 (HSV-1) latency in the nervous system are not well understood. To determine whether acute and latent HSV-1 infections of the nervous system are associated with oxidative damage, mice were inoculated with HSV-1 by the corneal route, and the extent of viral infection and oxidative damage in trigeminal ganglia and brain was assessed at 7, 90, and 220 days after inoculation. Abundant HSV-1 protein expression in the nervous system was observed in neurons and non-neuronal cells at 7 days after inoculation, consistent with viral replication and spread through the trigeminal and olfactory systems. Acute HSV-1 infection was associated with focal, neuronal and non-neuronal 4-hydroxy-2-nonenal- and 8-hydroxyguanosine-specific immunoreactivity, indicating oxidative damage. Rare HSV-1 antigen-positive cells were observed at 90 and 220 days after inoculation; however, widespread HSV-1 latency-associated transcript expression was detected, consistent with latent HSV-1 infection in the nervous system. HSV-1 latency was detected predominantly in the trigeminal ganglia, brainstem, olfactory bulbs, and temporal cortex. Latent HSV-1 infection was associated with focal chronic inflammation and consistently detectable evidence of oxidative damage involving primarily neurons. These results indicate that both acute and latent HSV-1 infections in the murine nervous system are associated with oxidative damage.

The herpes simplex virus type 1 immediate-early protein ICP0 is necessary for the efficient establishment of latent infection.

The immediate-early protein ICP0 of herpes simplex virus type 1 (HSV-1) is not essential for viral replication. However, ICP0 is important for efficient viral replication during the productive infection and for reactivation of latent HSV-1 in vivo. The in vitro model of HSV-1 latency in dorsal root ganglia neurons was used to examine the role of ICP0 in the individual steps that could lead to the appearance of a decreased reactivation phenotype of ICP0 mutant viruses. After establishment of latent infections in the neuronal cultures, induction of reactivation by nerve growth factor (NGF) deprivation resulted in the production of infectious virus with delayed kinetics and a burst size that was significantly decreased for the ICP0 mutants compared with wild-type HSV-1. The efficiency of establishment of latency with the ICP0 mutants was similarly decreased at least 10-fold, as measured by three criteria: (i) the percentage of neurons expressing the major latency-associated transcript during the latent infection, (ii) the amount of viral DNA detected in the neuronal cultures, and (iii) the percentage of neurons expressing ICP4 immunoreactivity after the induction of reactivation. The most striking finding was that ICP0 supplied by an adenovirus vector significantly restored the ability of an ICP0 mutant to establish latency and reactivation. These results strongly indicate a critical role for ICP0 in the establishment of the latent HSV-1 infection in the in vitro neuronal model.

Red blood cells as delivery system for recombinant HSV-1 glycoprotein B: immunogenicity and protection in mice

The immunotherapeutic potential of autologous red blood cells (RBC) coupled to the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) was examined with a mouse model of HSV-1 infection. C57BL 6 mice were immunized intraperitoneally with gB1s (0.05 μg per dose) linked to RBC, or mixed with Freund's complete or bound to AlPO 4 adjuvants (0.5 μg per dose). Mice immunized with RBC coupled gB1s were protected against lethal and latent HSV-1 infection, and developed an anti-HSV antibody response, as measured by ELISA and HSV-1 neutralization assays, similar or higher than that elicited by the same antigen in Freund's complete adjuvant, which suggested that autologous RBC coupled to gB1s may provide an effective and safe method of immunization against HSV infection.

Gamma interferon expression during acute and latent nervous system infection by herpes simplex virus type 1

This study was initiated to evaluate a role for gamma interferon (IFN-gamma) in herpes simplex virus type 1 (HSV-1) infection. At the acute stage of infection in mice, HSV-1 replication in trigeminal ganglia and brain stem tissue was modestly but consistently enhanced in mice from which IFN-gamma was by ablated monoclonal antibody treatment and in mice genetically lacking the IFN-gamma receptor (Rgko mice). As determined by reverse transcriptase PCR, IFN-gamma and tumor necrosis factor alpha transcripts were present in trigeminal ganglia during both acute and latent HSV-1 infection. CD4+ and CD8+ T cells were detected initially in trigeminal ganglia at day 5 after HSV-1 inoculation, and these cells persisted for 6 months into latency. The T cells were focused around morphologically normal neurons that showed no signs of active infection, but many of which expressed HSV-1 latency-associated transcripts. Secreted IFN-gamma was present up to 6 months into latency in areas of the T-cell infiltration. By 9 months into latency, both the T-cell infiltrate and IFN-gamma expression had cleared, although there remained a slight increase in macrophage levels in trigeminal ganglia. In HSV-1-infected brain stem tissue, T cells and IFN-gamma expression were present at 1 month but were gone by 6 months after infection. Our hypothesis is that the persistence of T cells and the sustained IFN-gamma expression occur in response to an HSV-1 antigen(s) in the nervous system. This hypothesis is consistent with a new model of HSV-1 latency which suggests that limited HSV-1 antigen expression occurs during latency (M. Kosz-Vnenchak, J. Jacobson, D.M. Coen, and D.M. Knipe, J. Virol. 67:5383-5393, 1993). We speculate that prolonged secretion of IFN-gamma during latency may modulate a reactivated HSV-1 infection.