Replication-Defective HSV-1 Effectively Targets Trigeminal Ganglion and Inhibits Viral Pathopoiesis by Mediating Interferon Gamma Expression in SH-SY5Y Cells

It has been widely believed that recurrence of herpes simplex keratitis (HSK) is due to the reactivation of herpes simplex virus type 1 (HSV-1) from latent sites in trigeminal ganglion (TG). However, there are also not effective vectors which could target TG for therapy. Replication-defective HSV-1 vector (rdHSV-IFNγ) was established by calcium phosphate co-transfection of complementing cells. We firstly infected rdHSV-IFNγ to SH-SY5Y, and detected IFNγ expression by western blot, evaluated 50 % cellular cytotoxicity (CC₅₀) by ELISA. Antiviral activity of rdHSV-IFNγ was examined by immunofluorescence and antiviral concentration of 50 % effectiveness (EC₅₀) assay. The rdHSV-IFNγ vector was immunized to Wistar rats to observe targeting function to TG. Kaplan–Meier survival analysis was utilized to assess security of rdHSV-IFNγ. RT-PCR and immunohistochemistry assay were employed to detect rdHSV-IFNγ localization in TG. Western blot was employed to detect IFNγ expression. rdHSV-IFNγ was successfully established, and performed an effective antiviral activity and higher security in SH-SY5Y. There were no significant differences of survival and corneal infection rate of rdHSV-IFNγ immunized rats among groups (P?>?0.05). RT-PCR and immunohistochemistry indicated that expression of glycoprotein D (gD) in TG could target TG and decreased following with times post immunization. Furthermore, IFNγ was expressed effectively in TG tissues. Our findings indicated that established rdHSV-IFNγ vector effectively transported therapeutic gene into TG tissues. The administration of replication-defective vector carrying therapeutic genes may become a promising tool in inhibition or reoccurrence of HSK in clinical.     

Chemical sympathectomy increases susceptibility to ocular herpes simplex virus type 1 infection

The cornea is one of the most highly innervated tissues in the mammalian host. We hypothesized changes to cornea innervation through chemical sympathectomy would significantly alter the host response to the neurotropic viral pathogen, herpes simplex virus type 1 (HSV-1) following ocular infection. Mice treated with 6-hydroxydopamine hydrobromide displayed reduced tyrosine hydroxylase-positive fibers residing in the cornea. Sympathectomized mice were also found to show a transient rise in virus recovered in infected tissues and succumbed to infection in greater numbers. Whereas there were no differences in infiltrating leukocyte populations including HSV-1-specific cytotoxic T lymphocytes in the infected tissue, an increase in substance P and a decrease in IFN-gamma levels in the trigeminal ganglion but not brain stem of sympathectomized mice were noted. Sympathectomized mice treated with the neurokinin-1 receptor antagonist L703,606 had delayed mortality implicating the involvement of substance P in HSV-1-mediated death.     

Cellular neuroimmunologic responses to ocular herpes simplex virus infection

Infectious herpes simplex virus type 1 (HSV-1) was cultivated from the trigeminal ganglion between days 3 and 21 after ocular infection. T lymphocytes were first seen 9 days after infection and were present in ganglia collected 21 days after corneal infection. Neuron cell bodies expressing cytoplasmic HSV-1 antigens were present in the ganglion by 6 days after infection and could be found up to 21 days after infection although the frequency was low and decreased with time. Neuron cell bodies containing nuclear viral DNA were seen with the same frequency as cells expressing cytoplasmic viral antigens. T lymphocytes were seen surrounding neuron cell bodies some of which contained either cytoplasmic HSV-1 antigens or nuclear HSV-1 DNA.     

Targets of herpes simplex virus type 1 infection in a mouse corneal model

Summary In animal models, spread of herpes simplex virus type 1 (HSV-1) from epithelial replication sites to the peripheral and central nervous system is known from analysis of individually dissected tissues. To examine virus spread in undissociated tissues, corneas of adult mice were inoculated with HSV-1. After 1 to 13 days groups of mice were perfused with formalin, and decalcified blocks of head and neck were embedded in paraffin. At intervals, serial sections were screened for HSV antigen. On days 1 and 2, viral antigen was restricted to cornea and conjunctiva but by days 3 and 4 was also seen in autonomic ganglia and the trigeminal system. On day 6, HSV antigen reached its maximum extent; infected sites included the trigeminal complex (ganglion, root, peripheral ophthalmic and maxillary branches and spinal nucleus and tract), ehtmoid sinus and olfactory buld, visual system, and autonomic ganglia (ciliary, pterygopalatine and superior cervical). Antigen progressively diminished on days 8 and 10, and was not detected on day 13. This method demonstrates a broader range of infected tissues and suggests a more complex pattern of HSV spread than has been previously recognized. Virus appears to reach the intracranial compartment by four different neural routes. When effects of higher and lower corneal inoculation doses were compared, a lower dose resulted in lower peak HSV titers in trigeminal ganglion and brain stem and later virus appearance in these tissues. Thus, dose may influence the kinetics of HSV spread from the peripheral inoculation site to the CNS.Supported in part by U.S.U.H.S. grant, R07396. the opinions or assertions contained herein are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or Department of Defense. There is no objection to its presentation and/or publication     

Herpes simplex virus type 1 strain KOS-63 does not cause acute or recurrent ocular disease and does not reactivate ganglionic latency in vivo

The virological, clinical, and histopathological manifestations of acute and experimentally reactivated infections of eyes and trigeminal ganglia have been studied following intranasal infection of rabbits with herpes simplex virus type 1 (strain KOS-63). All animals shed virus in nasal secretions, but only three shed virus in tear film during the first 12 days of infection. No animal developed clinical or histological evidence of corneal or retinal ocular disease at any time after infection. KOS-63 established trigeminal ganglionic latency; viral RNA, restricted to neuronal nuclei, was detected by in situ hybridization, and virus was recovered from co-cultivation cultures of nervous tissue, but not from cell-free homogenates. Reactivation of latent trigeminal ganglionic infection was attempted by intravenous administration of cyclophosphamide, followed by dexamethasone 24 h later. Injection of the drugs failed to reactivate KOS-63 latency; no animal shed virus in nasal or ocular secretions, and no animal developed gross or microscopic corneal lesions. In addition, viral antigens were not detected by immunofluorescence microscopy in ganglia from rabbits subjected to the drug protocol, and virus was only recovered from ganglia by in vitro co-cultivation reactivation techniques. The failure of KOS-63 to reactivate was not due to an inherent failure of populate and infect the ganglion, because the virus did not reactivate from ganglia that contained many latently infected cells. These studies demonstrate that, although KOS-63 is neuroinvasive and capable of establishing latency, it is virtually non-virulent for the eye, and cannot be reactivated by a systemic immunosuppressive trigger known to reactivate other HSV-1 strains.     

Exposure to herpes simplex virus,type 1 and reduced cognitive function

Herpes simplex virus, type 1 (HSV-1) causes cold sores, keratitis and rarely, fatal encephalitis. The infection is lifelong, with sensory ganglia serving as reservoirs of latent infection. Recently, exposure to HSV-1 has also been repeatedly associated with reduced cognitive function among healthy individuals without prior encephalitis. Though HSV-1 does not elevate risk for schizophrenia (SZ) per se, exposure is likewise associated with impaired cognitive functions among SZ patients. The range of cognitive changes observed in HSV-1 exposed persons has not been investigated systematically, nor is it known whether interaction between HSV-1 exposure and SZ related factors contributes to the impairment among SZ patients. Persons with or without schizophrenia/schizophreniform disorder (N = 298 total, DSM IV criteria) were assessed for HSV-1 exposure using serum HSV-1 antibody titers. The Penn Computerized Neurocognitive battery was used to assess eight cognitive domains with respect to accuracy and speed. There were no significant case–control differences in HSV-1 exposure. The SZ/schizophreniform disorder cases were significantly impaired in all cognitive domains compared with the controls. HSV-1 exposure was also associated with reduced cognitive function in the entire sample, but the magnitude of the effects and their patterns differed from the SZ related changes. Further, statistically significant interactions between HSV-1 exposure and SZ case status were not detected. HSV-1 exposure does not elevate risk for SZ, but it is associated with reduced function in specific cognitive domains regardless of SZ diagnostic status. An ‘epidiagnostic’ model for the association is proposed to explain the results.     

Chemokines and ocular pathology caused by corneal infection with herpes simplex virus

The role played by chemokines in disease process is an active area of research that continues to uncover new players. In this report we discuss the likely role of selected chemokines in the disease herpetic stromal keratitis (HSK). This lesion occurs as a sequel to herpes simplex virus infection and is currently accepted as an immunopathological process which primarily involves CD4+ T lymphocytes. In this review we discuss the events involved in HSK, the chemokine profile associated with this disease, and speculate on cellular activities and molecular events which characterize HSK as an immunopathological disease.     

A truncation mutation of the neurovirulence ICP22 protein produced by a recombinant HSV-1 generated by bacterial artificial chromosome technology targets infected cell nuclei

The major regulatory protein ICP22 is unique among the immediate early proteins of herpes simplex virus. Viruses deleted for ICP22 replicate well in actively dividing cells, but not in quiescent cells or certain rodent lines. Accordingly, ICP22 represents an understudied herpes simplex virus (HSV) neurovirulence marker which is absolutely essential for viral neurogrowth. We utilized the bacterial artificial chromosome methodology to create a novel ICP22 truncation mutant, termed HSV-1(BACX). The integrity of HSV-1(BACX) was confirmed by detailed polymerase chain reaction analyses and immunoblotting using anti-ICP22 antibody. HSV-1(BACX) showed a reduced replication capacity in rabbit skin cells, consistent with previous studies using ICP22-null viruses. Importantly, HSV-1(BACX) localized to nuclei of infected primate Vero cells in a manner similar to wild-type ICP22. Thus, HSV-1(BACX) will serve as a useful tool to decipher the unusual biological properties and functions of the ICP22 protein.     

Detection of herpes simplex virus mRNA in latently infected trigeminal ganglion neurons by in situ hybridization

Latent infection of the trigeminal ganglion with herpes simplex virus type 2 (HSV-2) was studied in guinea pigs by in situ DNA hybridization. Frozen ganglion sections from animals killed during the period of latent virus infection were studied under nondenaturing conditions. Some sections were treated with deoxyribonuclease (DNase) or ribonuclease (RNase) before incubation with HSV DNA probes. HSV probes consisted of viral DNA nick translated and labeled in vitro with tritiated nucleotides. Bacteriophage lambda DNA, similarly prepared, was used as a control probe. The lambda probe was negative in all situations, including HSV-2-infected monolayer cells in cell culture. HSV-2 probes produced heavy label and, therefore, evidence of hybridization with HSV-2-infected monolayer cells. When HSV-2 probes were incubated with latently infected ganglion sections, hybridization was detected in 71% of guinea pigs and 46% of ganglia. Label was seen only in neurons, and in positive ganglia 0.3 to 5% of neurons were labeled. The amount of label was markedly decreased by pretreatment of ganglion sections with RNase but not DNase, indicating that the DNA probes hybridized to HSV messenger RNA in the latently infected ganglia.     

Acute retinal necrosis caused by herpes simplex virus type 2 in children: reactivation of an undiagnosed latent neonatal herpes infection

Herpes simplex virus type 2 (HSV-2) is known to cause acute retinal necrosis (ARN). The availability of HSV-2-specific polymerase chain reaction tests for diagnostic analysis has greatly increased our ability to discriminate ARN caused by HSV-2 from ARN caused by either herpes simplex virus type 1 or varicella zoster virus (VZV). Of great interest, HSV-2 appears to be the most common cause of viral ARN in children and adolescents. Although a few children with ARN are known to have had neonatally acquired herpes infection, most children lack a history of known herpes disease. Thus, the origin of the HSV-2 infection is a mystery. The hypothesis of this review is that HSV-2 ARN in children and adolescents may be the first sign of a previously undiagnosed and asymptomatic neonatal HSV-2 infection, which has reactivated several years later from latency in a cranial nerve and entered the retina. The review brings together 7 previously published ARN cases, plus one new case is added. Thus, this review also expands the spectrum of complications from neonatal HSV-2 infection.     

Topical application of the cornea post-infection with plasmid DNA encoding interferon-alpha1 but not recombinant interferon-alphaA reduces herpes simplex virus type 1-induced mortality in mice.

A study was undertaken to compare the efficacy of recombinant interferon (rIFN)-alphaA to plasmid DNA encoding IFN-alpha1 against ocular herpes simplex virus type 1 (HSV-1) infection. The topical application of rIFN-alphaA (100-300 units/eye) onto the cornea of mice subsequently infected 24 h later with HSV-1 antagonized viral-induced mortality. The enhancement in cumulative survival in the rIFN-alphaA-treated mice correlated with a reduction of viral titers recovered in the eye and trigeminal ganglion (TG) at 3 and 6 days post-infection. The protective effect was site-specific such that when rIFN-alphaA was administered orally or intranasally, no efficacy against HSV-1 was observed. However, the protective effect was time-dependent. Specifically, when the rIFN-alphaA (100-1000 units/eye) was administered at 24 h post-infection, no protective effect was observed against HSV-1 compared to the vehicle-treated group. In contrast, plasmid DNA (100 microg/eye) containing the IFN-alpha1 transgene showed significant protection when topically applied 24 h post-infection. Although the transgene was found to traffic distal from the site of application (eye), including the trigeminal ganglion and the spleen where CD11b(+) and CD11c(+) cells express the transgene, the migration of the transgene did not correlate with efficacy. Collectively, the results suggest that naked DNA encoding type I IFN applied post-infection provides a greater degree of protection against ocular HSV-1 infection in comparison with recombinant protein effectively antagonizing viral replication and spread.     

Immunohistochemical identification of trigeminal ganglion neurons that innervate the mouse cornea: Relevance to intercellular spread of herpes simplex virus

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. Thus, the population of LD2/Bandeiraea Simplicifolia Isolectin neurons that innervates the cornea represents less than 1% of the total neurons in the ophthalmic division, although it constitutes about 20% of the infected cells in that division at three days after viral inoculation. We conclude that most of the LD2/Bandeiraea simplicifolia isolectin-positive ganglion cells that are infected with herpes at 3 days are infected as a result of HSV spread from nearby cells in the ganglion or proximal trigeminal root. Neurons labeled with antibodies to Substance P, calcitonin gene-related peptide, or α-SSEA-3 that are primarily infected may be sources of the cell-to-cell spread of virus. © 1993 Wiley-Liss, Inc.     

Intact TRL 9 and type I interferon signaling pathways are required to augment HSV-1 induced corneal CXCL9 and CXCL10

Herpes simplex virus type 1 ocular infection elicits a potent inflammatory response including the production of the chemokines, CXCL9 and CXCL10, in mice. Since HSV-1 nucleic acid is recognized by pattern receptors including Toll-like receptor (TLR) 9, we tested the hypothesis that TLR9 is necessary for the early augmentation of CXCL10 following HSV-1 infection. Similar to wild type controls, TLR9 deficient mice constitutively expressed CXCL10 in the cornea. Following infection or stimulation with the deoxycytidylate-phosphate-deoxyguanylate (CpG) motif, CXCL10 levels were significantly elevated in the cornea of wild type but not TLR9 or type I interferon receptor deficient mice. The reduced CXCL10 response in the cornea of TLR deficient mice was correlative with an increase in virus shedding and a reduction in neutrophil infiltration. This is the first report that shows enhanced CXCL10 expression following neurotropic viral replication requires both intact TLR 9 and type I interferon signaling pathways.