HSV-1 Genome Research Articles

Highly Efficient Regulation of Gene Expression by Tetracycline in a Replication-Defective Herpes Simplex Viral Vector

Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx™; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx™-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx™ for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases.

Oncolytic Viruses Derived from the γ34.5-Deleted Herpes Simplex Virus Recombinant R3616 Encode a Truncated UL3 Protein

Replication-competent herpes simplex virus (HSV-1) mutants are used in clinical trials in the experimental treatment of cancer. Mutants G207, HSV1716, NV1020, and Oncovex GM-CSF share in common a defect in one or both copies of the gene encoding the neurovirulence factor, ICP34.5, and are thus neuroattenuated. These viruses are acknowledged to differ from one another (a) in the specific types of mutations intentionally introduced during their derivation and (b) in the inherent genetic differences retained from the different parent strains used in their construction. Unintended mutations are expected to emerge at some low frequency during the selection for and passage of mutant viruses. Here we demonstrate that during the construction of the oncolytic virus R3616, a nonsense mutation arose in an untargeted region of the HSV-1 genome that resulted in a substantial truncation of the viral protein known as UL3. This report is the first published documentation that oncolytic herpesviruses developed and used in clinical trials contain adventitious mutations. The implications of these findings for the characterization and development of vectors proposed for use in clinical trials are discussed.

Functional inaccessibility of quiescent herpes simplex virus genomes

BackgroundNewly delivered herpes simplex virus genomes are subject to repression during the early stages of infection of human fibroblasts. This host defence strategy can limit virus replication and lead to long-term persistence of quiescent viral genomes. The viral immediate-early protein ICP0 acts to negate this negative regulation, thereby facilitating the onset of the viral replication cycle. Although few mechanistic details are available, the host repression machinery has been proposed to assemble the viral genome into a globally inaccessible configuration analogous to heterochromatin, blocking access to most or all trans-acting factors. The strongest evidence for this hypothesis is that ICP0-deficient virus is unable to reactivate quiescent viral genomes, despite its ability to undergo productive infection given a sufficiently high multiplicity of infection. However, recent studies have shown that quiescent infection induces a potent antiviral state, and that ICP0 plays a key role in disarming such host antiviral responses. These findings raise the possibility that cells containing quiescent viral genomes may be refractory to superinfection by ICP0-deficient virus, potentially providing an alternative explanation for the inability of such viruses to trigger reactivation. We therefore asked if ICP0-deficient virus is capable of replicating in cells that contain quiescent viral genomes.ResultsWe found that ICP0-deficient herpes simplex virus is able to infect quiescently infected cells, leading to expression and replication of the superinfecting viral genome. Despite this productive infection, the resident quiescent viral genome was neither expressed nor replicated, unless ICP0 was provided in trans.ConclusionThese data document that quiescent HSV genomes fail to respond to the virally modified host transcriptional apparatus or viral DNA replication machinery provided in trans by productive HSV infection in the absence of ICP0. These results point to global repression as the basis for HSV genome quiescence, and indicate that ICP0 induces reactivation by overcoming this global barrier to the access of trans-acting factors.

Enhanced long-term expression from helper virus-free HSV-1 vectors packaged in the presence of deletions in genes that modulate the function of VP16, U L46 and U L47

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off recombinant gene expression from HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus paradoxically, recombinant gene expression remains short-term in the absence of almost all (∼99%) of the HSV-1 genome. To resolve this paradox, we hypothesize that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. In an earlier study, we examined the effects on recombinant gene expression of five different proteins that affect the HSV-1 virion. We found that vectors packaged in the presence of mutated vhs or U S11 exhibited minimal changes in gene expression, vectors packaged in the presence of a mutated U S3 supported improved gene transfer (numbers of cells at 4 days), and vectors packaged in the presence of mutated U L13 or VP16 supported improved long-term expression. The capability of the VP16 transcriptional complex to reduce gene expression deserves additional study because VP16 is a powerful enhancer that interacts with a number of cellular and viral proteins. In particular, U L46 and U L47 are known to modulate the effects of VP16 on immediate early promoters. In this study, we examined expression from a HSV-1 vector that contains a neuronal-specific promoter and was packaged in the presence of deletions in U L46, or U L47, or both U L46 and U L47. In the rat striatum, each of these vector stocks supported both improved gene transfer (numbers of cells at 4 days) and improved long-term expression (2 months). Vectors packaged in the presence of a deletion in both U L46 and U L47 supported larger improvements in gene expression compared to vectors packaged in the presence of deletions in either gene alone. The implications of these results for strategies to improve long-term expression are discussed.

The Crystal Structure of the Herpes Simplex Virus 1 ssDNA-binding Protein Suggests the Structural Basis for Flexible, Cooperative Single-stranded DNA Binding

All organisms including animal viruses use specific proteins to bind single-stranded DNA rapidly in a non-sequence-specific, flexible, and cooperative manner during the DNA replication process. The crystal structure of a 60-residue C-terminal deletion construct of ICP8, the major single-stranded DNA-binding protein from herpes simplex virus-1, was determined at 3.0 A resolution. The structure reveals a novel fold, consisting of a large N-terminal domain (residues 9-1038) and a small C-terminal domain (residues 1049-1129). On the basis of the structure and the nearest neighbor interactions in the crystal, we have presented a model describing the site of single-stranded DNA binding and explaining the basis for cooperative binding. This model agrees with the beaded morphology observed in electron micrographs.

Effect of gene location on the evolutionary rate of amino acid substitutions in herpes simplex virus proteins

In an effort to understand the organization of genes in the herpes simplex virus (HSV-1) genome, I tested the idea that the location of a gene may be related to the evolutionary rate of amino acid sequence variation in the encoded protein. A measure of protein sequence divergence was calculated for homologous proteins in the U L region of six alphaherpesviruses including HSV-1, and this parameter was plotted against position in the HSV-1 genome. The results revealed a cluster of highly conserved proteins (U L27–U L33) encoded near the middle of U L. A similar analysis was restricted to HSV-1 and HSV-2 permitting an examination of U S proteins and proteins encoded in repeated regions at the segment ends. This analysis showed that U S proteins as a group are more highly divergent than those encoded in U L. A high degree of divergence was also observed in proteins coded at the segment ends including R L1 (γ 134.5), R L2 (α0), U L1 (glycoprotein L), U L56, U S1, and U S12. It is suggested that conserved proteins U L27–U L33 are encoded near the middle of U L to take advantage of a low local mutation rate. Highly divergent proteins are suggested to be encoded selectively in U S because of a comparatively rapid evolutionary rate with which genes can be introduced and removed from S in response to environmental variation.

During lytic infection herpes simplex virus type 1 is associated with histones bearing modifications that correlate with active transcription.

Herpes simplex virus type 1 (HSV-1) is a large (150-kb) double-stranded DNA virus that forms latent infections in neuronal cells of the human peripheral nervous system. Previous work determined that the HSV-1 genome is found in an ordered nucleosomal structure during latent infection. However, during lytic infection, it was unclear whether viral DNA was in a chromatin state. We examined HSV-1 during lytic infection using micrococcal nuclease digestion and chromatin immunoprecipitation. The HSV-1 genome is at least partially nucleosomal, although apparently not in a regular repeating structure. Analysis of histones associated with HSV-1, within both the promoter and the transcribed regions, revealed covalent amino tail modifications similar to those associated with active host mammalian genes. Certain of the modifications were detected in the temporal order expected of the immediate-early, early, and late gene classes. These data suggest that productive infection may be accompanied by acquisition of a permissive chromatin state.

806. HSV Based Genetic Selection System for Identification of Pain Signaling Regulators

Top of pageAbstract The vanilloid receptor type 1 (VR1, also known as TRPV1) is a calcium channel that plays a central role in pain signaling (nociception). The receptor is directly gated by heat, chemicals (e.g. capsaicin), and protons. In addition, VR1 is indirectly activated by NGF and bradykinin through the stimulation of protein kinase C (PKC) isoforms via G-protein coupled receptor mechanisms. PKC directly potentiates VR1 activity by phosphorylation of specific target residues. However, regulatory molecules and mechanisms involved upstream and downstream of the channel remain to be discovered. In the presence of capsaicin (a component of chili peppers), non-neuronal cells overexpressing the VR1 receptor undergo apoptosis as a result of calcium influx mediated excitotoxicity. Using the rationale of calcium influx induced excitotoxic cell death through the VR1 receptor, we developed an HSV-1 based selection system to dissect VR1 receptor biology with respect to its ligand interactions and regulation. Our aim is to identify novel genes or peptides that will specifically modulate VR1 activity that may be useful in the gene therapy based treatment of chronic pain states. First, we placed the VR1 cDNA under transcriptional control of the HSV-1 TK early promoter in the ICP4 locus of a replication defective HSV-1 genome. The genomic structure of the vector was confirmed by Southern blotting. Immunofluorescence for VR1 on infected complementing cell lines localized to the plasma membrane and perinuclear regions. We have developed a genetic selection system to identify regulators of VR1 activity from cDNA or peptide libraries using TkpVR1 replication as the gauge. Second, we tested the effect of a known agonist, capsaicin, on viral titers. In the presence of 40 μm capsaicin, TKpVR1 viral titers are reduced by greater than 100-fold at 48 hpi as compared to control virus. Low pH (6.8) similarly causes a 40-fold reduction in TKpVR1 viral titers when compared with control virus, due to proton mediated VR1 potentiation. Having established a drug induced genetic selection system for regulators of VR1 receptor activity, we have now initiated the process of screening libraries in order to identify novel regulatory proteins of VR1 channel activity within the framework of the selection system described above.

Examination of the potential interactions between herpes simplex virus vectors and replication-competent virus <I>in vitro</I> and <I>in vivo</I>

In this paper, we have studied the potential interactions of replication incompetent herpes virus vectors with replication-competent virus both in vitro and in vivo. This might be thought to be particularly important for the use of HSV as compared to other virus vectors, as humans often harbour latent HSV. A vector virus carrying a transgene could interact with endogenous wild-type virus when used in gene therapy procedures. For this study we constructed four recombinant viruses containing marker genes to allow interactions between replication competent and disabled vector viruses to be explored. Recombination of viruses under replicating conditions was assessed in vitro and in vivo in the peripheral nervous system following the inoculation of combinations of viruses into the hindpaw of BALB/c mice and the examination of virus content in the dorsal root ganglia. Recombination between the viruses, when co-administered was found to occur such that transgene-bearing replication-competent viruses were generated only when the transgene was inserted at a non-essential site in the HSV genome. When the transgene was inserted into an essential site in the disabled virus, or when disabled and non-disabled virus were administered separately to the same site, transgene-bearing replication-competent recombinants were not observed. In the central nervous system, the ability of disabled, LacZ containing virus to reactivate latent replication-competent virus was examined. CNS latency was established by infecting BALB/c mice with a replication competent, GFP containing virus by the intra nasal route. After the establishment of latency, disabled virus was injected intra-cerebrally. Reactivation could not be detected as evidenced by a lack of GFP expression and replicating virus even though robust LacZ expression from the incoming vector virus could be detected. A similar lack of reactivation occurred when vector virus was inoculated into the footpad following the establishment of latency in the PNS by a replication competent virus. These experiments show (i) that insertion of the transgene in an essential site of the viral genome prevents its incorporation into an hazardous replication-competent recombinant derivative, indicating that non-homologous recombination between disabled and replication competent viruses does not occur at the level of sensitivity of the in vitro/in vivo assays used here, (ii) even homologous recombination in vivo only occurs at detectable levels when vector and replication competent virus are co-administered, and (iii) inoculation of vector HSV into the nervous system is very unlikely to reactivate latent wild-type virus that may be present.

Herpes Simplex Virus Type-1: A Model for Genome Transactions

In many respects, HSV-1 is the prototypic herpes virus. However, HSV-1 also serves as an excellent model system to study genome transactions, including DNA replication, homologous recombination, and the interaction of DNA replication enzymes with DNA damage. Like eukaryotic chromosomes, the HSV-1 genome contains multiple origins of replication. Replication of the HSV-1 genome is mediated by the concerted action of several virus-encoded proteins that are thought to assemble into a multiprotein complex. Several host-encoded factors have also been implicated in viral DNA replication. Furthermore, replication of the HSV-1 genome is known to be closely associated with homologous recombination that, like in many cellular organisms, may function in recombinational repair. Finally, recent data have shed some light on the interaction of essential HSV-1 replication proteins, specifically its DNA polymerase and DNA helicases, with damaged DNA.

Herpesvirus quiescence in neuronal cells. V: forskolin-responsiveness of the herpes simplex virus type 1 alpha0 promoter and contribution of the putative cAMP response element.

The herpes simplex virus (HSV)-1 alpha0 promoter contains a putative cAMP response element (CRE) located at positions -68 to -60 with respect to the initiation of transcription. In this report, the authors examined the functionality of this element using (1) luciferase reporter gene assays in nerve growth factor-differentiated (ND)-PC12 cells and (2) virus-induced activation from quiescently infected (QIF)-PC12 cells. The putative alpha0 CRE was completely eliminated by digestion with the restriction enzyme Tsp45I followed by mung bean nuclease treatment. The mutated region was verified by DNA sequencing and was inserted into the alpha0-luciferase reporter plasmid (pRDalpha0-LUC) creating (pRDalpha0deltaCRE-LUC), and into the HSV-1 genome of strain 17(+)(alpha0deltaCRE). Insertion into both copies of the alpha0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRDalpha0-LUC and pRDalpha0deltaCRE-LUC plasmids responded similarly to forskolin (50 microM), with approximately 250% increases in luciferase activity compared to mock-treated cultures as measured 3 days following treatment. When QIF-PC12 cultures established with HSV-1 strain 17(+) and alpha0deltaCRE were treated with forskolin (50 microM) 17 days post infection, virus was detected in 9/24 (37.5%) and 13/24 (54.2%) of induced cultures by day 8 post treatment, respectively. In contrast, virus was detected in 0/23 and 1/24 (4.2%) of mock-treated cultures by day 8 post treatment for wild-type and mutant viruses, respectively. These findings indicate that the alpha0 promoter is forskolin responsive, the purported CRE of the alpha0 promoter does not confer forskolin responsiveness in ND-PC12 cells, and this element is not required for reactivation of HSV-1 from QIF-PC12 cells.

Regulation of Herpes Simplex Virus 1 Replication Using Tumor-Associated Promoters

To investigate use of transcriptional regulatory elements (promoters) for tumor-associated antigens to achieve HSV-1 replication preferentially in cells that overexpress the tumor-associated antigens. An important advantage of replicating viruses for cancer therapy is their ability to simultaneously destroy tumor cells by replication and release progeny virion to infect and destroy adjacent cancer cells. This strategy requires regulation of the viral life cycle to obtain robust replication in neoplastic cells and minimize replication in nonneoplastic cells. Promoters for the human carcinoembryonic antigen (CEA) and MUC1/DF3 tumor-associated antigens were characterized and cloned into HSV-1 mutants as heterologous promoters regulating expression of two different HSV-1 genes. Viral replication in tumor cells and cytotoxicity was quantified with in vitro assays. Antineoplastic efficacy was characterized in a flank tumor xenograft model. Several CEA promoters were cloned and characterized using luciferase reporter assays. The most specific promoter was used to construct and isolate two different HSV-1 mutants in which critical genes are regulated by this promoter (ICP4 and gamma(1) 34.5). Similarly, the promoter for the DF3/MUC1 tumor-associated antigen was cloned into a third HSV-1 mutant such that it regulates expression of gamma(1) 34.5. Regulation of ICP4 expression by the CEA promoter during HSV-1 infection overly attenuates viral replication. Regulation of gamma(1) 34.5 expression by either the CEA promoter or the MUC1/DF3 promoter during HSV-1 infection modulates viral replication, with preferential replication in cells that overexpress the corresponding tumor-associated antigen. A single intratumoral inoculation of an HSV-1 mutant with the MUC1/DF3 promoter regulating gamma(1) 34.5 expression results in significant antineoplastic activity in MUC1-positive pancreatic carcinoma xenografts as compared to mock inoculation. Promoters for tumor-associated antigens may be incorporated into the HSV-1 genome to regulate HSV-1 replication. The choices of HSV-1 gene and tumor-associated promoter are important determinants of success of this strategy. Because of its preferential replication in MUC1-positive tumors, an HSV-1 mutant with the MUC1/DF3 promoter regulating gamma(1) 34.5 expression will undergo further examination as a novel cancer therapy agent.

Secretion and dynamics of herpes simplex virus in tears and saliva of patients with Bell's palsy.

For clarification of the direct relationship between the reactivation of herpes simplex virus and the development of Bell's Palsy, a detection of the virus genome by deoxyribonucleic acid diagnostics and a quantitative analysis of its time-course change are both needed. The authors detected the HSV genome in specimens from patients with Bell's Palsy, quantified its number of copies, and examined time-course changes. The subjects were 16 patients with Bell's Palsy. The tear fluid and saliva from the submandibular gland and the parotid gland were separately collected from the affected and unaffected sides twice or more. A total of 244 specimens were subjected to extraction of deoxyribonucleic acid, polymerase chain reaction, and microplate hybridization. Herpes simplex virus-1 deoxyribonucleic acid was detected in 38 specimens (11.8%) from 5 patients (31%). The high detection (28.5%) was obtained within 2 weeks after onset. Detection at 3 weeks and later (2.8%) was significantly lower ( < 0.05). In three cases, deoxyribonucleic acid was also found on the unaffected side in the initial phase of the disease, but detection on that side (18.9%) was significantly lower than on the affected side (83.8%) ( < 0.01). The number of copies of the herpes simplex virus-1 genome was large on the affected side and early after the onset of the disease. The reactivation of herpes simplex virus-1 on the affected side is involved as a pathogenic factor of Bell's Palsy. A reactivation of herpes simplex virus-1 may be generated even on the unaffected side in the early phase of the disease. Herpes simplex virus deoxyribonucleic acid was not detected in any of the examined specimens collected from the remaining 11 cases. The need for constant study to clarify other causative factors of Bell's Palsy remains.

Identification and characterization of the UL56 gene product of herpes simplex virus type 2.

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.

Construction of Replication‐Defective Herpes Simplex Virus Vectors

Advances in identification and characterization of gene products responsible for specific diseases of the nervous system have opened opportunities for novel therapies using gene transfer vectors for gene replacement. Herpes simplex virus (HSV)-based vectors are particularly well suited for gene delivery to neurons of the central and peripheral nervous systems. The authors have developed methods to delete HSV-1 IE gene functions and to subsequently introduce foreign genes into the HSV-1 genome using homologous recombination. This unit describes methods for generating cell lines that complement multiple essential gene deletion mutants as well for generating such replication-defective virus recombinants and inserting foreign DNA sequences into replication-defective viral genomes, the last step in preparing a vector. Three support protocols describe methods for preparing virus stocks, titering virus, and preparing viral DNA.

Stromal Keratitis and Anterior Uveitis Due to Herpes Simplex Virus-2 in a Young Child

Background: An uncommon case of stromal keratitis and anterior uveitis due to herpes simplex virus type 2 (HSV-2) is reported. Case: The patient was a 3-year-old boy admitted for conjunctival injection of the right eye of unknown cause, accompanied by corneal opacity and anterior uveitis. Observations: High titers of antibodies against HSV and Epstein-Barr virus (EBV) were found in blood samples. Polymerase chain reaction (PCR) for the detection of HSV-1, -2, and EBV genome fragments was carried out using an anterior chamber sample as a template. An HSV-2 genome fragment was amplified by PCR. Administration of acyclovir and betamethasone was started, with the consequent elimination of corneal opacity, inflammatory cells, and keratic precipitates. Conclusion: PCR clearly showed that HSV-2 was the causative pathogen of the stromal keratitis and anterior uveitis in this young patient. Systemic EVB infection may induce systemic immunocompromised conditions that can lead to reactivation of HSV-2 followed by ocular disorders.

The transgenic ICP4 promoter is activated in Schwann cells in trigeminal ganglia of mice latently infected with herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of sensory ganglia, including those of the trigeminal ganglia. Latent viral infection has been hypothesized to be regulated by restriction of viral immediate-early gene expression in neurons. Numerous in situ hybridization studies in mice and in humans have shown that transcription from the HSV-1 genome in latently infected neurons is limited to the latency-associated transcripts. In other studies, immediate-early gene (ICP4) transcripts have been detected by reverse transcription-PCR (RT-PCR) in homogenates of latently infected trigeminal ganglia of mice. We used reporter transgenic mice containing the HSV-1(F) ICP4 promoter fused to the coding sequence of the beta-galactosidase gene to determine whether neurons in latently infected trigeminal ganglia activated the ICP4 promoter. Mice were inoculated via the corneal route with HSV-1(F). At 5, 11, 23, and 37 days postinfection (dpi), trigeminal ganglia were examined for beta-galactosidase-positive cells. The numbers of beta-galactosidase-positive neurons and nonneuronal cells were similar at 5 dpi. The number of positive neurons decreased at 11 dpi and returned to the level of mock-inoculated transgenic controls at 23 and 37 dpi. The number of positive nonneuronal cells increased at 11 and 23 dpi and remained elevated at 37 dpi. Viral proteins were detected in neurons and nonneuronal cells in acutely infected ganglia, but were not detected in latently infected ganglia. Colabeling experiments confirmed that the transgenic ICP4 promoter was activated in Schwann cells during latent infection. These findings suggest that the cells that express the HSV-1 ICP4 gene in latently infected ganglia are not neurons.

Effect of famciclovir on herpes simplex virus type 1 corneal disease and establishment of latency in rabbits

Famciclovir (FCV) is efficacious in the treatment of acute herpes zoster and recurrent genital infections but has not been used to treat ocular herpes simplex virus (HSV) infections. We evaluated the efficacy of orally administered FCV in treating HSV-1 epithelial keratitis and determined its effects on the establishment of latency and subsequent reactivation. Rabbits were inoculated with HSV-1 strain 17 syn+ and treated twice daily with increasing concentrations of FCV (60 to 500 mg/kg of body weight). This resulted in a significant, dose-dependent improvement in keratitis scores, as well as prolonged survival. Regardless of the dose of drug used, all groups exhibited the high rates of spontaneous and induced reactivation characteristic of 17syn+. The efficacy of 250 mg of FCV per kg was also compared to topical treatment with 1% trifluorothymidine (TFT). Although TFT treatment was more effective at reducing eye disease, FCV-treated rabbits had a better survival rate. Real-time quantitative PCR analysis of rabbit trigeminal ganglia (TG) demonstrated that FCV significantly reduced the HSV-1 copy number compared to that after treatment with TFT or the placebo but not in a dose-dependent manner. In summary, oral FCV treatment significantly reduces the severity of corneal lesions, reduces the number of HSV-1 genomes in the TG, improves survival, and therefore may be beneficial in reducing the morbidity of HSV keratitis in the clinic.

Enhanced reporter gene expression in the rat brain from helper virus-free HSV-1 vectors packaged in the presence of specific mutated HSV-1 proteins that affect the virion

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off promoters in HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus, recombinant gene expression remains short-term in the absence of ∼99% of the HSV-1 genome. To resolve this paradox, we hypothesized that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. This study evaluated expression from HSV-1 vectors, containing neuronal-specific promoters, that were packaged in the presence of specific mutated HSV-1 proteins that affect the virion. The mutated HSV-1 proteins that were examined included two protein kinases (U L13 and U S3), the virion host shut-off factor ( vhs), the transactivator of immediate early promoters (VP16), and a virion protein that affects RNA metabolism (U S11). Helper virus-free packaging could occur in the presence of each mutated protein alone or specific combinations of two or three mutated proteins. In BHK and PC12 cells, vectors packaged in the presence of each mutated protein increased (∼2-fold) the level of expression per cell, and vectors packaged in the presence of specific combinations of mutated proteins supported larger (4–7-fold) increases. In the rat striatum, vectors packaged in the presence of a mutated U S3 displayed enhanced gene transfer (13–18-fold increases in the number of cells at 4 days), and vectors packaged in the presence of mutated U L13 or VP16 enhanced long-term expression (2 months). Vectors packaged in the presence of mutated vhs or U S11 displayed minimal changes in expression.

Ｂｅｌｌ麻ひと単純ヘルペスウイルス

Bell's palsy is the most common cause of facial paralysis; it has an incidence of 20 to 30 per 100, 000 people. Many events such as viral infection, is chemia and autoimmune reaction have been proposed as causes of Bell's palsy. However, to date there have been no reports of conclusive evidence of the etiological agent responsible for Bell's palsysince Sir Charles Bell described the disease in 1830. Because the etiological agent remains unknown, treatment of this condition is empiric, varying from observation alone to the use of steroids and surgical decompression.Recent advancements in the molecular methods of in situ hybridization and polymerase chain reaction have revealed the existence of HSV-1 genomes in the involved facial nerve as well as the geniculate ganglion. In addition, animal experiments have shown that HSV-1 may induce facial paralysis and the beneficial effect of the antiviral agent of acyclovir for facial function has also been demonstrated. These findings provide the evidence that latently infected HSV-1 causes Bell's palsy when it reactivates and it is controversial whether we should continue using the term “Bell's palsy” to mean “idiopathic facial paralysis” or whether we should now recognize Bell's palsy as “herpetic facial paralysis”. However, currently, we have no reliable, widely available clinical test for the early identification of HSV-1 infection in Bell's patients. Methodological break-through will resolve this problem and data accumulation will datermine the percentage of patients with Bell's palsy in whom HSV-1 is the etiological agent.