Altered pathogenesis in herpes simplex virus type 1 infection due to a syncytial mutation mapping to the carboxy terminus of glycoprotein B.

A syncytial (syn) variant of herpes simplex virus type 1 strain 17 syn+ was selected by serial passage in heparin, a glycosaminoglycan which potently inhibits herpes simplex virus infectivity. This virus, 17 hep syn, is sixfold more heparin resistant than its parent. By using marker transfer techniques, its syn phenotype, but not heparin resistance, was mapped first to the BamHI G fragment (0.343 to 0.415 map units) and then to a 670-bp KpnI-PstI subclone (0.345 to 0.351 map units) encoding the carboxy terminus of glycoprotein B (gB). Three cloned syncytial recombinants were generated from cotransfections of 17 syn+ with either 17 hep syn BamHI-G or the 670-bp subclone. After footpad inoculation of mice, 17 hep syn was as virulent as its parent, despite reaching lower titers in feet, sciatic nerves, dorsal root ganglia, spinal cords, and brains. Animals infected with 17 hep syn or the gB recombinant viruses developed a unique pattern of disease that was strikingly different than that seen with wild-type virus: severe inflammation and edema of the inoculated limb and death without antecedent paralysis. Histopathologic examination revealed limitation of spinal involvement by 17 hep syn to the dorsal aspect of the cord and decreased virus-induced damage in the central nervous system. The genetically unrelated syn variant MP, in contrast, was avirulent and did not cause severe local inflammation. After intracerebral inoculation, 17 hep syn was highly virulent and replicated to high titers in the brain. Yet, unlike the parental virus, it resulted in an altered distribution of herpes simplex virus antigens, which were limited to the ependymal and subependymal regions surrounding the lateral ventricles. Despite their syncytial phenotype and pathogenic properties, the recombinant viruses, unlike 17 hep syn, were not heparin resistant. We conclude that a transferable alteration in the 670-bp carboxy-terminal portion of the glycoprotein gB gene of 17 hep syn results in both its syncytial phenotype and the unique pattern of disease that it causes but does not result in heparin resistance. These observations provide direct biological evidence for an important role for herpes simplex virus gB in pathogenic events both at the peripheral site of infection and within the nervous system.     

Vaccine therapy for ocular herpes simplex virus (HSV) infection: periocular vaccination reduces spontaneous ocular HSV type 1 shedding in latently infected rabbits.

Periocular vaccination of rabbits with preexisting herpes simplex virus type 1 (HSV-1) latent infection with recombinant HSV-2 glycoproteins B and D (gB2 and gD2) plus adjuvant significantly reduced ocular viral shedding. Rabbits were infected in both eyes with HSV-1 strain McKrae. Following HSV-1 infection and the establishment of latency (28 days postinfection), rabbits were given a periocular subconjunctival vaccination three times at 3-week intervals. Beginning 3 weeks after the final vaccination, tear films were collected daily and cultured to detect the presence of HSV-1 and determine the spontaneous HSV-1 ocular shedding rates. Periocular vaccination increased the mean HSV-1 serum neutralizing antibody titer to fivefold above that seen in mock-vaccinated latently infected rabbits. gB enzyme-linked immunosorbent assay (ELISA) antibody titers were increased approximately 8-fold, and gD ELISA antibody titers were increased 60-fold. These increases were all statistically significant (P < 0.0001). In two independent experiments, vaccination reduced the spontaneous shedding rate by approximately 2.5-fold (P < 0.0004). In addition, the percentage of eyes that never shed virus during the 6 week postvaccination test period increased threefold (20% in controls versus 60% in vaccinated animals; P < 0.007). These results show that spontaneous ocular shedding of HSV-1 in latently infected rabbits can be significantly reduced by local periocular vaccination. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1 ocular shedding. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans is possible.     

Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA

RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.     

Ultrastructural characterization of an early, nonstructural polypeptide of herpes simplex virus type 1.

An immunoperoxidase procedure was employed to study the expression of a large-molecular-weight, virus-induced polypeptide (VP175; molecular weight, 175,000) at the light and electron microscopic levels in Vero cells infected with herpes simplex virus type 1 or with tsB2, a DNA-negative, temperature-sensitive mutant of herpes simplex virus type 1. In cells infected with herpes simplex virus type 1 and in cells infected with tsB2 at the permissive temperature (34 degrees C), VP175 was found within the nucleus. The protein was detected as early as 2 h postinfection and, by 3 h postinfection, was generally distributed in a marginated pattern contiguous with, and extending from, the inner lamella of the nuclear membrane. At 6 h postinfection, protein accumulations were dispersed throughout the nucleus, and, by 9 h postinfection, these accumulations tended to be localized in a marginated pattern near the nuclear membrane. It was also noted that, at 9 h postinfection, under permissive conditions, VP175 was not found in association with nucleocapsids or enveloped particles. In contrast, in cells infected with tsB2 at the nonpermissive temperature (39 degrees C) and harvested at 6 or 9 h postinfection, accumulations of VP175 were identified not only within the nucleus, but also within the cytoplasm in the form of annular or globular aggregates. These aggregates consisted of a granular matrix and were not bound by membranes.     

Herpesvirus simiae (B virus) antibody response and virus shedding in experimental primary infection of cynomolgus monkeys.

Four cynomolgus monkeys (Macaca fascicularis) were inoculated in the lips and tongues with B virus. Virus shedding and antibody responses were monitored for up to 50 days postinfection. Virus was isolated from the oral cavities of all monkeys at 6 days postinfection despite the absence of observable lesions. Virus was not isolated from genital swabs or serum. Antibodies to both B virus and herpes simplex virus were detected by neutralization between days 8 and 12. Virus-specific IgM and IgG antibodies were measured by antibody capture radioimmunoassay. IgM was first detected on day 6; by contrast, IgG did not appear until day 12. Antibodies reactive in a competitive radioimmunoassay appeared by day 12 and peaked at 30 to 40 days postinfection. This study provides data on which to base the diagnosis of primary B virus infection in cynomolgus monkeys.     

Evidence for post-translational glycosylation of a nonglycosylated precursor protein of herpes simplex virus type 1.

Incubation of herpes simplex virus type 1-infected Vero and HEp-2 cells at a reduced temperature (34 degrees C) enhanced the detection of the nonglycosylated precursors (pgB97 and pgC75) to the gB and gC glycoproteins in the cytoplasmic and nuclear fractions. Relative to the fully glycosylated and high-mannose forms detected, the nonglycosylated precursors were the predominant components associated with the nuclear fraction of infected cells. Furthermore, addition of protease inhibitors to the fractionation buffers did not affect the distribution or abundance of the nonglycosylated precursors, suggesting that the presence of pgB97 and pgC75 was not the result of proteolysis. When infected Vero or HEp-2 cells were harvested at various times postinfection, the nonglycosylated precursors were detected after the initial appearance of the high mannose components (pgB110 and pgC105). In Vero cells, pgB97 and pgC75 were detected simultaneously at 8 h postinfection, whereas detection was not apparent in HEp-2 cells until 20 h postinfection. Conditions which favored detection of appreciable amounts of nonglycosylated precursors provided an unique approach to probe possible post-translational modifications in the absence of inhibitors of glycosylation. In nuclear fractions isolated from cycloheximide-treated HEp-2 or Vero cells, numerous discrete gC-immunoreactive bands migrating with decreased electrophoretic mobility relative to the nonglycosylated precursor pgC75 were observed. This series of one to four additional bands was eliminated by digestion with endoglycosidase H, and the appearance of these bands was blocked by the addition of tunicamycin. Collectively, the data suggest that high-mannose core oligosaccharides may be added to the nonglycosylated precursor of the gC glycoprotein of herpes simplex virus type 1 in a post-translational fashion.     

Proteins Specified by Herpes Simplex Virus: II. Viral Glycoproteins Associated with Cellular Membranes

Membranes prepared from HEp-2 cells infected with herpes simplex virus and free from soluble proteins, virus, ribosomes, and other cellular constituents were solubilized and subjected to electrophoresis on acrylamide gels. The electropherograms showed the following. (i) The synthesis of host proteins and glycoproteins ceases after infection. However, the spectrum of host proteins in membranes remains unaltered. (ii) Between 4 and 22 hr postinfection, at least four glycoproteins are synthesized and bound to the smooth cytoplasmic membranes. On electrophoresis, these glycoproteins form two major and two minor bands in the gel and migrate with proteins ranging from 50,000 to 100,000 daltons in molecular weight. (iii) The same glycoproteins are present in all membranes fractionated by density and in partially purified virus. The implications of the data are discussed.     

Differential effect of nucleoside analog triphosphates on ribonucleotide reductases from uninfected and herpes simplex virus-infected HeLa cells.

Effects of the triphosphates of eight pyrimidine nucleoside analogs (5-substituted, 2'-fluoroara-, and acyclonucleosides) and acycloguanosine were examined on the ribonucleotide reductases prepared from uninfected and herpes simplex virus (types 1 and 2)-infected HeLa cells. Of the analogs tested, E-5-propenyl- and E-5-(2-bromovinyl)-dUTP were more potent inhibitors than dTTP of the enzymes from virus-infected cells, whereas only the former compound showed this effect on the uninfected HeLa enzyme.     

Suppression of Herpes Simplex Virus Infection by Phosphonoacetic Acid

Disodium phosphonoacetate when administered orally or topically to mice experimentally infected with herpes simplex virus was able to significantly reduce the mortality associated with the agent. In addition, this compound was able to reduce herpesvirus lesions on the corneas of infected rabbits.     

In vivo kinetics of GITR and GITR ligand expression and their functional significance in regulating viral immunopathology

This report evaluates the role of interaction between glucocorticoid-induced tumor necrosis factor receptor (GITR) and GITR ligand (GITR-L) in the immuno-inflammatory response to infection with herpes simplex virus (HSV). Both GITR and GITR-L were transiently upregulated after ocular HSV infection, on antigen-specific T cells and antigen-presenting cells, respectively, in the draining lymph node (DLN). In addition, virus-specific T-cell responses in the DLN and spleen were enhanced by anti-GITR antibody treatment, an outcome expected to result in more severe inflammatory lesions. Intriguingly, the treatment resulted in significantly diminished T-cell-mediated ocular lesions. The explanation for these findings was that anti-GITR antibody treatment caused a reduced production of ocular MMP-9, a molecule involved in ocular angiogenesis, an essential step in the pathogenesis of herpetic keratitis. Our results are the first observations to determine in vivo kinetics of GITR and GITR-L expression after virus infection, and they emphasize the role of GITR-GITR-L interaction to regulate virus-induced immuno-inflammatory lesions.     

Overproduction of gamma interferon in B/Jas inbred rabbits with herpes simplex virus encephalitis.

Inbred rabbits of the B/Jas strain are highly susceptible to herpes simplex virus type-1 (HSV-1) encephalitis, developing seizures of encephalitis after intravenous injection of the KOS strain of the virus. Anti-viral interferon activity became detectable in the serum just prior to or at the onset of seizures, its level being lower in the serum than in the cerebrospinal fluid. The activity was of gamma interferon, as suggested by the acid instability and the inability of Mx protein induction. An immunohistochemical analysis of the brain tissues of encephalitic rabbits showed that MHC class I antigen was expressed on the microglia cells of inflamed lesions but not on these cells in uninflamed areas. These findings were discussed in correlation with the pathogenesis of herpetic encephalitis in the inbred rabbits.     

Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS.

It has been documented that KOS, a laboratory strain of herpes simplex virus type 1, is several orders of magnitude less neurovirulent than most other wild-type strains. Studies initiated to determine the functional nature of the block to neuroinvasiveness and to establish the genes involved have determined that, after footpad inoculation of mice, strain 17 syn+ induced neuropathologic signs (paralysis) at titers of 10(2) and yielded a PFU/50% lethal dose ratio of 10(4). In contrast, KOS was not lethal and did not induce paralysis at inoculation doses of 10(8) PFU. This reduced neurovirulence of KOS could not be explained by the lack of thymidine kinase activity, its inability to replicate in mouse cells maintained in culture at 38.5 degrees C, or its inefficient replication in nonneural tissues in vivo. Kinetic experiments tracing the virus through the nervous system after footpad inoculation showed that KOS was blocked at the level of the spinal ganglia. A cosmid library of strain 17 syn+ was utilized in recombination and in vivo selection experiments with strain KOS to establish the genomic region involved in 17 syn+ neuroinvasiveness. A cosmid clone containing the HindIII A fragment (0.25 to 0.53 map units) of strain 17 syn+ in mixed transfections with full-length KOS DNA yielded recombinants with enhanced neuroinvasiveness.     

Truncation of the carboxy-terminal 28 amino acids of glycoprotein B specified by herpes simplex virus type 1 mutant amb1511-7 causes extensive cell fusion.

Three amber mutations were introduced proximal to the syn3 locus of the herpes simplex virus type 1 glycoprotein B (gB) gene specifying gB derivatives lacking the carboxy-terminal 28, 49, or 64 amino acids. A complementation system that utilized gBs expressed in COS cells to complement gB-null virus K delta T was established. The 49- or 64-amino-acid-truncated gBs failed to complement gB-null virus K delta T, while the 28-amino-acid-truncated gB complemented K delta T efficiently. Mutant herpes simplex virus type 1 KOS (amb1511-7) specifying the 28-amino-acid-truncated gB fused Vero cells extensively.     

单纯疱疹病毒致病模型的研究

对单纯疱疹病毒（HSV）感染小白鼠致病特点进行了观察。小白鼠感染HSV第4天后开始发病,感染后2h血液内可分离出病毒,第48小时病毒血症水平和病毒检出率较高。不同组织病毒分布不同,脑、神经节在感染后第72小时病毒滴度较高,心、肝组织在第5天达到高峰。结果说明所建立的HSV致病模型可用于评价抗HSV药物。