Ruth Tal-Singer

Analysis of Individual Human Trigeminal Ganglia for Latent Herpes Simplex Virus Type 1 and Varicella-Zoster Virus Nucleic Acids Using Real-Time PCR

ABSTRACT Herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) establish latent infections in the peripheral nervous system following primary infection. During latency both virus genomes exhibit limited transcription, with the HSV-1 LATs and at least four VZV transcripts consistently detected in latently infected human ganglia. In this study we used real-time PCR quantitation to determine the viral DNA copy number in individual trigeminal ganglia (TG) from 17 subjects. The number of HSV-1 genomes was not significantly different between the left and right TG from the same individual and varied per subject from 42.9 to 677.9 copies per 100 ng of DNA. The number of VZV genomes was also not significantly different between left and right TG from the same individual and varied per subject from 37.0 to 3,560.5 copies per 100 ng of DNA. HSV-1 LAT transcripts were consistently detected in ganglia containing latent HSV-1 and varied in relative expression by >500-fold. Of the three VZV transcripts analyzed, only transcripts mapping to gene 63 were consistently detected in latently infected ganglia and varied in relative expression by >2,000-fold. Thus, it appears that, similar to LAT transcription in HSV-1 latently infected ganglia, VZV gene 63 transcription is a hallmark of VZV latency.

Herpes Simplex Virus Type 1 Glycoprotein E Domains Involved in Virus Spread and Disease

ABSTRACT Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.

Toxicity and neuronal infection of a HSV-1 ICP34.5 mutant in nude mice

HSV-1 mutants in the RL-1 gene encoding the ICP34.5 protein have been demonstrated to have diminished neurovirulence in brain yet replicate as efficiently as parental virus in transformed tissue culture cells. Thus they have been proposed as candidates viruses for human brain tumor therapies. Evaluation of their replicative properties and pathogenesis within the nervous system has been limited. As most patients undergoing therapies for brain tumors are likely to be immunocompromised, it will be important to understand the pathogenesis of these viruses in immunocompromised hosts. To this end, the lateral ventricle of nude mice was injected with high (2.5 x 10(7) PFU), medium (10(5) PFU), or low dose (10(3) PFU) HSV-1 variant-1716, which has a deletion in the RL-1 gene. Ten of 10 mice died within 2-3 days following the high titer infection. Six of 19 animals with medium titer infection died within 9 days, and viral antigens were seen in ependymal cells as well as neurons within the brainstem and thalamus. Although only two of 19 animals became moribund 18 days after medium titer viral infection, many neocortical and hippocampal neurons were positive for HSV-1 antigens. However, plaque-purified viral isolates recovered from brain homogenates of these animals demonstrated no increase in pathogenicity. Nine of 20 animals died following low dose infection; six of these animals, from which tissue was analyzed, all had many HSV antigen-positive neurons in the neocortex and hippocampus. These data imply that if this type of virus is used for human brain tumor therapy immunosuppressed patients may suffer from significant viral pathogenesis outside the tumor.

Herpes simplex virus 1716, an ICP 34.5 null mutant, is unable to replicate in CV-1 cells due to a translational block that can be overcome by coinfection with SV40

Herpes simplex virus (HSV) mutants lacking the gene encoding infected cell protein (ICP) 34.5 exhibit an attenuated phenotype in models of pathogenesis and have been used for experimental cancer therapy. Recently it was shown that the HSV ICP 34.5 protein functions to prevent the host cell-induced double-stranded RNA-activated protein kinase (PKR)-dependent translational block that normally occurs during virus infection. We now report that an HSV ICP 34.5 mutant called HSV-1716 is unable to replicate in the simian kidney cell-derived line CV-1, due to a translational block. Moreover, we find that this block can be overcome by simian virus 40 (SV40). This has been shown directly by infecting CV-1 cells with SV40 and HSV-1716 simultaneously, and indirectly via HSV-1716 infection of COS-1 cells (CV-1 cells transformed by an origin-defective mutant of SV40 that codes for wild-type T antigen). The translational block is restored when infections are done in the presence of the phosphatase inhibitor okadaic acid. These results support, but do not directly prove, contentions that HSV ICP 34.5 interacts with the PKR pathway to restore translation in non-permissive cells, and that SV40 large T antigen has a similar functional role, but acts downstream of the site of ICP 34.5 interaction (eIF2alpha) in the pathway. Study of this CV-1/COS-1 system should allow further clarification of the virus-host interactions that underlie the restricted replication of HSV-1 ICP 34.5 gene null mutants.

Differences in pathogenicity of herpes simplex virus serotypes 1 and 2 may be observed by histopathology and high-resolution magnetic resonance imaging in a murine encephalitis model.

The mouse model for herpes simplex-induced encephalitis (HSE) is an established preclinical tool for evaluating the efficacy of new therapeutic interventions. We evaluated the utility of high-resolution in vivo MRI in observing the progression of experimental HSE during the first week postinfection. Female BALB/c mice were inoculated intracerebrally with HSV-1 or HSV-2 by microinjection. Each animal was evaluated daily by high-resolution (4.7 Tesla) T2 weighted MRI and clinical disease scoring (neurological and behavioral). Lesions induced by a high dose of HSV-1 (1000 PFU) were detectable by MRI without administration of contrast agent whereas for low dose HSV-1 (100 PFU), administration of contrast agent was necessary to visualize the lesions in the brain. The correlation between the MRI and histologic results was excellent. No HSV-2 induced lesions were observed by MRI. Although both HSV serotypes caused similar clinical disease, significant type differences were found by histologic and MRI examinations. HSV-1 caused necrotizing meningoencephalitis, whereas HSV-2 induced mostly meningitis. The data indicate that in vivo high-resolution MRI may be useful to longitudinally evaluate HSV-1-related pathology in a mouse model of HSE and potentially could be used for monitoring the efficacy of anti-infective therapeutic approaches.