利用缺陷型单纯疱疹病毒载体在大鼠中枢神经系统…

本研究利用构建的Ｉ型单纯疱疹病毒（ＨＳＶ－１）扩增子质粒载体ｐＨＳＬ，在辅助病毒ＨＳＶ－１ｔｓＫ（许可温度３１℃）的辅助下，以首尾相连的连接全形成包装成复制缺陷型的ＨＳＶ－１假病毒颗粒，得到一种ＨＳＶ－１混合毒种ｄｖＨＳＬ。将ｄｖＨＳＬ接种体外培养的大鼠胚胎脊髓运动神经元和大脑皮质神经元后均可获得报告基因的表达；接种大鼠角膜后ｌａｃＺ基因三叉神经节中持续表达两个月；直接注射接种到大鼠前脑上质后，可     

单纯疱疹病毒1型载体

本文讨论单纯疱疹病毒1型的特点和作为基因治疗载体的单纯疱疹1型载体的构建方法及其应用。     

质粒型单纯疱疹病毒载体的构建及其在真核细胞中的表达

利用基因工程技术构建成功质粒型Ⅰ型单纯疱疹病毒（ＨＳＶ－１）载体ｐＨＳＶ，其中含有ＨＳＶ－１包装信号序列和ＨＳＶ－１复制起点，以及ＨＳＶ－１ＩＥ６８启动子。为验证其构建是否成功，在其ＩＥ６８启动子下游的多克隆位点上插入了Ｅ．ｃｏｌｉＬａｃＺ基因，构建成ｐＨＳＶＬ质粒。用ＨＳＶ－１温度敏感株（ｔｓＫ株）超感染传代细胞后，将这种质粒转染入该细胞，ｐＨＳＶＬ可在辅助病毒ＨＳＶ－１ｔｓＫ（允许温度为３１℃）存在的条件下被包装成病毒颗粒，由此得到的混合病毒含有ＨＳＶ－１ｔｓＫ和ｐＨＳＶＬ假病毒颗粒，收获后再感染培养的Ｖｅｒｏ细胞，原位检测β－半乳糖苷酶活性呈阳性。同时用ｐＨＳＶＬ病毒接种体外培养的ＳＤ大鼠胚胎脊髓运动神经元，用Ｘ－ｇａｌ原位检测β－半乳糖苷酶的活性，亦发现有阳性的神经元存在。说明载体ｐＨＳＶＬ携带的报告基因ｌａｃＺ在这两种真核细胞中均得以正确表达。报告基因可为其它目的基因所代替，可用于神经生理、病理以及神经系统疾病基因治疗的实验研究。     

Ⅰ型单纯疱疹病毒载体在中枢神经系统基因治疗中的应用

I型单纯疱疹病毒（HSV-1）是一种基因组为152kb的双链线状DNA病毒,由共价连接的长片断（L）和短片断（S）组成。HSV-1具有天然嗜神经性,可感染非分裂细胞,同时能进入感觉神经元突触末端并逆轴突到达胞体,然后在神经元中建立长期稳定静止的潜伏状态。     

单纯疱疹病毒扩增子载体转导的ATM基因在活体的异位表达(英文)

小脑性共济失调与血管扩张症 (A-T)是一种常染色体隐性遗传性疾病 ,具多效表型。此病的特征是小脑变性、免疫缺陷、生长迟缓、早熟性衰老、染色体不稳定、肿瘤易患体质和对辐射的过度敏感。目前尚未发现有效的方法阻断此病的进行性发展。基因治疗是此病的潜在希望。ATM是此病的致病基因 ,编码 ATM蛋白激酶。本研究将 ATM c DNA完整的读码框架克隆进 1型单纯疱疹病毒 (HSV-1)扩增子载体 (p TO-ATM)。培养的 A-T细胞经 p TO-ATM病毒载体转导后 ,用免疫组织化学方法证明了 ATM在这些细胞的异位表达。为了进一步研究在活体应用此载体系统的可能性 ,选择大鼠脑作为实验靶区。先用免疫组织化学方法观察了内源性 ATM在成年大鼠前脑和小脑的正常分布 ,发现在尾壳核有低水平的 ATM出现 ,而在其余脑区 ,ATM水平则显著地高于尾壳核区。将 p TO-ATM病毒载体注射到成年大鼠的一侧尾壳核 ,3 d后在注射区 (包括针道周围的区域 )观察到密集的 ATM免疫反应阳性神经元。本研究结果证明 ,HSV扩增子病毒载体可以稳定地运载相当大的 c DNA片段 ,转基因在离体和活体均能有效地表达。本研究为使用基因治疗的方法治疗 A-T病人的神经变性迈出了第一步     

单纯疱疹病毒载体用于基因治疗的研究进展

基因治疗（gene therapy）是向功能缺陷或病变的细胞及组织引入互补的正常基因或治疗性功能基因,使其表达而达到治疗疾病的方法。它是伴随着基因工程技术和分子生物学的发展而逐渐形成的一种新型“分子治疗”。单纯疱疹病毒在基因治疗方面有着很广泛的研究,首先它不论是在体外还是体内都是一个高容量,高效表达的载体,可以承载其他基因,同时,它所含的TK基因,可以辅助抗病毒药更昔洛韦（Ganciclovir,0CV）,用于肿瘤的治疗。本文将就这两个方面做一综述。     

单纯疱疹病毒载体用于基因治疗的研究进展

基因治疗(gene therapy)是向功能缺陷或病变的细胞及组织引入互补的正常基因或治疗性功能基因,使其表达而达到治疗疾病的方法。它是伴随着基因工程技术和分子生物学的发展而逐渐形成的一种新型“分子治疗”。单纯疱疹病毒在基因治疗方面有着很广泛的研究,首先它不论是在体外还是体内都是一个高容量,高效表达的载体,可以承载其他基因,同时,它所含的TK基因,可以辅助抗病毒药更昔洛韦(Ganciclovir,GCV),用于肿瘤的治疗。本文将就这两个方面做一综述。     

生殖道单纯疱疹病毒感染

单纯疱疹病毒（Herpes simplex virus,HSV）属嗜神经性的脱氧核糖核酸病毒,按其抗原性不同分为二类：Ⅰ型（HSV-1）主要侵犯口咽、扁桃体、眼、皮肤等部位;而Ⅱ型（HSV-2）主要侵犯生殖器部位,都通过上皮细胞传播,通过皮肤的破损,迁移到神经细胞,并在此潜伏。HSV-1主要潜伏在三叉神经节,发病时表现为口周面部的皮肤损害;HSV-2主要潜伏在腰骶神经节。但这两种病毒均可导致口周面部和生殖系统的感染。     

单纯疱疹病毒Ⅱ型小片段型特异基因的表达及其意义

目的 为我国单纯疱疹病毒Ⅱ型的基因工程疫苗提供早期实验研究资料。方法 采取PCR和蛋白质原核细胞表达方法。结果 自我国病毒储备株中成功地克隆出了单纯疱疹病毒Ⅱ型小片段特异基因，并获得高效表达。采用该抗原包被，自生殖系统感染者恢复期血清中均能查到针对该重组抗原的IgG抗体。结论 单纯疱疹病毒Ⅱ型小片段型特异基因的成功表达，为我国单纯疱疹病毒Ⅱ型感染者的特异性诊断和单纯疱疹病毒Ⅱ型疫苗的研究打下了基础。     

Spread of herpes simplex virus type 1 in the central nervous system during experimentally reactivated encephalitis

Because many of the features of reactivated herpes simplex virus type 1 (HSV-1) central nervous systems (CNS) infections in vivo are incompletely understood, we used an animal model to study the development of the morphological, ultrastructural, radiological and immunological changes which occurred during acute and experimentally reactivated diseases. Rabbits were intranasally inoculated with HSV-1, and their latent trigeminal ganglionic and CNS infections were reactivated by intravenous injection of cyclophosphamide and dexamethasone. Technetium brain scans were performed to localize areas of blood-brain barrier breakdown, and cerebrospinal fluid (CSF) was analysed for IgG content by radial immunodiffusion assays. Nervous system tissues were studied by in situ hybridization and by immunofluorescent, light and electron microscopic techniques. Diffuse uptake of technetium was observed as HSV-1 spread transsynaptically into the brain during the acute phase of infection, and viral antigens and nucleic acids were detected in both the CNS olfactory and trigeminal systems. During latency, viral RNA was detected in the nuclei of neurons within the CNS olfactory cerebral and entorhinal cortices, indicating that HSV-1 became latent within the same CNS structures that were involved during the acute phase of infection. Following drug-induced reactivation, the brain scans revealed a more focal breakdown of the blood-brain barrier, and both neurons and neuronal processes in the entorhinal and olfactory cortices contained viral nucleic acids which correlated with the ultrastructural presence of HSV-1 virions. During the reactivated phase of infection a marked increase in the CSF IgG index occurred without an increase in the CSF: serum albumen ratio indicating a prompt intrathecal response in infected rabbits as compared to controls. To some extent, the CSF IgG index reflected the degree of histopathological damage.     

Targeting the central nervous system with herpes simplex virus / Sleeping Beauty hybrid amplicon vectors

The pursuits of sustainable treatments for diseases and disorders that afflict the central nervous system (CNS) have proven challenging for the field of viral vector-based gene therapy. However, recent advances in viral vector technology coupled with efficient delivery methods have opened up new avenues that show promise at the preclinical testing stage. The development of the Herpes Simplex Virus/Sleeping Beauty (HSV/SB) hybrid vector represents such an advance for devising treatments targeting the CNS with its potential for stably integrating large transgenomic segments of DNA within the genomes of transduced cells. In utero administration of this hybrid vector into the embryonic mouse brain has revealed the capacity for widespread transgene dissemination due to the targeting of a neuronal precursor cell population. This unique feature has provided the means to stably express a transgene throughout the brain for prolonged periods, which is a prerequisite for the treatment of progressive CNS disorders. In this review we provide a comprehensive breakdown of the characteristics of the HSV/SB vector system and how it can be efficiently employed in the derivation of CNS-targeted gene therapeutic strategies.     

Targeted gene delivery to the nervous system using herpes simplex virus vectors

Neurotrophic factors are highly potent macromolecules with protean effects. Although they are highly effective in vitro and in animal models in vivo, they have not been successfully applied to the treatment of human disease. Our laboratories have developed recombinant herpes simplex virus (HSV)-based vectors, that we have demonstrated may be used to deliver and express neurotrophic factor genes in dorsal root ganglion neurons to protect against the development of neuropathy in animal models, without causing systemic side effects. In a similar fashion, we have demonstrated that a vector expressing proenkephalin to mediate the release of opioid peptides from afferent nerve terminals in the spinal cord can be used to produce a localized antinociceptive effect in animal models of pain. Targeted gene delivery using HSV-based vectors offers a means to utilize short-lived peptides to produce specific effects in the nervous system.     

Pathogenicity of glycoprotein C negative mutants of herpes simplex virus type 1 for the mouse central nervous system

A previous study from our laboratory showed that a mutant of herpes simplex virus type 1 (HSV-1), strain KOS-321, carrying a deletion in the structural gene for glycoprotein C (gC) had reduced pathogenicity for the mouse central nervous system when compared to the wild-type virus (Kümel et al., 1985). In this study, eight additional gC negative (gC-) mutants derived from KOS-321 were shown to vary widely in their ability to induce lethal encephalitis in female DBA/2 mice following intracerebral inoculation. This variation in virulence showed no correlation with thymidine kinase activity. One less virulent gC- strain, gC-39, was further studied to determine whether the neurovirulent phenotype could be restored by rescue of the gC gene using standard marker rescue cotransfection procedures. The resulting progeny contained 2% gC+ recombinant virions and was tested for its ability to cause encephalitis. Although this progeny had increased virulence, it was not attributable to the acquisition of the gC gene since passive immunization of mice with a pool of anti-gC monoclonal antibodies had no effect on the development of encephalitis and only gC- viruses were isolated from diseased brain tissues. In agreement with these findings, individual plaque-purified gC positive (gC+) virus recombinants were shown not to have been restored to the wild-type virus level of neurovirulence. It is concluded that gC is not a virulence determinant in this mouse model of HSV-induced encephalitis and that cotransfection procedures can induce additional mutations that affect viral pathogenesis.     

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA^r) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.     

Hemagglutination by herpes simplex virus type 1

Summary The McIntyre and HSZP strains as well as clinical isolate of herpes simplex virus type 1 were found to agglutinate C57B1/10su and CBA mouse red blood cells. The hemagglutinating activity was inhibited by antisera that neutralized the infectivity of the virus.     

Autonomic nervous system involvement in experimental genital infection by herpes simplex virus type 2

Summary Peroxidase-antiperoxidase technique and histology were employed to elucidate the peripheral routes involved in HSV-2 progression from vagina towards the central nervous system in mice. 12 week-old female Balb/c mice were intravaginally infected with 5 × 10⁵LD₅₀ of HSV-2. Sixty per cent of animals developed vulvovaginitis, perigenital alopecia and hind-limb paresia. Death occurred at 9–11 days post-infection. Colon dilatation and urinary bladder distention were observed in all cases. Complete transversal sections from vulva to kidneys were obtained of each animal, including the spinal cord in situ. Herpes antigen were regularly detected in vulvovaginal epithelium, intramural, perigenital and perivesical small nerves. Besides, their invariable presence in Auerbach's plexus and sympathetic ganglia, strongly suggests preferential autonomic nervous system involvement in the progression of HSV-2 intravaginal infection towards the spinal cord.With 7 Figures     

Marker rescue of temperature-sensitive mutants by defective DNA of herpes simplex virus type 1.

High-density, defective DNA (?_CsCl = 1.732 g/cm³) produced in infections of the 15th through 20th undiluted serial passage of herpes simplex virus type 1 (HSV-1), Strain KOS, was purified by preparative CsCl density gradient ultracentrifugation. Purified defective DNA expressed no discernible cytopathic effects when transfected into receptive cell monolayers. Cleavage of defective DNA preparations with EcoRI restriction endonuclease revealed four major and three minor bands upon electrophoresis in agarose gels. Restriction endonuclease HindIII failed to cleave defective DNA.Marker rescue studies revealed that of 13 temperature-sensitive mutants representing 12 complementation groups, only mutants in group B were rescued by defective DNA. These studies demonstrate, therefore, that defective DNA is enriched for at least part of the B cistron and suggest that this cistron is located in the region of the viral genome from which defective DNA is generated.     

Expression of entry receptor nectin-1 of herpes simplex virus 1 and/or herpes simplex virus 2 in normal and neoplastic human nervous system tissues

Herpes simplex virus 1 and/or Herpes simplex virus 2 (HSV) are important pathogens of human nervous system (NS) and genetically modified HSV strains have been proposed as vectors for gene therapy targeting the brain and brain tumors. Nectin-1 is an immunoglobulin-like adhesion molecule that participates in the formation of synapses and serves as an entry receptor for HSV. The expression pattern of nectin-1 in normal human NS and brain tumors is not well understood. To better understand the nectin-1 expression in normal and neoplastic human NS, immunohistochemistry was used to detect the nectin-1 expression in sections of normal human brain, spinal cord and trigeminal and dorsal root ganglia (n=10) and in sections of primary NS neoplasms (n=22). In normal human NS, nectin-1 was detected in the soma and processes of central and peripheral neurons, in ependymal cells, choroid plexus epithelial cells, vascular endothelial cells and meningothelial cells. Oligodendrocytes, astrocytes, vascular smooth muscle cells, and Schwann cells showed variable immunoreactivity. Among tumors, schwannoma, fibrous meningioma, and medulloblastoma were nectin-1 negative. Oligodendroglioma, ependymoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme and meningothelial meningioma showed weak focal nectin-1-positivity. Ganglion cells of ganglioglioma were strongly positive. These studies provide novel information about the expression of nectin-1 in normal and neoplastic NS, and thus may lead to a better understanding of cell targeting by HSV during HSV-induced neurological disease and during a HSV-based gene therapy.