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。     

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。     

单纯疱疹病毒I型胸苷激酶基因部分缺失重组质粒的构建？…

目的：构建ＨＳＶ－１型胸苷激酶基因部分缺失的重组质粒并进行序列测定。方法：以ｐＵＣ１８／ＴＫ重组质粒ＤＮＡ为模板,用ＰＣＲ方法扩增ＴＫ５＇端５０３ｂｐ基因片段,并将扩增的片段克隆入载体ｐＵＣ１８中（ｐＵＣ１８／ＴＫ１）;用ＰｓｔＩ和ＨｉｎｄⅢ双酶切ｐＵＣ１８／ＴＫ,获得ＴＫ３＇端３８３ｂｐ片段,将此酶切片克隆入ｐＵＣ１８／ＴＫ１中,并进行测序。结果：构建成重组质粒ｐＵＣ１８／ＴＫ＾－。经酶切鉴定获     

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     

利用缺陷型单纯疱疹病毒载体在大鼠中枢神经系统表达外源基因

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，以首尾相连的连接体形式被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基因在三叉神经节中持续表达两个月；直接注射接种到大鼠前脑皮质后，可在注射部位的局部表达报告基因近两个月。研究结果表明上述缺陷型单纯疱疹病毒载体可作为神经系统基因转移的工具，应用于神经系统生理、病理及神经系统疾病基因治疗的实验研究     

单纯疱疹病毒Ⅰ型基因治疗载体的快速构建

背景：单纯疱疹病毒Ⅰ型载体因具有独特的优点目前被广泛应用,但其构建尚缺乏一种快速有效的方法。 目的：利用Cre/Loxp高效重组系统构建单纯疱疹病毒Ⅰ型载体。 方法：分离单纯疱疹病毒HSV-1,将含Cre重组酶的c66-SV40-cre质粒转染Vero细胞,构建一株带有Loxp位点的重组HSV-1框架载体HSVLoxp。构建穿梭载体pShuttle- SV40-Cre-Loxp-IRES及重组单纯疱疹病毒Ⅰ型载体HSV-GDNF,用HAT培养基筛选出阳性毒株后用GDNF引物做PCR鉴定,扩增培养后测定滴度。 结果与结论：成功构建pHV-TK-GFP质粒,并在Vero细胞内发生重组,分离出缺失了Us3基因的重组病毒HSVtk-Loxp-GFP01。成功构建HSV-1框架载体HSVLoxp及穿梭载体pShuttle- SV40-Cre-Loxp-IRES,成功获得GDNF基因,并将其转移到了HSV-1基因组上,成功构建了表达GDNF的单纯疱疹病毒HSV-1载体,测定其滴度约为2.25×10⁶ IU/mL。     

单纯疱疹病毒载体转基因治疗神经疾病的潜力

基因治疗人类神经系统疾病是最近才提出来的并且已取得成功〔1,2〕。目前,国外已建立了许多病毒载体,如单纯疱疹病毒(HSV)、腺病毒、反转病毒、腺病毒相关病毒等载体,但主要是用于体外试验和动物。现已成功地把外源基因插入到上述病毒载体。而这种外源基因可以激活神经系统的一些特殊区域。从病毒在神经元细胞建立潜在感染的能力来看,HSV特别适合于神经元产生基因。病毒载体用于治疗某些疾病,如恶性脑胶质瘤、帕金森氏病及已知的单基因疾病和脑缺血等具有很大的潜力。本文着重综述病毒载体的以下几个方面。1　病毒载体途径的总则〔3〕基…     

单纯疱疹病毒I型胸苷激酶基因在大肠杆菌中的融合表达

用ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切已构建的含单纯疱疹病毒Ｉ 型胸苷激酶（ ＨＳＶ１ ＴＫ） 基因的ｐ ＵＣ１８／ＴＫ 质粒，将切出的ＴＫ 基因片段克隆入原核表达载体ｐ ＷＲ４５０１ 中，构建成ＨＳＶ１ ＴＫ 基因重组表达质粒ｐ ＷＲ４５０１／ＴＫ。以ＨＳＶ１ ＴＫ 特异性引物ＴＫ１ Ｆｏｒ 和ＴＫ２ Ｒｅｖ 进行ＰＣＲ 鉴定可扩增出预期的５０３ ｂｐ 的ＴＫ 基因编码区部分序列；ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切质粒ｐ ＷＲ４５０１／ ＴＫ， 可切出约１１５０ｂｐ 的ＤＮＡ 片段， 表明重组质粒中已插入ＨＳＶ１ ＴＫ基因片段。用ＩＰＴＧ 诱导ｐ ＷＲ４５０１／ＴＫ／ＪＭ１０９ ，表达出相对分子质量（ Ｍｒ） 约为９７ ０００ 的ＴＫ 和β半乳糖苷酶（ Ｍｒ５５ ０００） 融合蛋白。薄层扫描显示，该融合蛋白含量占菌体蛋白总量的２７ ．４７ ％ 。     

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.     

Identification of structural protein-protein interactions of herpes simplex virus type 1

In this study we have defined protein-protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument-tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566-9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35-pUL37 (capsid-tegument), pUL46-pUL37 (tegument-tegument) and pUL49 (VP22)-pUS9 (tegument-envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs.     

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 UL 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 (UL27-UL33) encoded near the middle of UL. 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 UL. A high degree of divergence was also observed in proteins coded at the segment ends including RL1 (gamma(1)34.5), RL2 (alpha0), UL1 (glycoprotein L), UL56, U(S)1, and U(S)12. It is suggested that conserved proteins UL27-UL33 are encoded near the middle of UL 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.     

Acute ascending necrotizing myelitis in Okinawa caused by herpes simplex virus type 2

Summary A case of rapidly progressing ascending myelitis was necropsied. Necrosis was present throughout the whole length of the spinal cord and involved both the grey and white matter randomly. The perivascular lymphocytic infiltration in the spinal cord in the present case was more pronounced than that in the previously reported two cases of necrotizing myelopathy associated with malignancy. Using immunoperoxidase staining the presence of herpes simplex virus type 2 (HSV 2) antigen was demonstrated. Electron microscopic examinations revealed large numbers of HSV particles in the spinal cord. HSV 2 may be a common aetiological agent of necrotizing myelopathy and myelitis in Okinawa, an HSV 2 endemic area. In the present case, the necrosis was mainly found in the spinal cord but was also observed, to a very limited extent, in the brain.     

Application of shRNA-containing herpes simplex virus type 1 (HSV-1)-based gene therapy for HSV-2-induced genital herpes

HSV-1-based vectors have been widely used to achieve targeted delivery of genes into the nervous system. In the current study, we aim to use shRNA-containing HSV-1-based gene delivery system for the therapy of HSV-2 infection. Guinea pigs were infected intravaginally with HSV-2 and scored daily for 100 days for the severity of vaginal disease. HSV-2 shRNA-containing HSV-1 was applied intravaginally daily between 8 and 14 days after HSV-2 challenge. Delivery of HSV-2 shRNA-containing HSV-1 had no effect on the onset of disease and acute virus shedding in animals, but resulted in a significant reduction in both the cumulative recurrent lesion days and the number of days with recurrent disease. Around half of the animals in the HSV-2 shRNA group did not develop recurrent disease 100 days post HSV-2 infection. In conclusion, HSV-2 shRNA-containing HSV-1 particles are effective in reducing the recurrence of genital herpes caused by HSV-2.     

Immunization with a replication-defective herpes simplex virus 2 mutant reduces herpes simplex virus 1 infection and prevents ocular disease

Ocular infections with herpes simplex virus 1 can lead to corneal scarring and blindness, with herpes keratitis being the major infectious cause of blindness. There is currently no clinically approved vaccine and nearly all developmental vaccines are targeted against HSV-2 and genital herpes. We tested the ability of an HSV-2 replication-defective virus, a genital herpes vaccine candidate, to protect against HSV-1 corneal infection. Immunization with HSV-2 dl5-29 reduced viral replication in the cornea, prevented ocular disease and reduced latent infection by the HSV-1 strain. Therefore, this HSV-2 replication-defective mutant strain may have applications for prevention of herpes keratitis and genital herpes due to HSV-1 infection.     

Development and pathogenic evaluation of recombinant herpes simplex virus type 1 expressing two fluorescent reporter genes from different lytic promoters

To develop means to explore viral gene expression in ganglia without laborious histological sectioning and staining, we created a two color fluorescent recombinant HSV-1, in which enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) are expressed from the glycoprotein B (gB) and glycoprotein C (gC) promoters respectively. We show that this virus retained growth and pathogenic capacity both in vitro and in vivo compared to wild type HSV-1; established latent infections with similar genome copy number in trigeminal ganglia (TG); induced a similar HSV-specific CD8⁺ T cell infiltrate; did not induce CD8⁺ T cells reactive to EGFP or RFP; and reactivated from latency with normal kinetics in ex vivo TG cultures. Fluorescent EGFP expression in plaques surrounding neurons preceded RFP expression and provided highly sensitive detection of reactivation and different stages of infection in ex vivo TG cultures. Expression of both EGFP and RFP in neurons was readily detectable in whole mounts of TG excised during acute infection and following invivo sodium butyrate-induced reactivation from latency. This virus constitutes a useful reagent for monitoring lytic viral promoter activity in sensory neurons in vivo and in vitro.     

单纯疱疹病毒性脑炎的脑电图分析

目的探讨脑电图（ＥＥＧ）对单纯疱疹病毒性脑炎（ＨＳＥ）提供诊断依据和评价疾病严重程度、疗效和预后的意义。方法：对４２例ＨＳＥ的ＥＥＧ行回顾性分析。结果：４２例中３７例ＥＥＧ异常,早期敏感性为８２％。结论：ＨＳＥ之ＥＥＧ早期敏感性高,对诊断有指导作用,可作为推断本病严重程度及治疗效果的依据之一