7例水痘患者水痘带状疱疹病毒的分离与鉴定分析

目的对7例临床水痘患者的疱疹液样本,进行水痘带状疱疹病毒(varicella-herpes zoster virus,VZV)的分离及鉴定分析。方法对7例临床水痘患者的疱疹液,用2BS细胞进行病毒分离及病毒滴度检测;用特异性引物分别对病毒分离株及疫苗株(Oka株)的ORF68、ORF54、ORF38进行聚合酶链反应(Polymerase Chain Reaction,PCR)扩增及测序,用DNAMAN软件对病毒分离株的ORF68序列与Dumas株序列进行比对鉴定,并对病毒分离株和Oka株的ORF54、ORF38进行限制性片段长度多态性(restriction fragment length polymorphism,RFLP)分析。结果从7例临床水痘患者的疱疹液样本中,分离到4株VZV病毒株;病毒分离株的细胞病变(cytopathic effect,CPE)及病毒滴度随传代次数的增加而增强;4株病毒分离株的ORF68序列与Dumas株完全一致;ORF54、ORF38的RFLP结果显示,4株病毒分离株均为BglⅠ+PstⅠ+型,Oka株为BglⅠ+PstⅠ-型。结论成功分离的4株病毒分离株均为VZV野生毒株。     

水痘-带状疱疹病毒的基因分型与分子进化

水痘-带状疱疹病毒(Varicella-zoster virus,VZV)又称人类疱疹病毒3型,属疱疹病毒科,与单纯疱疹病毒HSV-1、HSV-2一起归入α亚科。人类是其唯一的自然宿主,对其普遍易感。VZV引起的原发感染表现为水痘,并在宿主的感觉神经节内潜伏,再激活时可引起带状疱疹。近年来VZV分子流行病学的研究涉及流行病学、病毒学、生物信息学等相关领域,通过监测、研究VZV的基因变异,区分疫苗株或野生株引起的感染,探讨世界范围内各VZV病毒株的系统发育关系和各遗传支之间的分子进化史。现将近年来有关VZV不同的地理分布和遗传支进化的研究状况综述如下。     

水痘-带状疱疹病毒疫苗的评价与研究进展

水痘-带状疱疹病毒(VZV)属疱疹病毒α亚科,即人类疱疹病毒3型,为双链DNA病毒。原发感染可引起具有高度传染性的全球流行性疾病——水痘;潜伏病毒的再激活感染可引发典型的疼痛性皮肤病——带状疱疹及不典型的内脏器官感染。日本和美国分别自1987年和1995年开始实行给全体儿童预防接种水痘减毒活疫苗(vOka)后,两国儿童的水痘发病率和病死率显著降低。但VZV疫苗的不良反应,包括二次传播和突破感染等时有发生,因此有必要研发更为有效、安全的新型疫苗。本文就VZV相关疫苗的有效性、安全性及其新型疫苗的研究进展进行综述。     

以BAC为基础的水痘-带状疱疹病毒的感染性克隆技术及其应用

水痘-带状疱疹病毒（VZV）属于疱疹病毒科α亚科,其原发感染为水痘,潜伏再度激活则引起带状疱疹。目前对其基因功能和疫苗的减毒机制尚不十分清楚。细菌人工染色(BAC)是一种新的用于大分子DNA克隆的载体系统,它具有容量大、遗传稳定、操作简单等优点。将VZV全基因组克隆至BAC系统构建成VZV的感染性克隆,并利用现代基因修饰技术可极大促进对该病毒的研究。就近年来以BAC为基础VZV感染性克隆技术的建立和应用做一综述。     

利用接种人体组织的SCID小鼠研究疱疹病毒的发病机理

人细胞巨化病毒(HCMV)和带状疱疹病毒(VZV)属于疱疹病毒家族。对于有免疫力的宿主,HCMV很少引起疾病症状,但对于有免疫损害和成长中的胎儿,HCMV是引起感染性疾病和死亡的主要病因。VZV感染可引起水痘和带状疱疹。由于这些种属特异性疱疹病毒不能感染其他动物,没有动物模型可用于发病机理的研究。移植了人免疫组织的严重联合免疫缺陷小鼠(SCID-hu)为这项研究提供了一个有价值的模型。我们用HCMV或VZV感染SCID-hu以调查在人胎儿胸腺/肝脏组织的发病机理。HCMV临床分离株能在SCID-hu小鼠的移植组织中复制达到较高的滴度。然而…     

疫苗株病毒引起1例成人水痘的病原分离与鉴定

本文旨在探讨1例由疫苗株病毒引起成人水痘的病例,以提高对水痘疫苗二次传播的认识。从1名23岁女性水痘患者皮肤水疱中采集水疱液,接种至人胚胎成纤维细胞,3d后观察到细胞发生病变效应。用水痘-带状疱疹病毒gE`糖蛋白单克隆抗体间接免疫荧光法鉴定,结果阳性。测序分析显示,分离到的毒株其疫苗相关的单核苷酸多态性位点与Oka疫苗株一致,为疫苗株病毒。回顾性调查显示,患者没有水痘史和疫苗接种史,病毒可能来自1名与患者直接接触的接种疫苗后发生带状疱疹的儿童。     

水痘—带状疱疹病毒分离株核衣壳的形态特征

本文用超萍切片电镜技术对水痘－带状疱疹病毒（VZV）分离株J1的核衣壳进行了形态学研究。结果表明,在病毒感染后5小时即可观察到细胞核内大量的病毒核心相关颗粒和少量核衣壳。在细胞核内和细胞浆内均可见到病毒基质或毒浆结构。VZVJ1株具有三种类型的核衣壳,命名为A型、B型、C型核衣壳。A型具有电子致密核心,B型的核心呈颗粒状,C型具有电子透明核心。三种核衣壳大小一致,直径75－100nm,核心为35－55nm。将VZV的核衣壳与疱疹病毒科其它成员作了比较分析,并对各种核衣壳在病毒成熟过程中的作用进行了探讨。     

10株水痘带状疱疹病毒的分离鉴定与生物学特性

用Vero-E6细胞从12例水痘及带状疱疹病人水疱液中分离到10株病毒,分离阳性率为83.3%.10株病毒均具有使感染细胞圆缩、融合、脱落等典型的VZV局灶性细胞病变(CPE)特点,CPE随着传代次数增加而加快.用带状疱疹恢复期病人血清作间接免疫荧光染色镜检,可见典型的感染细胞核内荧光块.10株病毒感染细胞制成的抗原片,检测5例带状疱疹病人急性期及恢复期血清,荧光抗体滴度均有4倍以上增高.VZV对Vero细胞敏感;对沙鼠肾,胎兔肾,胎豚鼠肾、肺、脾原代单层细胞不敏感;对乳小白鼠不敏感.毒种在-20℃保存一周内死亡;但在30%脱脂牛奶、20%小牛血清及10%山梨醇Eagle's液中,液体或真空冷冻干燥-100℃可保存二年以上.     

水痘一带状疱疹病毒分离株核衣壳的形态特征

本文用超薄切片电镜技术对水痘一带状疱疹病毒(VZV)分离株J_1的核衣壳进行了形态学研究。结果表明,在病毒感染后5小时即可观察到细胞核内大量的病毒核心相关颗粒和少量核衣壳。在细胞核内和细胞浆内均可见到病毒基质或毒浆结构。VZVJ_1株具有三种类型的核衣壳,命名为A型、B型、C型核衣壳。A型具有电子致密核心,B型的核心呈颗粒状,C型具有电子透明核心。三种核衣壳大小一致,直径75—100nm,核心为35—55nm。将VZV的核衣壳与疱疹病毒科其它成员作了比较分析,并对各种核衣壳在病毒成熟过程中的作用进行了探讨。     

水痘-带状疱疹病毒ORF7蛋白原核表达及单抗制备

水痘-带状疱疹病毒（Varicella-zoster virus,VZV）减毒活疫苗株（V-Oka）为混合毒株,仍具备感染和潜伏神经系统的能力,VZV第7个开放阅读框（Open reading frame7,ORF7）嗜神经因子敲除的毒株有望成为更安全的VZV减毒活疫苗株,本文重组表达VZV ORF7蛋白,制备其单克隆抗体（Monoclonal antibody,McAb）,建立ORF7荧光抗体检测方法,为新一代ORF7敲除的VZV减毒疫苗的检测提供方法学基础。本研究以V-Oka株orf7为扩增模板,构建重组表达质粒pET-28a-ORF7,转化至E.coli BL21(DE3),IPTG诱导表达后,镍离子柱亲合层析纯化表达蛋白,WB鉴定重组蛋白的活性,免疫BALB/c小鼠,应用杂交瘤技术制备抗VZV ORF7单克隆抗体,对单抗进行鉴定,选取一株稳定、特异、高效的单抗进行纯化并标记异硫氰酸荧光素（Fluorescein Isothiocyanate,FITC）。结果显示,重组ORF7蛋白以包涵体形式表达,分子量约29 kD,亲和纯化后纯度约为92.0%,盐酸胍复性蛋白可与VZV全病毒免...     

Detection of herpes simplex and varicella zoster viruses in clinical specimens using direct immunofluorescence and cell culture assays

Patients (33 in toto) with a clinical diagnosis of herpes infections (simplex, zoster or chickenpox) were investigated for the presence of herpes simplex virus (HSV) and varicella zoster virus (VZV) in skin samples, using direct immunofluorescence and cell culture assays. Five patients with nonherpetic vesiculobullous disorders were included as negative controls. Of the 33 patients, nineteen (57.6%) were positive for HSV or VZV and fourteen (42.4%) were negative. Five controls were all negative for HSV or VZV. Of the nineteen positive patients, HSV was isolated from eight (42.1%) patients, by both direct immunofluorescence and cell culture assays. VZV was isolated from eleven (57.9%) patients, eleven (100%) by direct immunofluorescence assay, and six (54.5%) by cell culture assays. HSV was isolated from one patient clinically diagnosed as chickenpox (VZV), but otherwise the positive laboratory results were concordant with the clinical diagnosis. For epidemiological studies, atypical cases and immunocompromised patients the clinical diagnosis should be confirmed in the laboratory.     

Analysis of immune function in herpes zoster patients: demonstration and characterization of suppressor cells

We sought to identify imbalances of immune regulatory cells that might contribute to the depression of cell-mediated immunity that occurs during an episode of herpes zoster. Peripheral blood mononuclear cells (PBMC) were obtained from patients with herpes zoster during the acute (less than 7 days after disease onset) and convalescent (more than 10 days after disease onset) phases of illness and from healthy seropositive donors. The PBMC were analyzed for: lymphoproliferative responses to varicella-zoster virus (VZV) antigens, Leu-3 (helper/inducer):Leu-2 (cytotoxic/suppressor) ratios, and percentages of suppressor cells as defined by coexpression of the Leu-2 and OKM1 antigens. Significantly depressed proliferative responses of VZV antigens and Leu-3:Leu-2 ratios, and increased percentages of Leu-2+ OKM1+ suppressor cells were observed in PBMC of acute phase herpes zoster patients as compared with the PBMC of convalescent patients or healthy donors. These differences were also observed in individual patients sequentially studied during both phases of disease. Cryopreserved acute phase PBMC suppressed the proliferative response of autologous convalescent phase PBMC to VZV antigens, but not to herpes simplex virus (HSV) antigens. The acute phase PBMC suppressor cell was radiation sensitive and was identified as a Leu-2+ cell by fluorescence-activated cell sorting. Thus, depression of cell-mediated immunity during the acute phase of herpes zoster was associated with a relative increase of lymphocytes expressing a suppressor cell phenotype and the activation of a radiosensitive Leu-2+ suppressor cell with some degree of antigen specificity.     

Mechanisms of varicella-zoster virus neuropathogenesis in human dorsal root ganglia

Varicella-zoster virus (VZV) is a human alphaherpesvirus that infects sensory ganglia and reactivates from latency to cause herpes zoster. VZV replication was examined in human dorsal root ganglion (DRG) xenografts in mice with severe combined immunodeficiency using multiscale correlative immunofluorescence and electron microscopy. These experiments showed the presence of VZV genomic DNA, viral proteins, and virion production in both neurons and satellite cells within DRG. Furthermore, the multiscale analysis of VZV-host cell interactions revealed virus-induced cell-cell fusion and polykaryon formation between neurons and satellite cells during VZV replication in DRG in vivo. Satellite cell infection and polykaryon formation in neuron-satellite cell complexes provide mechanisms to amplify VZV entry into neuronal cell bodies, which is necessary for VZV transfer to skin in the affected dermatome during herpes zoster. These mechanisms of VZV neuropathogenesis help to account for the often severe neurologic consequences of herpes zoster.     

Herpes Zoster Risk Reduction through Exposure to Chickenpox Patients: A Systematic Multidisciplinary Review

Varicella-zoster virus (VZV) causes chickenpox and may subsequently reactivate to cause herpes zoster later in life. The exogenous boosting hypothesis states that re-exposure to circulating VZV can inhibit VZV reactivation and consequently also herpes zoster in VZV-immune individuals. Using this hypothesis, mathematical models predicted widespread chickenpox vaccination to increase herpes zoster incidence over more than 30 years. Some countries have postponed universal chickenpox vaccination, at least partially based on this prediction. After a systematic search and selection procedure, we analyzed different types of exogenous boosting studies. We graded 13 observational studies on herpes zoster incidence after widespread chickenpox vaccination, 4 longitudinal studies on VZV immunity after re-exposure, 9 epidemiological risk factor studies, 7 mathematical modeling studies as well as 7 other studies. We conclude that exogenous boosting exists, although not for all persons, nor in all situations. Its magnitude is yet to be determined adequately in any study field.     

Upregulation of CXCL10 in human dorsal root ganglia during experimental and natural varicella-zoster virus infection

Varicella-zoster virus (VZV) reactivation causes herpes zoster, which is accompanied by an influx of lymphocytes into affected ganglia, but the stimulus for this infiltrate is not known. We report that VZV infection of ganglia leads to increased CXCL10 production in vitro, in an explant ganglion model and in naturally infected dorsal root ganglia (DRG) during herpes zoster. Lymphocytes expressing the receptor for CXCL10, CXCR3, were also observed throughout naturally infected ganglia during herpes zoster, including immediately adjacent to neurons. This study identifies VZV-induced CXCL10 as a potential driver of T lymphocyte recruitment into DRG during herpes zoster.     

Genome-Wide Analysis of T Cell Responses during Acute and Latent Simian Varicella Virus Infections in Rhesus Macaques

Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.     

Evaluation of Chosen Cytokine Levels among Patients with Herpes Zoster as Ability to Provide Immune Response

Aim and Background

Herpes zoster is a viral disease caused by the reactivation of varicella–zoster virus (VZV) which remained latent in the cranial nerve or dorsal root ganglia. Cell-mediated immunity is known to decline with age as part of immunosenescence and can lead to the reactivation of VZV. Whereas herpes zoster is usually mild in healthy young persons, older patients are at increased risk for complications. In the present study we investigated the serum cytokine profile (IL-17, IL-23, IL-21, IL-4, IL-12), representing cellular and humoral immunity and assessed the level of VZV IgG antibodies in patients with herpes zoster.

Methods

We investigated the serum concentrations of IL-17, IL-23, IL-21, IL-4, IL-12 and the level of VZV IgG antibodies in 23 patients with herpes zoster who did not develop superinfection. The control group was represented by 21 individuals in similar age with no inflammatory and infectious diseases. Cytokine and antibodies levels were measured by ELISA method. Statistical analysis was performed using the ROC curve (receiver operating characteristic), t-test, Welch’s t-test, and nonparametric tests with STATISTICA 10 software.

Results

In patients with herpes zoster, the serum level of IL-17, IL-23, IL-21, IL-4 and IL-12 as well as VZV IgG antibodies titer were statistically significantly increased compared to control group.

Conclusion

Our results confirm the broad activation of the immune system involving humoral and cell-mediated immunity.     

Varicella-zoster virus in actively spreading segmental vitiligo skin: Pathological,immunochemical, and ultrastructural findings (a first and preliminary study)

Segmental vitiligo (SV) is a unilateral subtype of vitiligo which is clinically characterized by a cutaneous depigmentation and histologically by a melanocyte loss from the epidermis and hair follicle reservoirs. To date, its pathogenesis remains a mystery. In many cases, this skin depigmentation shares several clinical features and dysfunctions with herpes zoster (HZ). So, for the first time, we examined whether any nucleus and cell fusion associated with a positive immunolabelling of varicella-zoster virus (VZV) and VZV mature virions could be found in SV skin samples as in herpes zoster (HZ). A total of 40 SV samples were used for histological and immunochemical studies. Control samples were obtained from three HZ, and 10 generalized vitiligo lesions. For ultrastructural study, three recent SV and one HZ as controls were recruited. Here, we report that nuclear fusion in epidermal cells were statistically associated with recent SV (p < .001), whereas syncytia formation was associated with long-lasting SV (p = .001). A positive detection of VZV antigen was statistically associated in the epidermis with recent SV and in the dermis with long-lasting SV (p = .001). Finally, the discovery of mature virions in 3/3 recent SV samples provides additional arguments for our viral hypothesis.     

Successful infection of the common marmoset (Callithrix jacchus) with human varicella-zoster virus.

The common marmoset, Callithrix jacchus, can be infected with human varicella-zoster virus (VZV), both wild-type strain KMcC and attenuated vaccine strain Oka/Merck. Infection was accomplished with either whole-cell-associated or cell extract VZV by combined oral-nasal-conjunctival application and was characterized by substantial and persistent anti-VZV antibody responses. The infectivity of VZV for marmosets was destroyed by treatment of inocula with heat or UV light. Diluted inocula with as few as 40 PFU/ml were infectious for marmosets. The lungs were demonstrated to be a major site of viral replication; both the presence of viral antigens and signs of pneumonia were demonstrated in lung tissues. Four serial passages of VZV KMcC were carried out in C. jacchus by a process of in vitro isolation and culturing of VZV from infected lung tissue and reapplication of the cultured isolates to fresh animals. The isolated viruses were identified as VZV both serologically and by restriction endonuclease analyses. The C. jacchus infectivity model should prove useful for determining the efficacy of subunit and live recombinant VZV vaccines as well as for the study of zoster.