聚合酶链反应(PCR)检测可疑HSK患者外周血及角膜上皮单纯疱疹病毒DNA

1　材料和方法1.1　材料　正常对照组20例,临床可疑单纯疱疹性角膜炎(HSK)患者50例(均为我院眼科中心住院或门诊患者).1外周血采集:肘静脉血2mL,取血清50μL备检.2角膜上皮细胞采集:用棉签擦拭病变处角膜上皮细胞(含泪液成分),置入含1.5mL生理盐水的Eppendorf管中,待检.试剂:选用上海prormega公司提供的检测人单纯疱疹病毒(HSV-)试剂盒.本技术用HSV-DNA基因高保守区设计的3条引物,扩增片段分别为469bp和391bp,采用PCR检测其特异性DNA片段.1.2　方法　1DNA模板制备:外周血标本:取50μL裂解液于离心管中,加入20μL待检血清,100℃煮…     

Persistence in herpes simplex virus infections.

Diseases of man caused by the virus of herpes simplex fall into two broad categories. The primary disease occurs only once in any individual''s life and is caused by transmission of virus from an already infected human. Thereafter, the individual may be subject to recurrent herpetic disease, the manifestations of which are different from the primary disease. Recurrent disease varies in severity from trivial, to incapacitating and frankly lethal (as in diseases resulting from the virus''s neurotropic and oncogenic properties). The source of the virus in recurrent herpetic disease has never been conclusively resolved, but is almost certainly endogenous to the patient. Theories, case reports and experiments exist to show that endogenous virus may, in periods of clinical quiescence, be latent (or persistent) at the site of the recurrent lesions itself, or more remotely in nerve tissues related to the site of recurrence.     

干扰素治疗单疱病毒性角膜炎

【目的】探讨干扰素对单疱病毒性角膜炎的疗效。【方法】用干扰素给治疗组（30例）患者分别行结膜下、同侧耳前淋巴结区、同侧腋下淋巴结区注射与用聚肌胞对对照组（28例）患者行同样部位注射，进行疗效砒。【结果】治愈率：治疗组30例中，治愈26例，治愈率86．6％；对照组28例中，治愈18例，治愈率64．2％，两组差别有显著性（P〈0．05）。复发率：随访18～24个月，治疗组有效30例中，复发1例，复发率3．33％，对照组有效25例中，复发5例，复发率20．0％，两组差别有显著性（P〈0．05）。【结论】干扰素治疗单疱病毒性角膜炎具有治愈率高和复发率低的优点。     

Comparison of nested-polymerase chain reaction and virus culture for the diagnosis of genital herpes simplex virus infection

INTRODUCTION: This study was performed to compare nested-polymerase chain reaction (PCR) with viral culture as a diagnostic tool for genital herpes simplex virus (HSV) infection in a sexually transmitted infection (STI) clinic in Singapore. METHODS: 103 consecutive patients presenting with clinical features suggestive of genital herpes were enrolled in the study. Two swabs were obtained from each patient. On one swab, cell culture and typing was performed, and on the second swab, nested-PCR was performed and the infecting viral type was determined by using type-specific primers. RESULTS: 63 patients (61.2 percent) had a positive PCR test for HSV. Of these, 13 patients (20.6 percent) had HSV type 1 (HSV-1), 50 patients (79.4 percent) had HSV type 2 (HSV-2). HSV-1 and HSV-2 were detected by viral culture in only six patients and 24 patients, respectively. The sensitivity of cell culture compared to PCR was 46.1 percent for HSV-1 infection and 48 percent for HSV-2 infection. PCR further detected an additional 52.4 percent of HSV cases. The specificity of PCR was 100 percent. CONCLUSION: Nested-PCR has been shown in this study to be an effective diagnostic and typing method for HSV infection in a STI clinic in Singapore with its higher sensitivity and specificity to routine viral isolation in cell culture.     

单纯疱疹病毒性角膜炎对角膜表层上皮细胞的影响

目的:通过活体共聚焦显微镜观察单纯疱疹病毒性角膜炎(HSK)患者角膜表层上皮细胞变化及其与角膜神经知觉关系的相关性。方法:诊断单眼HSK的非急性期复查患者50例(100只眼),和50例未患过HSK健康体检者(100只眼)。行角膜知觉、活体共聚焦显微镜检查并对共焦显微镜图像进行分析、记录。结果:HSK患者患眼角膜表层上皮细胞数为1287.78±102.54cells/mm~2,其密度显著低于对侧眼角膜表层上皮细胞数的2425.18±202.13cells/mm~2和健康对照者角膜表层上皮细胞数2435.23±224.3cells/mm~2(P<0.05)。HSK患者患眼角膜表层上皮细胞面积为606.62±198.23mm~2,较对侧眼角膜表层上皮细胞面积415.36±58.13mm~2和健康对照者角膜表层上皮细胞面积409.21±63.20mm~2,显著增大,差异有统计学意义(P<0.05)。HSK患者患眼角膜知觉正常8例(8只眼),角膜知觉轻度异常34例(34只眼),角膜知觉重度异常8例(8只眼),角膜知觉度与HSK患者的角膜表层上皮细胞密度呈负相关(r=-0.90,P<0.05),与HSK患者的角膜表层上皮细胞面积呈正相关(r=0.81,P<0.05)。结论:HSK导致角膜表层上皮细胞密度降低,通过细胞体积增大来代偿,这些改变与角膜神经功能改变密切相关。     

单纯疱疹病毒潜伏和激活机制研究进展

单纯疱疹病毒（HSV,包括HSV-1和HSV-2）是引起多种疾病的重要病原,通常这些疾病具有反复发作和无法根治的特点。HSV在外周黏膜组织表面进行增殖式感染后,可进入感觉神经元并建立无复制的潜伏感染。潜伏的病毒还可以通过激活的方式重新进入复制周期,并回到外周组织进行复发感染。这种既能通过潜伏逃避宿主免疫攻击又能在激活过程中传播的能力是该病毒高超的生存策略,也是当前任何药物无法彻底消灭该病毒的根本原因。虽然HSV潜伏与激活的研究众多,且不断有新的研究进展,但由于潜伏和激活过程本身的复杂性和研究模型的局限性,其中很多具体过程相关机制依然不明确,甚至常有争议。本文重点梳理HSV-1潜伏和激活的主要研究成果,并讨论其发展趋势。     

单纯疱疹病毒感染诊断方法及标本选择的探讨

目的目的探讨单纯疱疹病毒（HSV）感染诊断方法及标本的选择对诊断结果的意义。方法选择117例疑似HSV感染的皮肤科门诊患者或神经内科脑炎脑膜炎患者血清，其中53例有明显疱疹症状者同时取疱液标本，78例脑炎脑膜炎患者同时采集脑脊液标本。应用ELISA法、免疫印迹法检测血清、脑脊液中HSV—IgM抗体，应用间接免疫荧光法（IIF）检测脑脊液、疱疹液HSV抗原。结果血清、脑脊液标本ELISA法和免疫印迹法检测HSV—IgM抗体阳性率分别为70．94％、6．41％和59．83％、3．85％。脑脊液、疱疹液TIF法检测HSV抗原阳性率分别是15．38％、67．93％。结论ELISA法与免疫印迹法检测HSV—IgM抗体阳性率无显著性差异（γ=0．017，P〉0．5）；脑脊液标本供检测HSV抗原与HSVIgM抗体的阳性率差异显著（γ=13．224，P〈0．05．）；对复发感染患者采用疱疹液抗原检测相对于采用血清检测抗体对诊断HSV感染更具有临床意义。在临床上，对不同病症病程的HSV感染，选择相应适宜的诊断方法及标本种类可取得更准确的检测结果。     

Characteristics of a macrophage culture persistently infected with herpes simplex virus type 1.

BACKGROUND: Persistence of herpes simplex type 1 (HSV-1) has been reported in sensory neurons, corneal epithelium, and lymphocytes, although other cell types such as macrophages should also be considered as hosts for HSV-1 persistence. Here we report the establishment and characterization of HSV-1 persistence in an immortalized murine macrophage-like cell line (P388D1). METHODS: The persistently HSV-1 infected culture (P388D1per) was obtained from surviving P388D1 macrophages infected with HSV-1 MP strain at multiplicity of 0.001. P388D1per was characterized by [corrected] extracellular production of viruses, cells expressing viral antigens, and cells releasing infectious viruses. Viral plaque size and cytophatic effect were determined in viruses (HSVA and HSVB) obtained from two different P388D1per passages. Host and viral proteins were detected in P388D1per and in P388D1 cells infected with HSV-1 by metabolic [35S]-methionine labeling assays. RESULTS: P388D1per culture was characterized [corrected] by cyclic production of infectious viruses from non-detectable to 10(6) TCID50/mL, [corrected] from 1.0 to 15.0% cells expressing viral antigens and macrophages released infectious viruses from 0.008 to 12.5%. Differences in viral plaque size and cytopathic effect morphology between HSVA, HSVB and HSV-1 were observed. Similar patterns of viral proteins were observed in P388D1per and in P388D1 infected with HSV-1. Nonetheless, the characteristic interference effect of HSV-1 on host protein synthesis was not observed in P388D1per culture. CONCLUSIONS: An HSV-1 persistently infected immortalized macrophage culture was established and characterized. Virus produced during persistence showed phenotypic alterations with respect to the original virus. P388D1per cell protein synthesis was not affected by the presence of HSV-1.     

宫颈单纯疱疹病毒感染的检测和分型

单纯疱疹病毒(HSV)分HSV-1和HSV-2两个血清型,两型病毒均能引起宫颈慢性炎症,并具有反复发作和致癌的特性,可对孕妇及围产儿造成严重后果．而且两型病毒的生物学特征及其引起的生殖器疱疹的病程和预后却不同．因此,对HSV感染的快速检测和准确分型,有助于生殖器疱疹的诊断、预后及流行病学研究．我们已建立了用于HSV检     

HSV-2感染ECV304的形态学变化

目的观察人脐静脉内皮细胞（ECV304）受到单纯疱疹病毒2型（HSV-2）感染后的连续病变过程及形态学特征。方法采用ECV304传代培养,接种HSV-2后用相差显微镜、组织染色观察贴壁生长细胞的病变全过程。结果接种HSV-2后,ECV304于1d逐渐出现细胞肿胀、变圆、胞浆颗粒增多粗大;2d逐渐出现细胞融合,细胞核着色变浅。结论HSV-2接种后在ECV304出现的形态学变化,造成细胞死亡的主要形式是细胞坏死,未见明显的细胞凋亡现象。     

宫颈活检组织中单纯疱疹病毒2型(HSV-2)抗原的检测意义

目的　探讨宫颈癌与HSV 2型感染的关系。方法　免疫组化法、ELISA。结果　用免疫组化法对 37例宫颈癌病理标本 ,10例宫颈不典型增生标本和 2 5例慢性宫颈炎标本进行HSV 2抗原的检测。宫颈癌组中有 32例检出HSV 2抗原 ,不典型增生组和慢性宫颈炎组分别有 7份和 9份检出HSV 2抗原。三组的抗原检出率分别是 86 4 %、70 0 0 %和 36 0 0 %。三组间相比较 ,宫颈癌组与慢性宫颈炎组之间 ,不典型增生组与慢性宫颈炎组之间 ,结果均有显著性差异 (p <0 .0 5 ) ,宫颈癌组与不典型增生组抗原阳性率比较 ,差异无显著性 (p >0 .0 5 )。结论　HSV 2感染与宫颈癌有密切关系。     

单纯疱疹病毒Ⅰ型胸苷激酶基因及更昔洛韦系统对S-D大鼠的毒副作用

旨探讨将单纯疱疹病毒Ⅰ型胸苷激酶基因载体生产细胞（pLTKcSN／VPC）脑内注射和腹腔注射更昔洛韦（GCV）在大鼠体内的急性系统性毒副作用，以及载体生产细胞（VPC）在大鼠脑内的存在时问。S-D大鼠24只，雌雄各半，随机分为2组。第1组颅内注射pLTKcSN／VPC，细胞总量3ｘ106/只，7天后腹腔注射GCV30mg·kg-1·d-1，共7天。GCV用药结束后1周处死动物，进行备脏器组织学检查及TK序列的聚合酶链反应（PCR）和原位杂交检测。第2组，采用脑内注射等量整合有β半乳糖苷酶基因（β-gal／nVPC），注射后的9、14、21和30天各处死3只动物，取注射针道周围脑组织行冰冻切片x-gal组化染色，观察VPC在大鼠脑内的生存时限。结果：第1组动物的各脏器组织学病理改变主要为脑内手术部位蛛网膜下腔出血、个别动物有心肌间质灶性炎症和肝细胞脂肪变性。TK序列聚合酶链反应（PCR）检测动物体内各脏器均未检出TK序列。x-gal组化染色见9天和21天两个时问点分别有1只动物脑内有阳性细胞存在。本文结果提示：pLTKcSN／VPC及GCV系统对大鼠脑及全身各脏器有轻微非特异性急性毒副作用，pLTKcSN／VPC在大鼠体内的生存时间约为3周。为pLTKCSN／VPC及GCV系统的临床试验治疗恶性脑胶质瘤提供了脑内注射PLTK。SN／VPC的安全性依据。     

Frequency of asymptomatic shedding of herpes simplex virus in women with genital herpes

Twenty-seven women with recurrent genital herpes simplex virus infection underwent daily home culturing to detect asymptomatic genital herpes simplex virus shedding. Asymptomatic herpes simplex virus shedding was documented on 1% of the days on which cultures were obtained. Asymptomatic shedding from the vulva was as frequent as asymptomatic cervicovaginal shedding, and 45% of asymptomatic episodes were identified only by positive results from vulvar cultures. All women who obtained samples on more than 100 days and 80% of women who obtained samples on more than 50 days had documented asymptomatic viral shedding, compared with only 6% of those who obtained samples for fewer than 25 days. Asymptomatic shedding was not related to contraceptive use or menstrual cycle. These data suggest that all women with recurrent genital herpes simplex virus infection should be instructed about the possible risk, albeit infrequent, of asymptomatically shedding virus from the genital tract.