白黎芦醇体外抑制水痘-带状疱疹病毒作用机制的研究

目的 应用构建的水痘-带状疱疹病毒(VZV)报告细胞系MV9G进一步研究白黎芦醇体外抑制VZV的作用机制.方法 将无细胞VZV直接感染MV9G细胞(CFVs直接感染)或将带细胞VZV与MV9G细胞共培养(CAVs共培养)以激发MV9G细胞表达报告基因萤火虫荧光素酶.在CFVs直接感染前或CAVs共培养不同时间点加入白黎芦醇,通过比较药物对CFVs或CAVs激发荧光素酶的抑制强度分析白黎芦醇直接灭活病毒、抑制病毒黏附和穿透、抑制病毒在细胞内复制及其时间点和可逆性:通过比较药物作用前后VZV即刻早期蛋白62(IE62)mRNA拷贝数和IE62表达强度变化分析白黎芦醇对IE62转录和表达的抑制作用.结果 白黎芦醇＞30.0 μg/ml时MV9G细胞三磷酸腺苷(ATPs)含量随药物浓度升高而逐渐降低,ATPs降低50％时白黎芦醇浓度(CD.)约60.3μg/ml.CFVs与白黎芦醇(25.0μg/ml)预混37℃水浴孵育2h后直接感染MV9G细胞,CFVs激发荧光素酶下降50％.MV9G细胞在含白黎芦醇培养基中37℃孵育2h后直接感染CFVs,CFVs激发荧光素酶随药物浓度升高而逐渐降低,但4℃孵育对无显著变化.在CAVs共培养中加入白黎芦醇后CAVs激发荧光素酶显著降低,药物抑制荧光素酶50％时浓度(IC.)约8.7 μg/ml.分别在CAVs共培养3、6、9、12、24、30和36 h时加入白黎芦醇,3～24h加药各组CAVs激发荧光素酶均显著高于对照组,但1h、3D h和36 h加药组与对照组间差异无统计学意义.CAVs共培养时撤除白黎芦醇后CAVs激发荧光素酶显著高于撤药前,尤以24h和72 h撤药组明显.VZV IE62 mRNA拷贝数和IE62抗体阳性细胞数随药物作用时间延长而逐渐降低.结论 白黎芦醇细胞毒性较强,MV9G细胞可耐受最高浓度为30.0μg/ml.白黎芦醇部分灭活CFVs、抑制CFVs穿透MV9G细胞但对CFVs黏附MV9G细胞无影响,以浓度依赖方式可逆性抑制CAVs细胞内复制.白黎芦醇可能通过抑制1E62基因的转录和表达而抑制VZV感染的早期阶段.     

水痘-带状疱疹病毒疫苗株ORF62碱基突变及ORF62编码IE62反式激活力的分析

目的 分析水痘-带状疱疹病毒(VZV)疫苗株(vOka)ORF62碱基突变及ORF62编码即刻早期蛋白(IE62)对VZV基因启动子的反式激活力.方法 分别以vOka及其亲本株(pOka)基因组DNA为模板,PCR扩增获得ORF62全序列,克隆到高效表达质粒pCAGGS中构建vOka和pOka ORF62表达质粒;采用双脱氧核苷酸链末端终止法对表达质粒中ORF62测序;应用基因转染瞬时表达技术比较vOka和pOka IE62在MeWo和CV1细胞中对VZV基因启动子的反式激活力差别.结果 成功构建vOka和pOka ORF62表达质粒pCAG-vOka62和pCAG-pOka62;测序结果发现,与pOka比较,vOka ORF62至少有9个碱基突变,其中106262、107136和107262位点碱基突变后分别产生Sma Ⅰ、BssH Ⅱ和Nae Ⅰ酶切位点.vOka IE62在MeWo细胞中对ORF4、ORF10和ORF61启动子的反式激活力低于pOka IE62,但在CV1细胞中对ORF9启动子的反式激活力高于poka IE62.结论 利用vOka ORF62碱基突变产生的酶切位点可鉴别vOka和pOka,vOka和poka IE62对VZV基因启动子的反式激活力差别可能与转染细胞类型有关.     

青藤碱抗肝癌作用机制研究北大核心CSCD

目的:探讨青藤碱对Hep G2人肝癌细胞株的增生和转移能力的多靶向作用机制。方法:运用噻唑蓝还原法(MTT)来检测青藤碱对Hep G2人肝癌细胞株的抗增殖作用,而同时运用Transwell法来检测药物对于细胞转移能力的作用变化;间接荧光标记法测定经过不同浓度的青藤碱作用后,细胞内活性氧水平的变化;利用酶抑制动力学方法,研究青藤碱对逆转录酶的抑制作用;再通过逆转录聚合酶链式反应和蛋白质印迹的实验来检测Hep G2细胞中凋亡蛋白水平的变化。结果:青藤碱对Hep G2人肝癌细胞株具有抗侵袭及转移的治疗作用。MTT检测结果显示青藤碱对于Hep G2人肝癌细胞抗增殖作用明显,半抑制浓度IC50为(15.35±2.43)μmol/L;而Transwell法的结果显示青藤碱对癌细胞有较好的抗转移能力;青藤碱是一种有效的逆转录酶抑制剂,IC50为(21.32±2.43)μmol/L;荧光标记法检测细胞内活性氧水平随青藤碱呈浓度依赖性升高;蛋白水平测定显示青藤碱上调Hep G2人肝癌细胞CASP3、CASP9、CAV1和下调SOX2的表达。结论:青藤碱可能是通过上调CASP3、CASP9、CAV1和下调SOX2的表达,进而抑制人肝癌细胞的增殖和转移,调节相关信号通路,最终在分子水平证明青藤碱对Hep G2人肝癌细胞的抑制作用。     

miR-21在白藜芦醇诱导A549肺癌细胞株凋亡中的作用

目的探讨白藜芦醇对A549肺癌细胞株凋亡的影响,并揭示miR-21参与其中的机制。方法以高糖DMEM培养基培养A549肺癌细胞株,给予100μmol/L的白藜芦醇,过表达(或不过表达)miR-21培养3 d。Real-time PCR检测miR-21表达;MTT法检测细胞活力;Hoechst染色观察细胞凋亡;Western blot检测细胞凋亡因子4(programmed cell death 4,PDCD4)的表达。结果白藜芦醇能抑制A549肺癌细胞株miR-21的合成,上调PDCD4的表达,抑制细胞生长,降低细胞活力,诱导A549细胞凋亡(P0.05)。过表达miR-21能显著抑制白藜芦醇的抗肿瘤作用,PDCD4表达变化不明显,细胞生长状态、活力以及凋亡等均无明显变化(P0.05)。结论白藜芦醇能通过抑制miR-21合成,降低PDCD4表达,诱导A549肺癌细胞株凋亡。     

清开灵注射液对登革病毒Ⅱ型的抗病毒作用研究

目的 检测清开灵注射液在细胞模型上抗登革病毒Ⅱ型(DENV-Ⅱ型)作用.方法 本实验以白纹伊蚊C6/36细胞为宿主细胞,阿昔洛韦(ACV)为阳性对照药物,通过观察细胞病变效应(CPE)和改良MTT法检测细胞存活率来测定药物的细胞毒性、药物对DENV-Ⅱ的直接灭活作用、以及药物抗DENV-Ⅱ对细胞的吸附和对DENV-Ⅱ在细胞内复制的抑制作用.结果 该药在体外对DENV-Ⅱ无直接灭活作用,也不能阻止其对细胞的吸附,但对病毒在细胞内的增殖有明显的抑制作用,且呈一定的剂量效应依赖性.结论 该药在体外有一定的抗DENV-Ⅱ感染作用.     

黄芩提取物对柯萨奇B3m病毒性心肌炎病毒复制及Fas配体表达的影响

病毒性心肌炎（MV）严重危害人类健康，现代研究证实，病毒的直接损伤与细胞介导的细胞毒作用（CMC）是MV心肌细胞损伤的主要原因.CMC致靶细胞损伤主要有两条途径——穿孔素和Fas／FasL途径。黄芩提取物具有明显的抗病毒和调节免疫的作用，但其对MV鼠心肌中柯萨奇病毒嗜心肌株CVB3m的复制及浸润细胞中Fas配体表达的影响，迄今尚未见文献报道，本研究对此作初步探讨。     

白藜芦醇对Hela细胞凋亡及Bcl-2和Fax基因表达的影响

目的观察白藜芦醇对人宫颈癌Hela细胞凋亡及Bcl-2、Fax基因表达的影响。方法实验设白藜芦醇用药组和空白对照组,应用流式细胞术检测药物作用24h后细胞凋亡和细胞周期进程情况;应用免疫组化法检测Heal细胞Bcl-2、Fax基因的表达情况。结果经不同浓度的白藜芦醇处理Heal细胞后,镜下可见到凋亡细胞,各组的凋亡率明显高于对照组(P0.01)。PCM分析发现各实验组S期细胞比例增高,G2/M期细胞比例减少,并呈剂量依赖性(P0.01);各实验组Heal细胞Bcl-2的表达均低于对照组。而Fax的表达均高于对照组(P0.01)。结论白藜芦醇对人宫颈癌Hela细胞有明显的抑制生长和促凋亡作用,凋亡途径可能与Fas表达上调、Bel-2表达下调有关。     

以分泌型荧光素酶为报告基因的单轮感染流感病毒的构建

目的构建以分泌型荧光素酶为报告基因的单轮感染流感病毒用于药物作用机制的快速鉴别。方法通过使用分泌型荧光素酶(Gluc)序列替代流感病毒HA基因,并利用表达质粒反式提供HA蛋白,借助甲型流感病毒WSN反向遗传学产毒系统,构建带有报告系统的单轮感染流感病毒。结果首轮接种单轮感染流感病毒的细胞上清中可以检测到Gluc信号,但检测不到病毒滴度,而在第二轮感染的细胞上清中检测不到Gluc信号,表明该单轮感染病毒在感染过程中能够分泌Gluc蛋白并且不能产生具有感染性的子代病毒颗粒。通过对复制动力学和时相的观察,明确了单轮感染病毒的生活周期,同时结合4种已知作用机制的抗流感药物的阻断实验结果,证明该系统可以用来快速、有效地鉴别药物的作用机制。结论成功构建了以分泌型荧光素酶为报告基因的单轮感染流感病毒,该系统可以用于抗病毒药物作用机制的快速鉴别。     

人胚胎干细胞对肝癌HepG2 细胞体外抑制作用的研究

目的:研究体外共培养情况下人胚胎干细胞H9 对肝癌HepG2 细胞的抑制作用。方法:建立人胚胎干细胞(H9) 与肝癌HepG2 细胞共培养体系,显微镜下观察胚胎干细胞对肿瘤细胞生物学行为的影响,流式细胞仪检测H9 细胞对肿瘤细胞的凋亡与周期的影响,Transwell 小室法检测H9 细胞对HepG2 细胞侵袭和迁移的影响,基因芯片分析共培养后HepG2 细胞全基因组的表达谱变化。结果:结果发现共培养过程中,肝癌HepG2 细胞生长受到抑制,随共培养时间延长,细胞数量逐渐减少,出现老化或凋亡迹象;流式细胞术检测发现HepG2 细胞凋亡率显著增加,细胞周期被阻滞于G0/ G1 期;Transwell 实验发现HepG2 细胞侵袭、迁移力均降低;基因芯片结果发现HepG2 细胞全基因组表达谱发生了显著变化,差异基因涉及多条信号通路。结论:人胚胎干细胞H9 体外对人肝癌细胞HepG2 有一定程度的抑制作用。     

Meningoradiculoneuritis due to acyclovir-resistant varicella zoster virus in an acquired immune deficiency syndrome patient

Varicella zoster virus (VZV) is recognized as one of the major viral pathogens reactivated in patients with the acquired immune deficiency syndrome (AIDS). We report the case of meningora-diculoneuritis in an AIDS patient, associated with the isolation in the cerebrospinal fluid (CSF) of a thymidine kinase (TK)-deficient, acyclovir (ACV)-resistant strain of VZV. Although the virus was sensitive in vitro to phosphonoformate (PFA), the patient did not improve during PFA therapy and finally died. Several VZV strains isolated from this patient (including two isolates from the patient's CSF) were analyzed for their TK activity and subsequently the viral TK gene was sequenced showing a major deletion leading to a truncated protein. Their susceptibility to several antiviral agents including ACV, PFA, (E)-5-(2-bro-movinyl)-2′-deoxyuridine (BVDU), 9-β-D-ara-binofuranosyladenine (vidarabine), (S)-1-(3-hy-droxy - 2 - phosphonylmethoxypropyl) cytosine (HPMPC), and (S)-9-(3-hydroxy-2-phosphonyl-methoxypropyl)adenine (HPMPA) was evaluated. All the virus strains isolated from this patient remained sensitive to HPMPA and HPMPC, pointing to the potential usefulness of these acyclic nucleoside phosphonates for the treatment of ACV-resistant VZV infections in immunocom-promised patients. © 1994 Wiley-Liss, Inc.     

Occurrence and characterization of acyclovir-resistant herpes simplex virus isolates: report on a two-year sensitivity screening survey.

For the past 2 years, a survey network was established for the screening of acyclovir (ACV)-resistant clinical isolates of herpes simplex virus (HSV). Among 889 strains tested for in vitro ACV sensitivity, 14 HSV-1 and 6 HSV-2 were resistant to ACV concentrations exceeding 3 micrograms/ml. These resistant isolates were most often obtained after prolonged ACV treatment of severely immunocompromised patients. For five patients, the emergence of ACV-resistant virus correlated with treatment failure. In particular, a decrease in the in vitro sensitivity to ACV was observed for eight successive HSV-1 isolates from one immunodeficient patient undergoing therapy. All ACV-resistant isolates were studied for their sensitivity to different antiherpetic compounds and showed various cross-sensitive and -resistant patterns. The examination of viral populations by plaque autoradiography procedures frequently revealed their heterogeneity in terms of thymidine kinase (TK) phenotype and allowed the detection of various proportions of TK-positive (TK+), TK-deficient (TKD), or TK-altered (TKA) viruses. Our data underline the importance of monitoring the emergence of drug-resistant virus during the course of antiviral therapy, and the need for the detection and characterization of TK mutants in clinical specimens. The routine examination of drug sensitivity of HSV isolates provides useful information to clinicians for the management of ACV treatment in the hope of preventing ACV-resistant mutants from becoming predominant in mixed viral populations.     

Transmission of cell-free and cell-associated HIV-1 through breast-feeding

BACKGROUND: Transmission through breast-feeding is an important cause of infant HIV-1 infections in developing countries; however, its mechanism remains largely unknown. We have explored the association between cell-free virus (CFV) and cell-associated virus (CAV) levels in breast milk (BM), as reflected by viral RNA and proviral DNA, respectively, and the risk of infant HIV-1 infection after 6 weeks postpartum. METHODS: Sixty-one HIV-positive mothers who transmitted HIV-1 by BM were matched to 61 HIV-positive nontransmitting mothers based on their infant's age at sample collection. CFV and CAV were quantified in a single milk specimen per mother preceding the infant's first HIV-positive result. RESULTS: After adjusting for maternal CD4 cell counts and disease stage, each 10-fold increase in CFV or CAV load was associated with an almost 3-fold increase in BM transmission. Whereas CAV load was predictive of transmission before and after 9 months postpartum, CFV was a significant predictor of transmission occurring only after 9 months. Phylogenetic analyses of the C2 to C5 env region showed that 85% of infants (11 of 13 infants) harboring viruses that clustered with CFV in their mother's milk were infected after 9 months postpartum. CONCLUSION: A reduction in milk CAV and CFV loads might significantly decrease HIV-1 transmission by breast-feeding.     

Susceptibility to acyclovir of herpes simplex virus isolates obtained between 1977 and 1996 in Japan

The susceptibility of genital herpes to acyclovir (ACV) in immunocompetent women was examined, as was the frequency of ACV-resistant viruses by analyzing 56 clinical isolates in Japan between 1977 and 1996. The mean susceptibilities of herpes simplex virus (HSV) type 1 and type 2 were 0.13+/-0.74 and 0.42+/-0.14 microg/ml, respectively, assessed by the 50% inhibitory concentration of plaque formation. The susceptibility to ACV of clinical isolates did not changed since 1977, and also that of nine pairs of HSV-1 and HSV-2 isolates was not affected by ACV treatment. In order to characterize the change in the virus population, the quantitation of the ACV-resistant virus in 10(4) plaque forming units (PFU) of clinical isolates was adopted. The mean frequencies of ACV-resistant viruses per 10(4) PFU for all strains of HSV-1 and HSV-2 were 0.31+/-0.41 and 9.74+/-14.83, respectively, and were not influenced by ACV treatment. Additionally, the phenotypes of ACV-resistance were not influenced by ACV treatment, and more than 90% of ACV-resistant viruses were found to be thymidine kinase-deficient. This study characterized clinical isolates with respect to ACV susceptibility as a population and the quantitative and qualitative characterization of ACV-resistant virus in the virus population of clinical isolates was also studied. The susceptibility of isolates from genital lesions, the frequency of ACV-resistant viruses, and also the phenotypic characterization of ACV-resistant viruses was maintained between 1977 and 1996, even after the introduction of ACV treatment for genital herpes in Japan.     

Surveillance network for herpes simplex virus resistance to antiviral drugs: 3-year follow-up

Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains. We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3900 isolated strains among 3357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.     

Simplified test for detecting the resistance of herpes simplex virus to acyclovir

The detection of herpes simplex virus (HSV) antigen by means of an enzyme amplified ELISA was investigated for rapid screening of acyclovir (ACV) resistance. Vero cell monolayers were inoculated in the presence of different concentrations of ACV. When cytopathic effect was present, the culture supernatants were tested by ELISA. The absorbance values were found to correlate with the results of virus yield and plaque reduction assays. The comparison between absorbance values obtained in the presence of 10 microM ACV and in the absence of drug provided the basis for a simplified sensitivity test. The use of a single ACV concentration allowed discrimination between ACV-resistant and ACV-sensitive reference strains, the detection of ACV-resistant virus mixed in the proportion of 10% with ACV-sensitive virus, and a study of the emergence of an ACV-resistant virus population in serial samples taken from experimental rabbit keratitis. The simplified susceptibility assay is a sensitive and convenient method for rapid screening of HSV resistance to ACV.     

In vitro and in vivo evaluation of a novel antiherpetic flavonoid, 4′-phenylflavone, and its synergistic actions with acyclovir

The development of therapeutic agents for preventing herpes simplex virus (HSV) infections has become urgently necessary because of the increasing incidence of this virus and its role as a cofactor in the transmission of human immunodeficiency virus infection. We have evaluated the antiviral activities of a series of natural and synthetic flavonoids and found that a synthetic flavonoid, 4'-phenylflavone, showed the highest activity against acyclovir (ACV)-sensitive and ACV-resistant strains of HSV-1, as well as HSV-2, with a selectivity index of 213, 35 and 55, respectively. Although the attachment and penetration of HSV-1 to host cells and the synthesis of viral proteins were not inhibited, the infectivity of the virus and the amount of progeny virus released were reduced by 4'-phenylflavone treatment in a dose-dependent manner. 4'-Phenylflavone plus ACV synergistically inhibited the replication of HSV-1. This flavonoid also showed efficacy in vivo and potentiated the antiherpetic effect of ACV in a mouse model of genital herpes. Our results suggest that 4'-phenylflavone might be useful as a candidate for the development of novel antiherpetic therapeutics.     

Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization.

BACKGROUND: Widespread and frequent use of acyclovir (ACV) for treatment, suppressive therapy and prophylaxis of herpes simplex virus (HSV) infections and its over the counter availability may be associated with emergence of HSV resistance. OBJECTIVES: To determine the prevalence of ACV-resistant HSV isolates in different patient groups between 1999 and 2002 in the Netherlands. STUDY DESIGN: A total of 542 isolates, 410 HSV-1 and 132 HSV-2, from 496 patients were screened for reduced susceptibility to ACV. A newly developed ELVIRA HSV screening assay was used that allowed a high throughput screening. The genotypic analysis of the HSV thymidine kinase gene was performed to identify resistance-associated mutations. RESULTS: Thirteen isolates, 8 HSV-1 and 5 HSV-2, from 10 patients (2%) were found resistant to ACV. A single ACV-resistant strain was identified among isolates from 368 immunocompetent patients (0.27%; 95% confidence interval [CI], 0.007%-1.5%), whereas in nine isolates from 128 immunocompromised patients resistant HSV was identified (7%; 95% CI, 3.26%-12.93%). The highest frequency of ACV-resistant HSV was associated with bone marrow transplantation: four patients out of 28 (14.3%) shed resistant virus. In addition, resistant virus was obtained from two HIV-positive patients, one patient with a hematological malignancy and two patients on immunosuppressive drugs. Further testing showed that none of the isolates was resistant to foscarnet. Several new mutations were identified in the thymidine kinase gene of these resistant isolates, and their effect on ACV-resistance is discussed. CONCLUSIONS: Our study shows that the prevalence of ACV resistance is low in immunocompetent patients (0.27%), whereas ACV-resistant HSV infections occur relatively frequently in immunocompromised patients (7%; P < 0.0001). This emphasizes the need for drug susceptibility monitoring of HSV infections in immunocompromised patients with persisting infections despite antiviral therapy.     

Inhibitory effect of acyclovir on the growth of Inoue-Melnick virus isolated from cerebrospinal fluid

Inoue-Melnick virus (IMV) was isolated from the cerebrospinal fluid of patients with chronic neurologic disorders. The effect of acyclovir (ACV), 9-(2-hydroxyethoxymethyl)-guanine, on the growth of IMV was studied in human diploid cell cultures. ACV markedly inhibited the multiplication of all three IMV serotypes in vitro with the same inhibitory effect. By passage of IMV in the presence of ACV in the medium, ACV-resistant mutants of three IMV serotypes were easily isolated.