关键词： Randomized controlled trial   Papillomavirus vaccines   Diphtheria-tetanus-acellular pertussis-inactivated poliovirus vaccine   Adolescent   Female   Immunology   Adverse effects  

摘要： Purpose: Many countries recommend human papillomavirus (HPV) vaccination in female adolescents at an age when other vaccines are routinely administered. This open, randomized, multicenter study (108464/NCT00426361) evaluated coadministration of HPV-16/18 AS04-adjuvanted vaccine with diphtheria-tetanus-acellular pertussis-inactivated poliovirus vaccine (dTpa-IPV). Methods: Healthy females aged 10-18 years were randomized to receive HPV vaccine at months 0, 1, and 6 (n = 248), HPV vaccine coadministered with dTpa-IPV at month 0 and HPV vaccine at months 1 and 6 (n = 255), or dTpa-IPV at month 0 followed by HPV vaccine at months 1, 2, and 7 (n= 248). Immunogenicity was evaluated at months 0, 1, and 7 or 8 (depending on group). Vaccine reactogenicity and safety were also assessed. Results: Coadministered dTpa-IPV and HPV vaccine was noninferior to dTpa-IPV alone in terms of seroprotection against diphtheria (99.2% and 100%), tetanus (100% and 100%) and poliovirus types 1, 2, and 3 (>= 99.6%), and geometric mean antibody concentrations (ELISA Units/mL) for pertussis toxoid (84 vs. 75), filamentous hemagglutinin (612 and 615) and pertactin (426 and 360) at month 1. Coadministered dTpa-IPV and HPV vaccine was noninferior to HPV vaccine alone in terms of sero-conversion rates for HPV-16 (99.5% and 100%) and HPV-18 (99.5% and 100%) and geometric mean antibody titers (ELISA Units/mL) for HPV-16 (15,608 and 18,965) and HPV-18 (6,597 and 6,902) at month 7. Coadministration was generally well tolerated. The reactogenicity of dTpa-IPV and the first dose of HPV vaccine was similar. Conclusions: Results from this study support coadministration of the HPV-16/18 AS04-adjuvanted vaccine with dTpa-IPV vaccine in females aged 10-18 years. (C) 2010 Society for Adolescent Medicine. All rights reserved.

关键词： MICROBIAL ecology   RESEARCH   VIRUS diseases -- Prevention   VIRUSES   POLIOVIRUS   CELL culture   FLOW cytometry   PLAQUE assay technique   RESEARCH -- Methodology   IMMUNOSPECIFICITY  

摘要： The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2A(pro)) as an indication of infection. Quantification of the infectious-virus titers was resolved by using flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2Apro were infected with different titers of PV1. After incubation at various time points, cells were harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.

关键词： T cells   LYMPHOCYTES   POLIO   VACCINATION   ENTEROVIRUSES   PREVENTIVE medicine   IMMUNIZATION   COMMUNICABLE diseases -- Prevention   GESTATIONAL age  

摘要： Background. The antigen-specific T cell responses of preterm infants to immunization are not well understood. The aim of the present study was to compare the T cell responses of preterm infants after inactivated poliovirus vaccination with those of term infants. Methods. We prospectively enrolled 2-month-old preterm (gestational age, <= 33 weeks) and term (gestational age, >= 37 weeks) infants to receive 3 doses of diphtheria-tetanus toxoids-acellular pertussis-hepatitis B virus-inactivated poliovirus vaccine. Whole blood and peripheral blood mononuclear cells (PBMCs) were stimulated with poliovirus vaccine, and memory T cell activation was analyzed by flow cytometry and lymphoproliferation, respectively. Levels of poliovirus neutralizing antibodies were measured in serum. Results. We enrolled 33 preterm and 50 term infants. Preterm infants had fewer circulating CD4(+)CD45RO(+) memory (P=.005) and CD4(+)CD69(+)IFN-gamma(+) cells activated by staphylococcus enterotoxin B at 2 (P=.015) and 7 (P=.05) months of age. After immunization, preterm and term infants had comparable frequencies of poliovirus-specific CD4(+)CD45RO(+)CD69(+)IFN-gamma(+) memory T cells (P=.79). PBMCs from preterm infants had diminished poliovirus-specific lymphoproliferation (P<.001). Although all infants developed seroprotective poliovirus antibody titers, serotype 1 titers were lower among preterm infants (P=.03). Conclusions. Preterm infants develop poliovirus-specific T cell responses that are comparable to those of term infants. However, they demonstrate nonspecific and poliovirus-specific functional T cell limitations, suggesting that investigations into whether T cell differences remain as preterm infants mature are warranted.

关键词： Enterovirus 2B   Viroporin   Protein-lipid interaction   Pore-forming peptide   Viral protein-induced apoptosis  

摘要： Non-structural poliovirus 2B protein induces plasma membrane permeabilization and has been recently implicated in triggering apoptosis via the mitochondrial pathway. Here we describe that the pore-forming P3 peptide, based on the 2B amphipathic domain, translocates through the plasma membrane of culture cells and targets mitochondria. Cell permeabilization by P3 versions of different lengths, together with peptide uptake analyses supported an internalization mechanism dependent on P3 capacity to interact physically with lipid bilayers and establish permeating pores therein. Internalized P3 was found associated with mitochondria, but contrary to the parental 2B protein, the short peptide did not affect the morphology or cell distribution of these organelles, nor induced apoptosis. We conclude that P3 constitutes a mitochondriotropic sequence, which is however devoid of 2B pro-apoptotic activity. (C) 2009 Elsevier B.V. All rights reserved.

关键词： Poliovirus   RNA-dependent RNA polymerase   3Dpol   Mutation rate   3AB  

摘要： The fidelity of poliovirus RNA-dependent RNA polymerase (3Dpol) was determined using a modified alpha-complementation assay. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. Various conditions including high and low MgCl2, replacing MgCl2 with MnCl2, skewed nucleotide pools, and the presence of poliovirus protein 3AB were analyzed. The assay included RNA synthesis by 3Dpol on an RNA template that coded for a region of the alpha peptide of beta-galactosidase (lacZ-alpha). The product of this reaction was used as a template for a second round of 3Dpol synthesis and the resulting RNA was reverse transcribed to DNA by reverse transcriptase. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on lacZ-alpha. Although 3AB strongly stimulated 3Dpol synthesis as expected, no change in fidelity was detected. Changes in MgCl2 also showed little effect. Mutation rates of similar to 9 x 10(-5) (similar to 1 error per 11,000 Incorporations) were estimated for these conditions. In contrast, MnCl2 or skewed nucleotide pools were highly mutagenic resulting in lowered fidelity. (c) 2009 Elsevier B.V. All rights reserved.

关键词： enteroviruses   integrated cell culture quantitative PCR   UV disinfection  

摘要： This study demonstrated the applicability of integrated cell culture-quantitative RTPCR (ICC-qRTPCR) for the simultaneous quantification of coxsackievirus, echovirus, and poliovirus in disinfection studies. Buffalo green monkey cells were inoculated with a 10-fold dilution series of mixed enteroviruses and incubated prior to qRTPCR quantification. Optimal assay conditions included three post infection washes and a 24-hour post infection incubation period based on successful differentiation between infectious and noninfectious viruses and significant and consistent viral replication rates. Ultraviolet disinfection studies were performed to validate the ICC-qRTPCR assay. Using the optimized assay, three-log microbial inactivation was achieved at UV doses of 30-44, 28-42, and 28-29 mJ/cm(2) for coxsackievirus B6, echovirus 12, and poliovirus 1, respectively. These results compare favorably to side-by-side assessments using conventional cultural techniques and values previously reported in the literature. This indicates that ICC-qRTPCR is a practical alternative for the simultaneous quantification of enteroviruses in disinfection studies.

关键词： Ⅰ型脊髓灰质炎病毒   VP1编码区基因特征   实时荧光定量   逆转录-聚合酶链反应   型内鉴定  

摘要： 脊髓灰质炎(脊灰)是主要发生于儿童的一种急性传染病,可以通过脊灰病毒灭活疫苗(Inactivated Poliovirus vaccine, IPV)或口服脊灰减毒活疫苗(Oral Poliomyelitis Attenuated Vaccine, OPV)来预防。全球消灭脊灰计划开始于1988年,1999年时全球Ⅱ型脊灰野病毒的传播已被阻断；截止2009年底,除阿富汗、印度、尼日利亚、巴基斯坦四个国家外Ⅰ型和Ⅲ型本土脊灰野病毒的传播也已被阻断。 中国自1995年以来未发现本土脊灰野病毒,2000年包括中国在内的世界卫生组织(WHO)西太平洋地区(WPRO)宣布进入无脊灰状态,中国目前仍使用OPV维持无脊灰状态。OPV株在人类肠道中复制时能发生变异,一些变异可以导致强神经毒力的回复,引起疫苗相关的脊髓灰质炎(VAPP)。未免疫人群会受到服苗者排泄到外环境中的变异的疫苗病毒感染,而造成其循环,在人群中循环到一定程度(VP1编码区核苷酸变异率≥1%),就称为脊髓灰质炎疫苗衍生病毒(VDPV)。而且,尽管中国目前处于无脊灰状态,但由于Ⅰ型脊灰野病毒仍在个别国家和地区流行,因此仍存在脊灰野病毒输入的危险。为了研究工型脊灰病毒(PV)的基因特征,为我国维持无脊灰状态提供依据,本文对中国2009年急性弛缓性麻痹(Acute Flaccid Paralysis, AFP)病例监测系统中分离到的57株Ⅰ型PV的VP1编码区采用逆转录-聚合酶链反应(Reverse Transcription Polymerase Chain Reaction, RT-PCR)法进行扩增,并对PCR产物进行核苷酸序列测定和分析,未发现PV野毒株或疫苗衍生PV；但发现5株高变异株,其中分离于两例AFP病例的两株高变异株的核苷酸序列同源性100%。同时57株Ⅰ型PV的VP1区序列比对结果表明,两个核苷酸(nt2747和nt2749)是突变热点。根据江西省和湖北省两株高变异株的流行病学及实验室检测结果,不能排除它们之间发生了短时间的循环,有待于深入研究。同时,突变热点的存在提示毒株受到选择压力的影响,容易回复突变成野生型,从而带来一系列的神经毒力等表型方面的变化。 采用世界卫生组织推荐美国疾病预防与控制中心研发的实时荧光定量逆转录(Reverse Transcription, RT) PCR法对57株Ⅰ型PV中6株代表性毒株进行型内鉴定和疫苗衍生脊灰病毒(Vaccine-derived Polioviruses, VDPVs)筛选,并将检测结果与毒株的VP1区核苷酸序列测定结果进行比较分析。结果显示,实时荧光定量RT-PCR无法鉴别6株病毒中的5株Pre-VDPVs,作为WHO推荐使用的新型型内鉴定方法,实时荧光定量RT-PCR法是否适用于中国PLN (poliomyelitis laboratory network)还有待大样本脊灰病毒实时荧光定量RT-PCR法的回顾性和前瞻性研究。

关键词： 原发性免疫缺陷病   脊髓灰质炎病毒   肛门周围脓肿  

摘要： 研究背景 截至2009年底,全球共报告41例免疫缺陷相关的疫苗衍生脊髓灰质炎(脊灰)病毒(Immunodeficiency-associated Vaccine-Derived Poliovirus, iVDPV)患者。病例主要来自中、高收入国家,但近年来不断在中、低收入国家发现新的病例。2005年安徽省发现了我国首例iVDPVs病例。为评估原发性免疫缺陷病(Primary Immunodeficiency, PID)患者排出VDPV的风险,近年来多国在PID患者中开展了排出脊灰病毒的筛查,结果显示这类人群排出脊灰病毒的风险非常低。但由于均是在中、高收入国家开展筛查工作,这些国家已多年未使用口服脊灰减毒活疫苗(Oral Poliomyelitis Attenuated Live Vaccine, OPV),因此在使用OPV的中、低收入国家开展此类筛查将更有意义。 研究目的 调查PID患者携带VDPV的状况,确定排毒持续时间； 调查肛门周围(肛周)脓肿患儿免疫状况,以及脊灰病毒携带状况。 研究方法 本研究采用现况调查的方法对PID人群中排出脊灰病毒的状况进行研究。通过治疗PID的专科医院、在医院中就诊的反复发作肛周脓肿的患儿和急性弛缓性麻痹(Acute Flaccid Paralysis,AFP)病例监测系统报告的脊灰病毒阳性的病例等三个途径搜集到的确诊和疑似PID患者,按照标准对粪便标本检测脊灰病毒,了解排泄脊灰病毒型别和持续时间。 研究结果 本调查通过专科医院共搜索到的PID确诊病例153例,在这些病例中未检测出VDPV病例,仅检测出非脊灰肠道病毒(non-polio enterovirus, NPEV)7例,脊灰病毒阳性的3例,其中1例VP1区与疫苗病毒相比有5个核苷酸变异。 筛查到肛周脓肿病例587例,其中IgG, IgA, IgM3项检测中有2项低于标准值的疑似PID病例有57例,占10.03%,其中未发现排出脊灰病毒。 调查山东省63例脊灰病毒分离阳性的AFP病例,未发现PID患者。 结论 通过在我国PID人群中开展排出脊灰病毒情况的调查,尚未发现排出VDPV的病例,仅发现个别病例排出脊灰疫苗病毒和NPEV;在肛周脓肿患者和脊灰病毒分离阳性的AFP病例中也未发现确诊的PID患者。

关键词： 口蹄疫病毒   脊髓灰质炎病毒   牛腹泻病毒   病毒基因组复制   持续性感染   氯化铵   单细胞实时荧光定量RT-PCR  

摘要： 正链RNA病毒(Positive-stranded RNA viruses)是以RNA为遗传物质且病毒RNA可直接作为mRNA翻译产生病毒蛋白的一类病毒,其中许多成员是严重威胁与损害人类健康的病原体,或是造成经济动物患病死亡的致病因子,如丙型肝炎病毒、脊髓灰质炎病毒、SARS病毒、登革热病毒、口蹄疫病毒、牛病毒性腹泻-粘膜病病毒(简称牛腹泻病毒)等。目前,对这些危害较大的病毒基本上都缺乏快速有效的防治方法。病毒基因组的复制是病毒生存和繁殖的基础,是病毒生命周期不可或缺的重要环节,所以研究单股正链RNA病毒基因组的复制是防止相关致病因子(病原体)蔓延与传播的基础。 口蹄疫病毒(Foot-and-mouth disease virus,FMDV)是一种单股正链RNA病毒,以负链RNA作为中间体进行复制,感染敏感细胞系(如仓鼠肾细胞系BHK-21或猪肾细胞系PK-15)会引发急性感染。为了更好地研究病毒感染,需要检测单个急性感染细胞中病毒RNA量而不仅仅检测群体细胞的病毒量。在本研究中,我们建立了一种链特异性的单细胞实时荧光定量RT-PCR技术,其技术包括显微操作分离单细胞、单细胞裂解与高灵敏度的实时荧光定量RT-PCR三部分。该技术用于检测FMDV基因组RNA,总计224个急性感染FMDV的单细胞样品被分离并进行定量检测,其中185个样品为阳性(阳性率为82.6%),阳性细胞中病毒载量从几十至几十万拷贝数不等,最高达约1000000拷贝数/细胞。结果表明,在FMDV感染过程中不是每个细胞都被病毒感染上,且群体细胞中单个细胞的病毒载量存在很大的差异。该技术实现了在单细胞水平上研究病毒的复制及其与宿主细胞的关系,为阐明病毒的复制机制和进化奠定了基础,也为病毒感染细胞生物学的深入研究提供一种新的方法。另外,分离细胞和裂解细胞的技术具有广泛适用性、能用于任何其他细胞(悬浮细胞与贴壁细胞)的分离与预处理,为在单细胞水平上进行的各种研究奠定基础。 病毒基因组RNA起始复制的方式有两种：非引物依赖和引物依赖,口蹄疫病毒(FMDV)和脊髓灰质炎病毒(PV)属引物依赖,牛腹泻病毒(BVDV)是以非引物依赖起始复制。在本研究中,单细胞实时荧光定量RT-PCR技术被用于检测FMDV、PV、BVDV正链RNA与负链RNA以研究正链RNA病毒的复制规律。PV感染Vero细胞和FMDV感染BHK-21细胞、PK-15细胞的单细胞定量数据(+RNA与-RNA的对数)都具有较好的线性相关性,经过数学模型的推导分析发现一条负链可能是对应合成一条正链,只是起始+RNA量和用于合成负链的正链比例上存在一定的差异。而BVDV感染BKC细胞的单细胞定量数据表明,+RNA与-RNA的对数基本不具线性相关性,建模初步分析结果为一条负链可能是对应合成一条正链,但起始+RNA量和用于合成负链的正链比例上与FMDV和PV差异非常大。 弱碱(如NH4Cl)进入酸性的胞内体中提高与细胞内吞作用相关的细胞器的pH值,阻止依赖于低pH值的蛋白质构象变化从而影响病毒基因组释放,降低病毒感染后的增殖量。本研究用NH4Cl维持培养感染FMDV的BHK-21细胞系,存活细胞经显微操作单细胞分离、单细胞克隆培养传代,共获得17株阳性克隆细胞株。随机选取一株阳性细胞(BHK-Op)常规传代,采用实时荧光定量RT-PCR,透射电子显微镜,western blot等方法研究病毒持感细胞特性。BHK-Op48细胞切片电镜观察,病毒粒子存在于细胞质中弥散的小囊泡结构内(每一个小囊泡内仅有几个病毒粒子),直径20-25nm,不同于急性感染细胞中病毒粒子的分布,后者主要位于近核膜的囊泡中(囊泡中存在大量病毒粒子)；持感病毒感染其敏感细胞不能使之形成蚀斑但TCID50实验结果表明其毒力较野生型病毒强；序列比对结果显示,持感病毒基因组中有15个基因编码的蛋白突变全部位于非结构蛋白区,与之前的研究结果大相径庭,其中3C蛋白编码区两个保守位点的突变恰好位于其RNA结合结构域,与病毒复制相关,可能是其不能形成蚀斑的原因之一。体外持续感染的建立为研究病毒与细胞共进化以及病毒与受体相互作用提供有效模型。

关键词： 脊髓灰质炎病毒   二乙烯亚胺   灭活   Sabin株   佐剂  

摘要： 随着全球消灭脊髓灰质炎(脊灰)目标的临近,疫苗衍生脊灰病毒(Vaccine-derived Poliovirus, VDPV)的流行、疫苗相关麻痹型病例(Vaccine Associated Paralytic Poliomyelitis, VAPP)的发生等对无脊灰状态成果的巩固威胁很大。在消灭脊灰的最后阶段,应尽快消灭脊灰野毒株,并使用脊髓灰质炎灭活疫苗(Inactivated poliovirus vaccine, IPV)替代目前广泛使用的脊髓灰质炎减毒活疫苗(Oral poliovirus vaccine, OPV)来维持无脊灰状态,消除自然界的活病毒。考虑到疫苗生产的生物安全性等因素,WHO鼓励研制Sabin株脊髓灰质炎灭活疫苗(Sabin IPV)。 本论文旨在针对Sabin II型病毒,寻找一种与传统灭活剂-甲醛相比较,对病毒的抗原破坏更小,且更安全的病毒灭活剂；并进一步探索Sabin IPV中添加佐剂对免疫效果产生的影响,探讨Sabin IPV最适剂量和佐剂应用的意义。 本研究第一部分进行了二乙烯亚胺(BEI)对脊髓灰质炎病毒Ⅱ型Sabin株的灭活效果观察。通过在不同温度下采用4种浓度的BEI及甲醛对脊髓灰质炎病毒Ⅱ型Sabin株进行灭活,测定活病毒含量和D抗原含量,并进行病毒灭活验证试验。结果在37℃条件下,0.002 mol/L浓度的BEI能在48h内完全灭活病毒,灭活后的病毒液D抗原含量回收率为90.6%,显著高于甲醛灭活的回收率(60.6%)。经验证灭活彻底,无活病毒存在。BEI对脊髓灰质炎病毒Ⅱ型Sabin株的D抗原破坏小,能较好的保持疫苗的有效成分。 本研究的第二部分四组不同剂量的Sabin IPV疫苗与佐剂Al(OH)3配伍后免疫Wistar大鼠,通过检测各组大鼠免疫后血清中和抗体水平、特异性IFN-γ的产生情况以及免疫后各组大鼠CD3/CD4/CD8分子表达的差异,以确定使用Al(OH)3佐剂时Sabin IPV的最适剂量。通过微量中和实验检测Sabin株脊髓灰质炎特异性中和抗体发现,疫苗不同剂量产生的中和抗体GMT水平基本是抗原含量高的组高于抗原含量低的组,添加Al(OH)3佐剂组的中和抗体水平也是高于未添加佐剂组的中和抗体水平。ELISPOT测定针对Sabin株脊髓灰质炎病毒的特异性IFN-γ的结果显示,添加佐剂实验组产生的斑点要多于未添加佐剂组,2-①实验组三个型别所产生的斑点数都最多。流式细胞仪检测各组大鼠CD3/CD4/CD8分子表达差异的结果显示,1-①实验组CD3+CD4+/CD3+CD8+比值最高。综上所述,添加佐剂对三个型别的SabinIPV的免疫反应有增强的效果；综合实验结果可认为Ⅰ型采用30 DU/dose,Ⅱ型采用16 DU/dose,Ⅲ型22.5 DU/dose剂量时较为适宜。