利用人支气管上皮细胞系分离和培养人博卡病毒

目的 研究人博卡病毒(HBoV)在呼吸道感染中致病性及机理.方法 利用人支气管上皮细胞系进行HBoV分离及培养,通过电镜对培养细胞中HBoV的形态及感染细胞进行研究,并采用逆转录PCR(RT-PCR)方法,检测HBoV在细胞中转录产物mRNA,同时对扩增产物进行测序及分析.结果 将含有HBoV阳性痰液标本无菌接种人支气管上皮细胞系,出现2例致使细胞发生明显的病变(CPE).电镜观察在细胞质中散在六角形或圆形的病毒粒子,大小18～26 nm之间,大部分是20nm.RT-PCR扩增产物经电泳检测与预期结果相符是295 bp,扩增产物测序结果与GenBank中HBoV序列比较,核苷酸同缘性99%,氨基酸同缘性100%.结论 HBoV感染人支气管上皮细胞后能够在该细胞内增殖,并产生特征性生物学改变.本研究为今后HBoV致病性及免疫应答谱研究奠定基础.     

儿童急性呼吸道博卡病毒感染的病毒载量与临床特征相关性研究

目的 研究急性呼吸道感染患儿人博卡病毒（ human bocavirus,H BoY）病毒载量与临床特征的相关性。方法 对2009年l1月至2010年12月间956例呼吸道感染的患儿及251例健康对照组儿童鼻咽部抽吸物、咽拭子采用PCR法进行HBoV检测,进而对阳性样本进行实时荧光定量PCR法测定博卡病毒DNA载量,并结合患儿的临床检查进行综合分析。结果 实验组与对照组HBoV阳性率存在显著差异,下呼吸道感染病例HBoV的病毒载量水平与上呼吸道感染病例及对照组儿童差异均有统计学意义,上呼吸道感染病例与对照组儿童病毒载量无统计学意义,重症下呼吸道感染患儿与普通下呼吸道感染患儿HBoV的病毒载量无统计学差异,HBoV混合感染与独立感染患儿病毒载量亦无统计学差异。结论 博卡病毒常年均可引起发病,是儿童呼吸道感染的重要病原体之一,但可能不是儿童急性呼吸道感染的唯一因素。HBoV病毒载量并不能独立反映临床疾病感染的严重程度。     

兰州地区呼吸道感染患儿中人类博卡病毒1～3型检测与临床研究

目的了解兰州地区急性呼吸道感染患儿中人博卡病毒1—3型（HBoV1～3）感染的临床及分子流行病学特征。方法收集兰州大学第一医院2009年12月至2010年11月急性呼吸道感染患儿鼻咽分泌物及咽拭子标本524份,用：巢氏PCR扩增人博卡病毒（HBoV）NS1片段,检测HBoV1～3;同时PCR检测常见呼吸道病毒。结果524份标本中检出HBoV43例,检出率为8．2％,仅次于鼻病毒、呼吸道合胞病毒、副流感病毒3型;混合感染率为69．8％。其中人博卡病毒1型（HBoV1）在下呼吸道感染中检出率显著高于上呼吸道感染的检出率;2例人博卡病毒2型（HBoV2）患儿都出现胃肠道症状,与标准株GU048662．1的核苷酸同源性分别为99％和100％;1例人博卡病毒3型（HBoV3）与标准株HM132056．1的核苷酸同源性为99％。结论本地区儿童急性呼吸道感染中博卡病毒感染以HBoV1为主,首次检出HBoV3;人博卡病毒与其他病毒有较高的合并感染。人博卡病毒是本地区儿童急性呼吸道感染的重要病原之一。     

南京95例肝病患者中3种人细小病毒感染的检测与分析

目的 建立人细小病毒B19、人博卡病毒(HBoV)及人细小病毒4(PARV4)分子检测方法,并应用于南京95例肝病患者血浆中三种人细小病毒的感染情况分析.方法 分别建立人细小病毒B19、HBoV及PARV4感染的巢式PCR方法,确定其灵敏度与特异性;并对南京市某医院95例住院肝病患者血浆中三种人细小病毒的感染情况进行检测,并对检测结果进行统计学分析.结果 建立的三种巢式PCR方法特异性好,检测灵敏度均可达10个拷贝.95例肝病患者血浆中检出B19感染2例(2.1％),HBoV感染9例(9.5％),且发现在5例药物性肝炎患者中检出HBoV阳性3例,但未检出PARV4.结论 人细小病毒B19、人博卡病毒(HBoV)及人细小病毒4(PARV4)巢式PCR检测方法灵敏特异.从肝病患者血浆中检出了B19及HBoV.     

重症急性呼吸道感染儿童中人博卡病毒的分型检测与基因进化分析

目的 了解重症急性呼吸道感染住院儿童中人博卡病毒（HBoV）的感染状况,流行病学特征及其进化特征.方法 采用巢式PCR的方法,对来自北京儿童医院重症急性呼吸道感染住院儿童的259份鼻咽抽吸物,进行人博卡病毒（HBoV）分型检测与测序,同时进行了合并感染检测、流行病学、临床特点及基因多态性分析.结果 共检出56份人博卡病毒感染阳性标本,阳性率为21.6％,[95％ CI（16.0％～27.3％）,P＜0.0001],其中2岁以下儿童感染率较高.与其他呼吸道常见病毒的合并感染率为94.6％.HBoV阳性产物测序分析发现,人博卡病毒1型占96.4％ （54/56）,2、3型各1份.HBoV阳性株分型区（VP1/VP2）序列变异不明显.结论 人博卡病毒是儿童急性呼吸道感染常见的病原体,以I型最为常见,分型区（VP1/VP2）序列较保守.HBoV在重症急性呼吸道感染儿童中是否起到真正的致病作用还需进一步的研究.     

儿童博卡病毒感染的临床特点

目的了解儿童博卡病毒(Human bocavirus,HBoV)感染的临床特征,探讨博卡病毒感染的疾病相关性。方法收集从2005年10月至2006年2月因急性呼吸道感染住院儿童鼻咽抽吸物(nasopharyngeal aspirates,NPA)148份,并采集了2份博卡病毒呼吸道样本阳性患者的血清。采用聚合酶链式反应(PCR)扩增的方法,对鼻咽抽吸物及血清标本进行了检测,并进行序列测定。结果从148份急性呼吸道鼻咽抽吸物中检出11份博卡病毒感染阳性,阳性检出率为7.4%,所获得的2例博卡感染血清核酸扩增也呈阳性。该11例博卡病毒阳性患者临床主要表现为支气管炎,支气管肺炎或肺炎症状,部分患者伴随有腹泻症状。结论博卡病毒感染患者均有下呼吸道感染表现,博卡病毒可能是引起下呼吸道感染的一个重要病原;博卡病毒血清阳性提示,博卡病毒有可能引起病毒血症;博卡病毒感染除表现下呼吸道症状外,有可能引起肠道等其他相关症状。     

博卡病毒VP_2基因在昆虫细胞sf9中表达及临床标本筛查

目的 表达人博卡病毒(HBoV)VP_2蛋白,用于临床标本HBoV的筛查.方法 将VP_2基因重组于杆状病毒基因组,重组的杆状病毒感染昆虫细胞sf9,用HA抗体进行Western Blot鉴定重组融合VP_2蛋白表达.通过电镜观察重组蛋白颗粒.以VP_2蛋白作为抗原对176例临床样本进行免疫印迹筛查.结果 在昆虫细胞中VP_2蛋白的表达量约占细胞总蛋白的60%以上,VP_2蛋白相对分子质量为60×10~3.电镜观察博卡病毒VP_2蛋白形成病毒样颗粒(VLP),其大小在20mm左右;采用免疫印迹技术从临床标本中筛查出4例患者HBoV阳性,阳性率达到2.28%(4/176).结论 本研究成功的在昆虫细胞中表达了博卡病毒VP_2蛋白;表达VP_2蛋白具有一定抗原性,为开展人博卡病血清学研究方法建立提供基础.     

人博卡病毒研究进展

2005年，瑞典的Allander等人^[1]采用宿主DNA消除、随机PCR扩增、高通量测序和生物信息学知识相结合的方法，在呼吸道感染患儿的鼻咽抽吸物中发现了一种新的细小病毒——人博卡病毒（hu．man bocavirus，HBoV）。随后，世界上许多国家陆续报道了在呼吸道感染患儿中检测出HBoV，     

上海地区人博卡病毒在儿童急性呼吸道感染的研究CSCD

目的了解上海地区人博卡病毒（HBoV）在儿童急性呼吸道感染中的流行情况和临床特点。方法上海地区在2012年1月至2012年12月共收集271例急性呼吸道感染患儿的鼻咽抽吸物,采用巢式聚合酶链反应（Nested PCR）方法检测人博卡病毒NS1基因、并经测序确认,对所获得的基因序列进行同源性和进化分析,博卡病毒阳性样本同时检测鼻病毒、呼吸道合胞病毒、腺病毒等8种呼吸道相关病毒。结果271例标本中共检出HBoV阳性31例,检出率11.4%;21例存在混合感染,全年均有检出,阳性患儿中位年龄17个月（4个月-4岁）,诊断包括上呼吸道及下呼吸道感染,临床表现包括发热、阵发性咳嗽、咳痰、腹泻、呕吐、咽充血、湿罗音等,无死亡病例,门诊患者检出率明显高于住院患者;序列分析表明其中29例为HBoV1、2例为HBoV2、与参考株的核苷酸同源性为99%-100%,氨基酸同源性96%-100%。结论HBoV1是上海地区急性呼吸道感染患儿中的重要病原,HBoV2在该地区首次检出,临床症状及诊断无特异性。     

乌鲁木齐地区腹泻患儿中人博卡病毒感染分子流行病学调查

目的 了解新疆乌鲁木齐地区腹泻患儿中人博卡病毒1～4型(HBoV1 ～4)感染的分子流行情况.方法 收集新疆维吾尔自治区人民医院2011年1月至12月住院及门诊腹泻患儿粪便标本315例,用巢氏PCR扩增入博卡病毒(HBoV) NS1片段(518 bp),检测HBoV1～4型.结果 315份标本中,HBoV总阳性检出率为8.57％ (27/315),其中HBoV1、2、3、4型分别为2例、22例、3例、0例.除XJ1378外,其他26例HBoV均与参考株的核苷酸同源性达到98％ ～ 100％,但其中3例HBoV3型与大猩猩GBoV1型(HM145750.1)核苷酸同源性为92％,且系统进化显示HBoV3型NS1片段更接近于HBoV1型.HBoV感染呈全年散发,并无明显季节性.在性别、年龄及民族间均无差异.结论 本地区腹泻患儿中HBoV1 ～3型均有流行,且以HBoV2型为主要流行株.     

Characterization of the cytopathic effect in human bronchial epithelial cell after Human Bocavirus Infection (HBoV)]

OBJECTIVE: In this study, human bronchial epithelial cells were inoculated with positive sputum specimens of HBoV. After four days' infection, cytopathic effects (CPE) were observed by inverted microscopy. These viruses all cause typical cell damages such as rounded and shrivelled, fusion and fallout. These damages got quick following increased future degenerations. The other assay result of CPE within the infected cells were observed by inverted microscopy, have typical "owl's eye" plaque and above 90 percent hemadsorption within the infected cells by erythrocytes for hemadsorption technique. The typical fluorescence lump of nucleus within the infected cells was found by indirect immunofluorescence technique. CONCLUSION: Isolation and identification of HBoV could be done in the human bronchial epithelial cell, and we found some characterizing CPE in the human bronchial epithelial cell after HBoV infection. The above studies pave a way for studying pathogenicity of human bocavirus.     

Respiratory syncytial virus infection of human primary nasal and bronchial epithelial cell cultures and bronchoalveolar macrophages.

In adults, clinical symptoms caused by respiratory syncytial virus (RSV) are usually confined to the upper respiratory tract, whereas RSV infection in infants frequently causes bronchiolitis and pneumonia. The preferential localization of RSV infection to the upper airways may partially be due to protective immunity, but may also depend on a difference in susceptibility of epithelial cells from upper and lower airways, or on antiviral activities of bronchoalveolar macrophages (AM). In this study, we have compared the susceptibility of primary adult human nasal epithelium, primary adult human bronchial epithelium, a human bronchial epithelial cell line (BEAS-2B), and adult human AM to infection with RSV. The cell cultures were infected with multiplicities of infection (moi) of 1 and 0.1. Virus release into the supernatants was assayed at days 1, 2, 4, and 7, and the percentage of virus-positive cells determined by immunofluorescence at the same time points. Similar proportions of nasal epithelial cells (NE) and bronchial epithelial cells (BE) were infected with RSV. Approximately 50 to 75% (with moi 1) and 2 to 10% (with moi 0.1) of the cells were infected by 24 h; almost all the cells were RSV positive by day 4. However, BE released less infectious RSV than do NE. With moi 0.1, 10-fold less virus was released over 4 days of culture. By days 4 to 7, cytopathic effects (CPE) were maximal in all epithelial cell cultures, but CPE developed latest in BE infected with moi 0.1. AM were also productively infected with RSV, with peak virus production at day 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

涎腺导管上皮细胞体外感染HCMV模型的建立

目的　建立涎腺导管上皮细胞体外人巨细胞病毒 (HCMV)感染模型。方法　用免疫细胞化学方法初步鉴定涎腺导管上皮细胞 (HSG)角蛋白 8(cytokeratin 8,CK8)和角蛋白 18(CK18)抗原的表达 ;观察HCMV感染HSG后导致的病变 ;观察宿主细胞中病毒颗粒的超微结构 ;用RT PCR及nest RT PCR检测HCMVIE1(即刻早期 1) /IE2基因的转录 ;用免疫细胞化学法检测HCMV感染细胞中IE1/IE2蛋白的表达。结果　呈上皮样贴壁生长的HSGCK8和CK18阳性表达 ;HCMV感染后 12小时 ( 12h .p .i.)即可见细胞病变 ,6 0h .p .i.CPE已极为明显 ,72h .p .i.绝大部分细胞出现病变。感染的HSG细胞在细胞核、内质网、细胞质中出现数量不等的 3种不同形态的疱疹病毒样颗粒。宿主细胞中可检测到HCMVIE1/IE2的转录以及IE1/IE2蛋白的表达。结论　成功建立了涎腺导管上皮细胞体外HCMV感染模型     

A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence

The species specificity of human adenoviruses (HAdV) almost precludes studying virulence and tropism in animal models, e.g. rodent models, or derived tissue and cell culture models. However, replication of HAdV type 5 (HAdV-C5) has been shown after intravenous injection in swine. In order to study adenovirus replication in airway tissue propagation of bronchial epithelial cells from porcine lungs was established. These primary cells proved to be fully permissive for HAdV-C5 infection in submerged culture, demonstrating efficient HAdV genome replication, infectious viral particle release (1.07×10(8) TCID(50)/ml±6.63×10(7)) and development of cytopathic effect (CPE). Differentiation of porcine bronchial epithelial cells was achieved at the air-liquid interface on collagen I coated 0.4μm polyester membranes. Morphology, expression of tubulin and occludin, the development of tight-junctions and cilia were similar to human bronchial epithelial cells. Infection with HAdV-C5 from the basolateral side resulted in release of infectious virus progeny (2.05×10(7) TCID(50)/ml±2.39×10(7)) to the apical surface as described recently in human bronchial epithelial cells, although complete CPE was not observed. Differentiated porcine bronchial epithelial cells hold promise as a novel method for studying the virulence and pathophysiology of pneumonia associated HAdV types.     

Persistent parainfluenza type 1 (6/94) infection of brain cells in tissue culture

Summary A state of persistent infection with parainfluenza type 1 virus (6/94 strain) was established in cultures of human and bovine brain cells. Following primary infection of human brain cells, viral cytopathic effect (CPE) and hemadsorption (HAD) depended on the multiplicity of infection. After persistent infection was established the virus rapidly became cell-associated; no CPE occurred and no viral antigen was detectable by HAD, immunofluorescence (FA), or immunoprecipitation. Infectious virus could be recovered only by fusion or cocultivation. This was in marked contrast with infected bovine brain cells, where, following primary infection, little or no CPE occurred. A productive infection rapidly evolved and persisted without CPE, but with 100 per cent HAD and FA positive cells.With 6 Figures     

巨细胞病毒感染后晚期mRNA表达与细胞病变相关性的研究

目的 研究巨细胞病毒(HCMV)感染后晚期mRNA的表达与致细胞病变作用(CPE)及感染细胞的超微结构变化。方法 用HCMV AD169株感染人胚肺成纤维细胞，通过半定量RT-PCR检测HCMV晚期mRNA的表达水平，同时动态观察CPE的改变，电镜观察细胞的超微结构变化。结果 HCMV晚期mRNA在感染后12h开始表达，随时间的延长水平逐渐升高；而CPE在48h后才出现，并逐渐加重。电镜下可见内质网早期囊腔扩张，晚期呈空泡变，线粒体肿胀，线粒体嵴数目减少，甚至缺失，感染晚期细胞核中有大量成熟待出壳的病毒核衣壳。结论 细胞超微结构变化与HCMV晚期mRNA表达密切相关，在病毒循环中起重要作用。晚期mRNA可作为观察治疗。HCMV活动性感染的临床疗效指标。     

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium

Summary. Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor. Reeived December 2, 1996 Accepted March 4, 1997     

Abortive infection of human diploid cells by murine cytomegalovirus

Inoculation of human diploid cells (WI-38) with murine cytomegalovirus (MCMV) did not result in the synthesis of any infectious virions. However, morphological changes typical of the cytopathic effects (CPE) of MCMV were detectable within 12 hr of infection. The CPE included rounding, swelling, and detachment of cells. The nuclei of infected cells were enlarged, and intranuclear inclusions were visible by May Grunwald-Giemsa staining and by the indirect fluorescent-antibody test. All cells infected at a multiplicity of infection of 3 showed CPE, and these cells could not be passaged successfully. Cell lysates and exhausted media from infected WI-38 cultures did not produce any CPE in WI-38 cells. The virus absorbed to WI-38 cells with the same efficiency as to mouse embryo fibroblast cells (MEF). Samples of MCMV in which virus infectivity for MEF cells had been inactivated by ultraviolet irradiation or by exposure to 56 C failed to produce any of the above signs. MCMV-specific CPE did not occur in the presence of actinomycin D (1 mug/ml) or puromycin (20 mug/ml), but 5'-fluoro-2'-deoxyuridine at 1 x 10(-4)m did not prevent CPE or the development of intranuclear inclusions.     

Poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing

The replication of type 1 poliovirus in 13 established human blood cell lines differing in the differentiation stage and cell lineage was investigated. Three T (CCRF-CEM, CCRF-HSB-2, and Molt-3) and three B (Raji, CCRF-SB, and RPMI 8226) cell lines showed no cytopathic effects (CPE) or virus production. CPE associated with virus production were detected in the other seven cell lines: HL-60, ML-1, and KG-1 (granulocytic lineage), U-937 and THP-1 (monocytic lineage), K-562 (erythroid lineage), and Molt-4 (T cell lineage). These susceptible cell lines greatly differed in the speed at which the CPE progressed. The progression of CPE was faster in relatively well-differentiated cell lines such as HL-60 and U-937, independently of the multiplicity of infection, than in less differentiated cell lines such as K-562, KG-1, and THP-1. Thus, for the same lineage, the speed at which CPE progressed became proportionally higher with subsequent differentiation stages. In the K-562 cell culture, CPE were not observed until at least 5 days postinfection (p.i.), while more than 80% of HL-60 cells were killed within 3 days p.i. There were no significant differences between infected HL-60 and K-562 cells in the efficiency of infection determined at 8 hr p.i. by the indirect immunofluorescent technique, the rate of virus growth, or the amount of viral capsid protein synthesized. This indicated that there were similar viral replication cycles in the two cell lines. These observations suggest that the killing function of the virus is expressed more slowly in K-562 cells than in HL-60 cells.