Expression of Early and Late Adenoviral Proteins in Fatal Adenovirus Bronchopneumonia

The localization and distribution of three adenoviral proteins, hexon, E1 A, and 55-kDaE1B, in 16 cases of fatal adenovirus bronchopneumonia in infants and children, are described. The proteins were immunohistochemically demonstrated in paraffin sections using monoclonal antibodies followed by the avidin-biotin-peroxidase method. The hexon antigen was present in inclusion-bearing bronchial, bronchiolar, and alveolar cells, mainly in the so-called rosette cells, as well as in necrotic debris in necrotizing areas. E1A antigen was also recognized in cells with nuclear inclusions where the reaction decorated the inclusion, nuclear chromatin, and cytoplasm but distributed mainly in alveolar cells and to a lesser extent in bronchial and bronchiolar cells. The 55-kDa E1B protein was extensively present in “activated,” reactive-appearing, nuclei of bronchial, bronchiolar, and alveolar epithelial cells and in the cytoplasm of rare cells having nuclear inclusions. These activated nuclei did not stain for the other two antigens. “Smudge” cells reacted poorly or not at all with any of the antibodies. The reactivity found produced a sort of complementary pattern between the hexon-positive, inclusion-containing cells and the 55-kDa E1B-positive, inclusion-noncontaining cells. The relationships of present findings and uirologic data are discussed.     

实时荧光定量PCR快速检测腺病毒方法的建立与评价

目的建立针对人腺病毒Hexon基因的实时荧光定量Taq Man PCR检测方法,用于腺病毒感染的早期诊断及病毒核酸定量分析。方法根据人腺病毒Hexon基因高度保守区设计引物和Taq Man探针,建立人腺病毒实时荧光定量Taq Man PCR快速检测方法。对方法的灵敏度及特异性进行评价;选取3个浓度的标准品,进行5次重复检测,对方法的重复性进行验证;用该方法对90份临床标本进行检测。结果建立的检测方法对人腺病毒的检测与其它呼吸道病原无交叉反应;检测灵敏度为10拷贝/μl;对103、104、105拷贝/μl 3个浓度标准品分别进行5次重复检测,其Ct值的变异系数均小于5%。对临床90份发热病例的咽拭子标本检测的腺病毒阳性率为90%,与普通PCR检测法的结果(90%)一致;检测准确率达100%。结论本研究建立的Taq Man荧光定量PCR方法的特异性、灵敏度和重复性均好,适用于人腺病毒的日常监测和暴发疫情的应急诊断。     

Human adenovirus replication and persistence in hypertrophic adenoids and palatine tonsils in children

The role of human adenovirus (HAdV) infection in different acute diseases, such as febrile exudative tonsillitis, conjunctivitis, and pharyngoconjunctival fever is well established. However, the relationships, if any, of HAdV persistence and reactivation in the development of the chronic adenotonsillar disease is not fully understood. The present paper reports a 3-year cross-sectional hospital-based study aimed at detecting and quantifying HAdV DNA and mRNA of the HAdV hexon gene in adenoid and palatine tonsil tissues and nasopharyngeal secretions (NPS) from patients with adenotonsillar hypertrophy or recurrent adenotonsillitis. HAdV C, B, and E were detectable in nearly 50% of the patients, with no association with the severity of airway obstruction, nor with the presence of recurrent tonsillitis, sleep apnea or otitis media with effusion (OME). Despite the higher rates of respiratory viral coinfections in patients with HAdV, the presence of other viruses, including DNA and RNA viruses, had no association with HAdV replication or shedding in secretions. Higher HAdV loads in adenoids showed a significant positive correlation with the presence of sleep apnea and the absence of OME. Although this study indicates that a significant proportion (~85%) of individuals with chronic adenotonsillar diseases have persistent nonproductive HAdV infection, including those by HAdV C, B, and E, epithelial and subepithelial cells in tonsils seem to be critical for HAdV C production and shedding in NPS in some patients, since viral antigen was detected in these regions by immunohistochemistry in four patients, all of which were also positive for HAdV mRNA detection.     

Human adenovirus type 7 outbreak in Police Training Center, Malaysia, 2011

In March 2011, an outbreak of acute respiratory disease was reported at the Kuala Lumpur (Malaysia) Police Training Centre. Approximately 100 trainees were hospitalized and 5 were admitted to the intensive care unit. Three of these 5 trainees died. Human adenovirus type 7 was identified as the etiologic agent.     

黄芪甲苷体外抗腺病毒作用研究

目的研究黄芪甲苷体外抗腺病毒作用。方法采用细胞病变效应(CPE)抑制实验和噻唑蓝(MTT)比色法观察黄芪甲苷对人腺病毒3型(HAdV3)的抑制作用,激光共聚焦显微镜荧光分析检测黄芪甲苷在病毒生物合成阶段对HAdV3六邻体(hexon)蛋白表达的影响。结果 CPE及MTT结果表明黄芪甲苷对腺病毒有直接灭活作用,同时能够抑制腺病毒的复制和吸附,病毒抑制率与药物浓度呈正相关,但在细胞保护作用方式下黄芪甲苷不能阻断病毒进入细胞。在生物合成阶段黄芪甲苷组与病毒对照组比较hexon蛋白的表达明显降低。结论黄芪甲苷在体外具有抗腺病毒作用,黄芪甲苷抗腺病毒作用可能与其在生物合成阶段抑制hexon蛋白的表达有关。     

重组人3型腺病毒六邻体蛋白纯化及抗原性检测

目的纯化重组人3型腺病毒六邻体蛋白并检测其抗原性。方法在原核表达系统中高效表达重组人3型腺病毒六邻体蛋白。利用Ni-NTA琼脂糖亲和层析柱进行蛋白纯化。用纯化的重组六邻体蛋白免疫新西兰家兔,利用免疫印迹技术检测其血清中抗体效价以鉴定该蛋白的免疫原性和反应原性。结果得到纯化的重组人3型腺病毒六邻体蛋白,该蛋白免疫后产生高效价抗体,该抗体能与重组六邻体蛋白、含有该蛋白的重组工程菌株以及人3型腺病毒发生特异性的免疫印迹反应,证明该重组六邻体蛋白具有良好的抗原性和3型腺病毒特异性。结论所获得的重组人3型腺病毒六邻体蛋白具有良好的抗原性和特异性,为开发基因工程疫苗奠定了基础。     

Cross-reactivity between human adenoviruses in delayed-type hypersensitivity

The aim of this study was to determine the antigen responsible for the induction of delayed-type hypersensitivity (DTH) by human adenoviruses (Ads). The estimation of DTH was based on measurement of the extent of swelling of the hind footpads of mice. CsCl density gradient-purified human Ad serotype 6 (Ad6) induced DTH in a dose-dependent manner. In Ad6-sensitized mice, DTH could be elicited by serotypes belonging to the same species of human Ads (types 1 and 5) and by a serotype (type 3) belonging to another species. Latex particles coated with purified hexon antigen prepared from Ad5 had the capacity to sensitize mice and elicit a DTH reaction. We suggest that, for serotypes belonging to species C, the cross-reactive highly conserved T cell epitope of the hexon protein might be responsible for the DTH induction, and furthermore the same epitope might result in the cross-reactivity between serotypes 3 and 6. The possible importance of these data is discussed in relation to human gene therapy through the application of Ad vectors.     

人腺病毒55型六邻体蛋白同源建模及B细胞抗原表位预测

目的 预测人腺病毒55型六邻体蛋白B细胞抗原表位.方法 使用DNAStar软件Protean模块,PSIPRED在线服务器和SWISS-MODEL在线服务器联合预测人腺病毒55型六邻体蛋白B细胞抗原表位.结果 构建了人腺病毒55型六邻体蛋白三级结构模型和预测得到5个候选的人腺病毒55型六邻体抗原表位.结论 成功预测人腺病毒55型六邻体蛋白B细胞抗原表位,为进一步研制疫苗和检测试剂奠定了基础.     

人5型腺病毒六邻体蛋白单克隆抗体的制备及鉴定

目的制备抗人5型腺病毒(HAdV5)六邻体(Hexon)蛋白的单克隆抗体并对其进行鉴定。方法用纯化的Hexon蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合,经ELISA间接法筛选和克隆化培养,获得4株分泌抗HAdV5的Hexon蛋白单克隆抗体的杂交瘤细胞。纯化获得单克隆抗体,使用ELISA和Western blot鉴定单克隆抗体的敏感性和特异性。结果 ELISA和Western blot结果表明这4株单克隆抗体与HAdV5的Hexon蛋白可以特异性结合。单克隆抗体株4A9-HRP标记和8D6建立的ELISA夹心法具备较高灵敏度和特异性。结论获得4株抗HAdV5的Hexon蛋白的单克隆抗体,为HAdV5相关的疫苗研制和HAdV5感染性疾病的早期快速诊断奠定了基础。