cDNA探针检测柯萨奇B_3病毒RNA

应用光敏生物素标记ｃＤＮＡ探针作打点杂交检测柯萨奇Ｂ＿３病毒（ＣＶＢ３）－ＲＮＡ，并采用薄层层析扫描仪反射扫描杂交信号和计算机整合反时峰面积的手段，对所测ＣＶＢ３－ＲＮＡ进行半定量。结果显示反射峰面积与进行自身杂交的ｃＤＮＡ含量（ｒ＝０．９８１，Ｐ＜０．００２５）及与病毒稀释度（ｒ＝０．９９７，Ｐ＜０．００５）呈有显著意义的直线正相关，提了薄层层析扫描结合核酸杂交的确可对ＣＶＢ３－ＲＮＡ进行半定量分析，也可为研究其他病毒或非病毒核酸提供新的检测方法，值得推广。     

基于荧光共振能量转移技术的快速检测CA16型手足口病病原体新方法的建立及其评价

目的：基于荧光共振能量转移（FRET）技术建立一种柯萨奇病毒A组16型（CA16）手足口病病原体快速检测的新方法,并对检测效果进行评价,使其达到疾病爆发流行期间大样本量检测的要求。方法：采用SDS-PAGE凝胶电脉技术和蛋白浓度定量试剂盒测定CA16鸡卵黄抗体（CA16-IgY）纯度及蛋白水平,采用间接ELISA法检测抗体效价及特异性,采用紫外可见光谱（UV-Vis）、红外光谱（FTIR）和透射电子显微镜（TEM）等方法对所制备的金纳米粒子（AuNPs）及其生物探针（IgY-AuNPs）的尺寸、形貌和性能进行表征;基于FRET技术构建CA16检测体系,通过优化检测体系中IgY-AuNPs浓度、氯化钠（NaCl）用量和荧光恢复时间等指标,对检测方法的灵敏度、特异度和临床样本检测进行评价。结果：CA16-IgY纯度较高,抗体平均蛋白水平为12.15 mg·L^-1,抗体效价高达1：128 000,特异性良好;AuNPs及IgY-AuNPs的UV-Vis、FTIR和TEM观察结果,IgY已成功标记至AuNPs表面,提示已通过静电自组装成功制备了可以特异性识别CA16的IgY-AuNPs。基于FRET技术构建CA16检测体系,体系经优化后,确定IgY-AuNPs的最佳浓度为0.52×10^-3g·L^-1,NaCl的最佳用量为40 μL,荧光恢复最佳时间为90 min,建立的检测方法的标准曲线为I_525nm=15.452IgC-9.746,R²=0.993 2,检测限为1×10⁴ PFU·mL^-1,与临床检测金标准实时荧光定量PCR（qRT-PCR）法比较,一致率可达93.75%。结论：成功建立了CA16型手足口病病原体的快速检测新方法,可用于实验室和临床检测。     

济南市2009-2011年手足口病病原学及基因特征分析

手足口病(HFMD)是由多种肠道病毒(EV)引起的一种急性传染病.2008年5月我国将其纳入法定报告丙类传染病管理.2009年卫生部进一步要求提高其病原学检测比例.2010年山东省开始进行全年监测.本研究采用RT-PCR对2009-2011年济南地区发生的2066例HFMD患者标本进行分析,阐明重症病例其病原及基因特征.     

两种基因检测方法在病毒性心肌炎诊断中的应用

目的:阐明基因检测手段在病毒性心肌炎(VMC)病原诊断中的作用。方法:采用RT-PCR、地高辛标记CBV3 -cDNA探针对PCR扩增产物进行斑点杂交,检测血标本中的柯萨奇病毒B组病毒(CBV)。结果:RT-PCR阳性检出率:心肌炎组为48.33%.对照组4.41%(P<0.01)。斑点杂交阳性率:心肌炎组为51.67%,对照组4.41% (P<0.01)。检测CBV的两种方法阳性率无显著差异(P>0.05)。结论:两种方法特异性均高,敏感性无显著差异。     

Indications that maternal coxsackie B virus infection during pregnancy is a risk factor for childhood-onset IDDM

Summary In a population-based setting, we traced serum samples collected at time of birth from 55 mothers whose children later developed insulin-dependent diabetes (IDDM) and matched them pairwise to control subjects who gave birth at the same hospital during the same month. The sera were analysed for IgM antibodies to coxsackie B virus serotypes 2, 3 and 4 (CBV-2, 3 and 4) using a type-specific -antibody-capture radioimmunoassay. Despite a decreased power due to the close matching by time of birth we found a significantly higher frequency of CBV-3 IgM at delivery in mothers whose children later became diabetic compared to their matched control subjects. When using the presence of CBV-3 IgM as a risk factor the Mantel-Haenszel odds ratio estimate (95% confidence limits) was 2.57 (1.02; 7.31), p=0.043. For CBV-2 and CBV-4, respectively no significant difference was found between mothers of patients and control subjects. According to the odds ratio estimate for CBV-3 and the proportion of exposed mothers among patients estimated in this study the aetiological fraction for this risk determinant would be 27%. In conclusion, this study indicates that children of mothers who expressed CBV IgM at delivery are at increased risk for developing childhood onset IDDM. A fetal infection with CBV similar to rubella virus may initiate autoimmunity or cause persistent infection that may lead to progressive beta-cell destruction.Abbreviations IDDM Insulin-dependent diabetes mellitus - CBV coxsackie B virus - CBV-2,3,4 coxsackie B virus serotypes 2, 3 and 4     

汉黄芩苷对柯萨奇B3病毒诱导的病毒性心肌炎小鼠炎症反应的影响

目的:探讨汉黄芩苷对柯萨奇B3病毒(CVB3)诱导的病毒性心肌炎小鼠炎症反应的影响及其可能的调控机制。方法:用CVB3感染BALB/c小鼠构建病毒性心肌炎动物模型。取40只BALB/c小鼠将其随机分为4组:正常对照组、CVB3感染的病毒性心肌炎组、CVB3感染后给予汉黄芩苷处理的治疗组以及CVB3感染后同时给予汉黄芩苷和AKT激动剂处理的激动剂组,每组10只。于药物治疗7 d后处死各组小鼠。用HE染色检测前3组小鼠心肌组织内炎症细胞浸润情况。用ELISA检测前3组小鼠血清中白细胞介素1β(IL-1β)和IL-6含量。用Western blot实验检测前3组小鼠心脏组织中炎症因子蛋白表达水平以及AKT/NF-κB通路的活化情况。最后,通过Western blot实验检测全部4组小鼠心肺组织中AKT/NF-κB通路的活化情况。结果:与正常组相比,病毒性心肌炎小鼠心脏组织内存在大量炎症细胞浸润,而汉黄芩苷治疗显著降低CVB3病毒诱导的心脏组织炎症细胞浸润(P<0.05)。CVB3病毒感染后小鼠血清中IL-1β和IL-6含量较正常组显著升高(P<0.05),而给予汉黄芩苷治疗后小鼠血清中IL-1β和IL-6含量较病毒性心肌炎组显著降低(P<0.05)。Western blot检测结果表明,与病毒性心肌炎组相比,汉黄芩苷治疗显著降低心肌组织内炎症因子(IL-1β和IL-6)蛋白表达水平以及AKT和NF-κB蛋白的磷酸化水平(P<0.05);而给予AKT激动剂处理后,汉黄芩苷对NF-κB蛋白磷酸化水平的抑制作用显著减弱(P<0.05),且汉黄芩苷对炎症因子的下调作用也显著受到抑制(P<0.05)。结论:汉黄芩苷可通过调控AKT/NF-κB通路减轻病毒性心肌炎小鼠的炎症反应。     

Surmounting limited gene delivery into primary immune cell populations: Efficient cell type‐specific adenoviral transduction by CAR

Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication‐defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: 1614–1620] report the generation of transgenic mice that enable conditional Cre/loxP‐mediated expression of human CAR. The authors demonstrate that this R26/CAG‐CAR?1^StopF mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge.     

Coxsackie B virus IgM in children at onset of Type 1 (insulin-dependent) diabetes mellitus: evidence for IgM induction by a recent or current infection

Summary Thirty-five children with newly-diagnosed Type 1 (insulin-dependent) diabetes mellitus and their 47 siblings were investigated for the presence of IgM antibodies to Coxsackie B virus serotypes 1–5 (CBV1-5) with the aid of -antibody-capture radioimmunoassays. When a high cut-off value was used, 16 (46%) diabetic children and 16 (34%) siblings showed CBV-IgM. Of the siblings of diabetic patients positive for CBV-IgM, 11 (44%) were CBV-IgM-positive; the corresponding figure for the siblings of negative patients was five (26%). With a lower cut-off value, leading to a borderline titre, the frequency of IgM positivity increased in both the patients and siblings. When the borderline titres were included, the number of IgM-positive patients was 19 (54%) and the corresponding number of siblings was 29 (62%). Of the siblings of positive patients, 27 (93 %) showed CBV-IgM, and of the siblings of the negative patients, two (11 %) were CBV-IgM-positive. Sixteen (89 %) siblings of IgM-negative patients remained negative. Regarding the serotypes of CB V to which IgM was directed, CBV 4 was most frequent, followed by serotypes CBV 3, CBV 2, CBV 5 and CBV 1. There was a striking similarity between the individual diabetic child and his or her sibling(s) concerning this specificity of IgM. It is concluded that within most families with a newly-diagnosed diabetic child positive for CBV-IgM the same serotype(s) of the virus circulates and that the intrafamilial spread of virus is considerable. The results strongly indicate that the IgM detected was CBV-specific and caused by a recent or current CBV infection. It is highly probable that the same strain of virus was present in different members of the same family. Therefore, if diabetogenic CBV strains do in fact exist, additional factors must be of importance for the development of Type 1 diabetes in children infected with such a CBV strain but remaining non-diabetic.     

柯萨奇病毒与1型糖尿病发病关系研究进展

近年来的研究表明,1型糖尿病(T1D)的发生与人类肠道病毒的感染关系密切,尤其是桐萨奇B组病毒.一方面,病毒感染可诱发T1D,且与病毒数量、病毒株型、宿主微环境等因素密切相关.另一方面,病毒感染也能抑制T1D的发生.该文从病毒感染诱发T1D的相关因素及病毒感染抑制T1D发生的可能机制这两方面探究柯萨奇B组病毒与T1D发...