低硒与柯萨奇病毒B_(3/0)毒性突变

目的 :　体外观察低硒培养柯萨奇病毒非致病株 B3 / 0 ( CVB3 / 0 )的毒性突变。方法 :　筛选低硒胎牛血清 ,制备低硒细胞培养基 ,利用 CVB3 致病株和非致病株感染人脐静脉内皮细胞( human umbilical vein endothelial cells,HUVEC)后结局不同 ,先于体外连续传代观察的毒性突变情况 ,然后于敏感动物体内鉴定突变株的致病性。结果 :　CVB3 / 0 在低硒细胞培养液中培养的脐静脉内皮细胞 2 0次传代后 ,可出现细胞病变效应 ,该突变株接种 Balb/C小鼠后可使其心肌组织出现损伤。结论 :　 CVB3 / 0 非致病株不仅在低硒小鼠内可突变为致病株 ,在体外相对低硒 + H2 O2 培养基中培养的人脐静脉内皮细胞中也可发生。     

中药防治CVB_3病毒性心肌炎实验研究进展

CVB3是病毒性心肌炎的主要致病因素,目前西医对CVB3病毒性心肌炎的治疗尚无特效药物。近年来,中医药对CVB3的治疗取得了一定的成就。大量的动物实验表明,单味药和复方广泛用于CVB3的治疗,具有部分直接杀灭病毒、抑制病毒复制、抗氧化、保护心肌细胞,调节免疫功能以及参与心肌细胞的凋亡等。文章对近年中药防治CVB3病毒性心肌炎从实验研究的角度做一综述。     

清心抗炎饮治疗柯萨奇B病毒性心肌炎的疗效观察及护理

目的 观察清心抗炎饮治疗柯萨奇B病毒性心肌炎（简称CVB心肌炎）的效果及探讨相应的护理措施。方法 将111例患者随机分为治疗组和对照组,治疗组57例子清心抗炎饮口服并实施辨证护理,对照组54例予西药病毒唑口服及心血管病常规护理。观察1个疗程30d,比较分忻两组的疗效。结果 治疗组在临床症状及病毒学检查方面的改善明显优于对照组。结论 采用清心抗炎饮并实施辨证护理对CVB心肌炎具有良好的疗效。     

Protection of White Leghorn chickens by U.S. emergency H5 vaccination against clade 2.3.4.4 H5N2 high pathogenicity avian influenza virus

During December 2014–June 2015, the U.S. experienced a high pathogenicity avian influenza (HPAI) outbreak caused by clade 2.3.4.4 H5Nx Goose/Guangdong lineage viruses with devastating consequences for the poultry industry. Three vaccines, developed based on updating existing registered vaccines or currently licensed technologies, were evaluated for possible use: an inactivated reverse genetics H5N1 vaccine (rgH5N1) and an RNA particle vaccine (RP-H5), both containing the hemagglutinin gene of clade 2.3.4.4 strain, and a recombinant herpesvirus turkey vectored vaccine (rHVT-H5) containing the hemagglutinin gene of clade 2.2 strain. The efficacy of the three vaccines, alone or in combination, was assessed in White Leghorn chickens against clade 2.3.4.4 H5N2 HPAI virus challenge. In Study 1, single (rHVT-H5) and prime-boost (rHVT-H5 + rgH5N1 or rHVT-H5 + RP-H5) vaccination strategies protected chickens with high levels of protective immunity and significantly reduced virus shedding. In Study 2, single vaccination with either rgH5N1 or RP-H5 vaccines provided clinical protection in adult chickens and significantly reduced virus shedding. In Study 3, double rgH5N1 vaccination protected adult chickens from clinical signs and mortality when challenged 20 weeks post-boost, with high levels of long-lasting protective immunity and significantly reduced virus shedding. These studies support the use of genetically related vaccines, possibly in combination with a broad protective priming vaccine, for emergency vaccination programs against clade 2.3.4.4 H5Nx HPAI virus in young and adult layer chickens.     

鸡血藤醇提物体外抗病毒活性研究

目的 研究鸡血藤醇提物及其不同极性溶剂萃取物的体外抗柯萨奇B3病毒、单纯疱疹病毒Ⅰ型、呼吸道合胞病毒、甲型流感病毒以及乙型肝炎病毒的活性.方法 鸡血藤药材60%醇提物经石油醚、乙酸乙酯、正丁醇依次萃取后浓缩制得各不同极性溶剂萃取物,观察其药物毒性、病毒引起的细胞病变效应判断药物毒性及药物抗病毒效应.结果 鸡血藤醇提物无抗柯萨奇B3病毒和呼吸道合胞病毒活性,但具有抗甲型流感病毒、乙型肝炎病毒和单纯疱疹病毒Ⅰ型活性,且抗单纯疱疹病毒Ⅰ型效果显著.醇提物经初步萃取分离,得到5个不同极性溶剂萃取物,其中乙酸乙酯萃取物和水层留余物抗单纯疱疹病毒Ⅰ型活性最显著.结论 鸡血藤醇提物及其乙酸乙酯萃取物和水层留余物在体外对单纯疱疹病毒Ⅰ型具有明显的抑制作用,适用于抗单纯疱疹病毒Ⅰ型治疗药物的深入研究与开发.     

广枣总黄酮对CVB3病毒引起细胞凋亡的影响

目的:建立病毒性心肌炎的体外模型,用新的化学方法提取到含量较高的广枣总黄酮,研究广枣总黄酮抗病毒的效果.方法:化学法提取广枣总黄酮,高效液相法测定其含量.培养HeLa细胞和乳鼠心肌细胞,在HeLa细胞和心肌细胞水平上对柯萨奇B3病毒进行毒力滴定,并测定广枣总黄酮的药物毒性,建立体外病毒性心肌炎模型,通过细胞病变抑制实验...     

Chitosan-DNA基因免疫诱导粘膜特异性免疫应答及其抗CVB3感染作用

目的：构建新型粘膜基因疫苗，诱生CVB3VP1特异性粘膜免疫应答，探讨粘膜免疫抗CVB3感染的作用，为病毒性心肌炎的特异性防治奠定基础。方法：抽提CVB3 RNA，以RT-PCR扩增得VP1基因，插入真核表达载体pcDNA3中，构建质粒pcDNA3-VP1，以Chitosan多糖包裹形成Chitosan-DNA基因疫苗。将该质粒转染Hela细胞，观察其体外表达情况。以50辉DNA剂量的Chitosan-DNA疫苗滴鼻免疫BALB／C小鼠3次，检测CVB3特异性体液和细胞免疫应答；隔4周以5LD50活CVB3致死攻击小鼠，观察攻击后存活情况。结果：制备了直径为80-100nm的Chitosan-DNA复合物颗粒，体外转染证实Chitosan-DNA复合物中VPl的表达高于pcDNA3-VP1质粒-Lipofectamine的表达水平。该疫苗滴鼻3次免疫后，不仅诱生了高水平的IgG，而且诱生了高水平的粘膜IgA抗体，第6周抗体P/N值分别达3．5和3．2。特异性细胞免疫应答研究发现，该疫苗诱生了较强的VP1特异性CTL杀伤作用，并显著高于pcDNA3-VP1和pcDNA3组。CVB3攻击后，可保护33．3％小鼠长期存活，而pcDNA3和pcDNA3-VP1质粒滴鼻免疫对照组的平均存活天数分别为8．5和10．8天。病理学研究显示：Chitosan-DNA免疫小鼠心肌组织基本正常，而对照小鼠死亡前心肌显示大量的灶性坏死和炎性浸润。结论：Chitosan-DNA基因疫苗滴鼻免疫可诱生CVB3特异性粘膜IgA应答及CTL反应，具有一定的抗CVB感染的免疫保护力。     

A shine-dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 RNA

Translation initiation of coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. However, the details of ribosome-template recognition and subsequent translation initiation are still poorly understood. In this study, we have provided evidence to support the hypothesis that 40S ribosomal subunits bind to CVB3 RNA via basepairing with 18S rRNA in a manner analogous to that of the Shine-Dalgarno (S-D) sequence in prokaryotic systems. We also identified a new site within both the 18S rRNA and the polpyrimidine-tract sequence of the IRES that allows them to form stronger sequence complementation. All these data were obtained from in vitro translation experiments using mutant RNAs containing either an antisense IRES core sequence at the original position or site-directed mutations or deletions in the polypyrimidine tract of the IRES. The mutations significantly reduced translation efficiency but did not abolish protein synthesis, suggesting that the S-D-like sequence is essential, but not sufficient for ribosome binding. To determine how ribosomes reach the initiation codon after internal entry, we created additional mutants: when the authentic initiation codon at nucleotide (nt) 742 was mutated, a 180-nt downstream in-frame AUG codon at nt 922 is able to produce a truncated smaller protein. When this mutation was introduced into the full-length cDNA of CVB3, the derived viruses were still infectious. However, their infectivity was much weaker than that of the wild-type CVB3. In addition, when a stable stem-loop was inserted upstream of the initiation codon in the bicistronic RNA, translation was strongly inhibited. These data suggest that ribosomes reach the initiation codon from the IRES likely by scanning along the viral RNA.     

ROLE OF COXSACKIEVIRUS AND ADENOVIRUS RECEPTOR （CAR） IN CARDIOTOXICITY INFECTED BY COXSACKIEVIRUS B3

Objective To explore the role of coxsackievirus and adenovirus receptor（CAR） in cardiotoxicity infected by coxsackieviras B3. Methods A toxic cellular model was established in vitro by adding myocarditic coxsackievirus B3 （CVB3m） into the culture of neonatal mouse cardiomyocytes. 48 h later, the cardiomyocytes were divided into control, CVB3m, and CAR antibody ＋ CVB3m groups. CVB3m-mediated myocytopathic effect of above three groups was observed after further culturing for 48h. At the same time, the cardiomyocytes＇ viability of above three groups was assessed by MTT assay. Results The degree of cytopathic effect（CPE） of CAR antibody ＋ CVB3m group was significantly lower than CVB3m group （ P 〈 0. 01 ） and there was a significant increase in cell viability in CAR antibody ＋ CVB3m group compared with CVB3m group（ P 〈 0. 01 ）. No significant difference was found between CAR antibody ＋ CVB3m group and control group. Conclusion CAR antibody possesses a protective effect on CVB3m infected cardiomyoctyes, which indicates that CAR may play an important role in mediating cardiotoxicity infected by CVB3m.