For best viewing of the website please use Mozilla Firefox or Google Chrome.

2015年30卷5期

|     

Review

基于CRISPR/Cas9技术进行基因编辑控制HBV复制的研究进展

彭程, 陆蒙吉, 杨东亮

2015, 30(5): 317 doi: 10.1007/s12250-015-3660-x

[HTML全文] [PDF 2500 KB] Springerlink
HBV感染目前仍是一个全球性的问题在于当前的治疗方法不能完全清除病毒以达到完全治愈。因此,迫切需要发展新的治疗方法以有效清除HBV包括根除潜伏的病毒储备。近来发展的一种新的基因编辑技术CRISPR/Cas9可以高效诱导靶位点突变,被认为是疾病治疗的新型潜力工具,并有望用来根除入侵的病原包括HBV。本综述回顾近来在体内及体外实验中运用CRISPR/Cas9技术靶向HBV特异DNA片段以抑制病毒复制及诱导基因突变的研究进展,探讨CRISPR/Cas9作为一种新的技术成为HBV根治方法的优缺点以及未来发展的方向。

Dengue epidemiology and pathogenesis: images of the future viewed through a mirror of the past

2015, 30(5): 326 doi: 10.1007/s12250-015-3624-1

[HTML全文] [PDF 968 KB] Springerlink
Every year, millions of individuals throughout the world are seriously affected by dengue virus. The unavailability of a vaccine and of anti-viral drugs has made this mosquito-borne disease a serious health concern. Not only does dengue cause fatalities but it also has a profoundly negative economic impact. In recent decades, extensive research has been performed on epidemiology, vector biology, life cycle, pathogenesis, vaccine development and prevention. Although dengue research is still not at a stage to suggest definite hopes of a cure, encouraging significant advances have provided remarkable progress in the fight against infection. Recent developments indicate that both anti-viral drug and vaccine research should be pursued, in parallel with vector control programs.
Research Article

Human enteric viruses – potential indicators for enhanced monitoring of recreational water quality

2015, 30(5): 344 doi: 10.1007/s12250-015-3644-x

[HTML全文] [PDF 2174 KB] Springerlink ESM
Recreational waters contaminated with human fecal pollution are a public health concern, and ensuring the safety of recreational waters for public use is a priority of both the Environmental Protection Agency (EPA) and the Centers for Disease Control and Prevention (CDC). Current recreational water standards rely on fecal indicator bacteria (FIB) levels as indicators of human disease risk. However present evidence indicates that levels of FIB do not always correspond to the presence of other potentially harmful organisms, such as viruses. Thus, enteric viruses are currently tested as water quality indicators, but have yet to be successfully implemented in routine monitoring of water quality. This study utilized enteric viruses as possible alternative indicators of water quality to examine 18 different fresh and offshore recreational waters on O‘ahu, Hawai‘i, by using newly established laboratory techniques including highly optimized PCR, real time PCR, and viral infectivity assays. All sample sites were detected positive for human enteric viruses by PCR including enterovirus, norovirus genogroups I and II, and male specific FRNA coliphage. A six time-point seasonal study of enteric virus presence indicated significant variation in virus detection between the rainy and dry seasons. Quantitative PCR detected the presence of norovirus genogroup II at levels at which disease risk may occur, and there was no correlation found between enteric virus presence and FIB counts. Under the present laboratory conditions, no infectious viruses were detected from the samples PCR-positive for enteric viruses. These data emphasize both the need for additional indicators for improved monitoring of water quality, and the feasibility of using enteric viruses as these indicators.

一种基于体外重组的日本脑炎病毒反向遗传学系统的建立

杜瑞坤, 王曼丽, 胡志红, 王华林, 邓菲*

2015, 30(5): 354 doi: 10.1007/s12250-015-3623-2

[HTML全文] [PDF 1402 KB] Springerlink
日本脑炎病毒(Japanese encephalitis virus, JEV)是病毒性脑炎的重要病原,给人类健康带来极大威胁。反向遗传技术是病毒研究中的一种有效的手段,在病毒复制、病毒宿主相互作用以及疫苗研发等研究中应用广泛。然而JEV基因组cDNA在大肠杆菌中不能稳定复制,极大程度地阻碍了JEV反向遗传学系统的建立。本研究利用In-fusion克隆系统发展了一种基于体外重组的JEV反向遗传学系统。本系统通过缺失JEV结构基因将基因组截短形式构建到CMV启动子下作为基础质粒,该质粒可在大肠杆菌稳定复制。缺失的结构基因片段可通过融合PCR引入突变及扩增,之后与线性化的基础质粒体外连接后直接转染受纳细胞,培养3-5天后即可收获恢复病毒。比较病毒空斑形态及生长曲线表明,野生型恢复病毒与母株SA14几乎一致,说明该系统有着较高的保真性。利用该系统将E蛋白12个保守半胱氨酸(Cys)分别突变为丙氨酸(Ala),结果表明,任何Cys-Ala单点突变均不能产生感染性病毒粒子,而对照E蛋白107位Leu-Phe突变并不影响感染性病毒粒子的产生。这一结果表明Cys在病毒生命周期中具有重要作用。综上所述,我们构建了一种可以快速引入JEV结构蛋白突变的反向遗传学系统,为JEV结构蛋白的功能研究提供了一种有力手段。

Rotavirus VP7 Epitope Chimeric Proteins Elicit Cross-immunoreactivity in Guinea Pigs

赵冰心, 潘小霞, 滕玉梅, 夏文跃, 王晶, 文喻玲, 陈元鼎

2015, 30(5): 363 doi: 10.1007/s12250-015-3620-5

[HTML全文] [PDF 1340 KB] Springerlink
A组轮状病毒(RVA)VP7含有主要的病毒抗原表位。研究表明,以VP6作载体、携带外源抗原表位的嵌合蛋白拥有良好的抗原反应性和免疫原性。本研究以VP6作载体,构建了分别携带RVA VP7上3个不同抗原表位的嵌合蛋白,并对其免一特性进行了研究。结果显示,这些嵌合蛋白可与抗VP6蛋白抗体和抗SA11和抗Wa株RV抗体反应;这些嵌合蛋白的免疫豚鼠血清抗体可特异性识别RVA的VP6和VP7,可在体外保护哺乳动物细胞免受SA11和Wa病毒的感染;这些嵌合蛋白的免疫豚鼠血清抗体的中和活性显著高于载体蛋白的免疫豚鼠血清抗体。结果表明,这些以VP6为载体、携带VP7抗原表位的嵌合蛋白,具有一步开发预防RVA感染的新型疫苗的前景。

严重呼吸道合胞病毒感染的疫苗预防效果:meta分析

朱童娜, 张传龙, 于莉, 陈敬贤, 邱欢, 吕伟伟, 黄升海

2015, 30(5): 371 doi: 10.1007/s12250-015-3630-3

[HTML全文] [PDF 469 KB] Springerlink
呼吸道合胞病毒(Respiratory syncytial virus,RSV)是引起婴幼儿急性下呼吸道感染的一种常见病原体,然而目前尚没有有效的疫苗来预防RSV感染。本文主要进行评估预防性疫苗对RSV感染的影响。本文主要分析了1792篇从1973年1月至2015年9月已发表的关于RSV疫苗的随机临床研究,其中有13篇符合我们的选择标准。在这13篇研究中,11篇评估了RSV疫苗的有效性及其作用,4篇评估了疫苗中佐剂的作用。其OR值分别为0.31 (95%CI, 0.15-0.67) 和 0.62 (95%CI, 0.29-1.34)。通过分析我们发现RSV疫苗可以有效地降低RSV感染的风险,然而添加佐剂的疫苗与没有添加佐剂的疫苗相比却没有明显的作用。我们希望该研究能够为今后防治RSV感染提供更多精确的分析数据。

Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria

2015, 30(5): 386 doi: 10.1007/s12250-015-3618-z

[HTML全文] [PDF 2306 KB] Springerlink
The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.
Letter

猪嵴病毒引起的仔猪组织病理学变化

杨凡, 刘小琬, 周远成, 吕雯婷, 许思遥, 徐志文, 朱玲*

2015, 30(5): 396 doi: 10.1007/s12250-015-3608-1

[HTML全文] [PDF 2156 KB] Springerlink ESM
猪嵴病毒为单股正链RNA病毒。病毒粒子呈球形,无囊膜。PKoV在世界各地广泛流行。但是,关于PKoV是否引起组织病变目前尚不清楚。在此研究中,采集疑似病猪组织和粪便样品,经Vero细胞培养、蚀斑纯化分离得到两株表现出细胞适应性的PKoV病毒。本论文挑选20只10日龄SPF仔猪,随机分成3个实验组和1个对照组。选择1株PKoV病毒,实验组每只注射2mL病毒液,对照组每只注射等量无菌培养液,并分别在接种后的第2、第4、第6、第8和第10天,每组选择1只仔猪实行安乐死。通过临床特征、病理解剖和组织切片,观察到PKoV感染仔猪后所引起的组织病理学变化,尤其集中在肺脏、肾脏和小肠,其它组织均表现出轻微病变。PKoV感染后仔猪出现腹泻等症状,表明它可能是一种引起仔猪腹泻的病原。这一研究结果,为后期深入探讨PKoV的致病机理奠定了一定的基础。