Antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in Japanese encephalitis.
Antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in Japanese encephalitis.
复制标题
八胍树枝状大分子缀合的吗啉寡聚物在日本脑炎中的抗病毒和神经保护作用。
DOI：
10.1371/journal.pntd.0000892
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发表时间：
2010-11-23
影响因子：
3.8
通讯作者：
Basu, Anirban
中科院分区：
文献类型：
--
作者：
Nazmi, Arshed;Dutta, Kallol;Basu, Anirban
日本脑炎（JE）是由蚊子传播的黄病毒引起的，在整个东南亚和邻近地区流行。目前没有治疗干预措施可用于乙脑，从而使其成为世界上最可怕的脑炎之一。对抗病毒的有效方法是通过使用针对病毒基因组的反义分子来抑制病毒复制。八胍鎓树枝状聚合物缀合的吗啉代（或Vivo-Morpholino）是不带电荷的反义寡聚体，其可以通过胞吞作用进入活生物体的细胞，随后从内体逃逸到细胞的胞质溶胶/核区室中。我们推测，针对JEV基因组3′或5′非翻译区的特定区域产生的Vivo-Morpholinos，当在JE实验模型中施用时，将具有显著的抗病毒和神经保护作用。用JEV（GP 78株）感染小鼠，随后每天腹膜内施用吗啉代（5 mg/kg体重），进行多达五次处理。监测动物的存活率15天（或直至死亡），随后将其处死，并处理其脑进行免疫组织化学染色或蛋白质提取。从脑匀浆中进行的空斑试验和免疫印迹分析显示病毒载量和病毒蛋白表达减少，导致感染动物的存活率更高。脑切片硫堇染色观察神经保护作用。细胞因子珠阵列显示在吗啉代治疗后脑中促炎细胞因子水平降低，其在感染后升高。这对应于大脑中小胶质细胞活化减少。氧化应激减少，某些应激相关的信号分子被发现是积极调制后吗啉代治疗。体外研究还表明，在吗啉代处理后，感染性病毒颗粒的产生减少。在JE的鼠模型中，体内-吗啉代的施用有效地导致动物存活率增加和神经保护。因此，这些低聚物代表了一种潜在的抗病毒剂，值得进一步评价。日本脑炎（JE）是由一种黄病毒引起的，这种病毒通过属于库蚊属的蚊子传播给人类。JE的威胁笼罩着一个广阔的地理区域，包括大约100亿人。这种疾病之所以令人恐惧，是因为目前还没有特效的抗病毒药物。有报道称，其他研究人员已经表明，阻断病毒复制的药物可用作有效的治疗对策。体内吗啉代（MO）是合成产生的DNA或RNA的类似物，其可以被修饰以与基因组中的特定靶向区域结合。在这项研究中，作者提出，在乙脑动物模型中，专门设计的MO与乙脑病毒（JEV）基因组的特定区域结合，阻断活生物体细胞中病毒的产生。这导致受感染动物的死亡率降低。由于JEV的主要靶点是神经细胞，用MO治疗后的实验动物的脑分析显示出神经保护作用。对培养细胞的研究也支持MO的抗病毒作用。这些MO在动物体内的有效反义作用和在有效剂量下缺乏明显的毒性使其成为具有未来治疗乙脑应用的良好研究试剂。
Japanese encephalitis (JE), caused by a mosquito-borne flavivirus, is endemic to the entire south-east Asian and adjoining regions. Currently no therapeutic interventions are available for JE, thereby making it one of the most dreaded encephalitides in the world. An effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. Octaguanidinium dendrimer-conjugated Morpholino (or Vivo-Morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. We hypothesize that Vivo-Morpholinos generated against specific regions of 3′ or 5′ untranslated regions of JEV genome, when administered in an experimental model of JE, will have significant antiviral and neuroprotective effect. Mice were infected with JEV (GP78 strain) followed by intraperitoneal administration of Morpholinos (5 mg/kg body weight) daily for up to five treatments. Survivability of the animals was monitored for 15 days (or until death) following which they were sacrificed and their brains were processed either for immunohistochemical staining or protein extraction. Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals. Neuroprotective effect was observed by thionin staining of brain sections. Cytokine bead array showed reduction in the levels of proinflammatory cytokines in brain following Morpholino treatment, which were elevated after infection. This corresponded to reduced microglial activation in brain. Oxidative stress was reduced and certain stress-related signaling molecules were found to be positively modulated following Morpholino treatment. In vitro studies also showed that there was decrease in infective viral particle production following Morpholino treatment. Administration of Vivo-Morpholino effectively resulted in increased survival of animals and neuroprotection in a murine model of JE. Hence, these oligomers represent a potential antiviral agent that merits further evaluation. Japanese encephalitis (JE) is caused by a flavivirus that is transmitted to humans by mosquitoes belonging to the Culex sp. The threat of JE looms over a vast geographical realm, encompassing approximately 10 billion people. The disease is feared because currently there are no specific antiviral drugs available. There have been reports where other investigators have shown that agents that block viral replication can be used as effective therapeutic countermeasures. Vivo-Morpholinos (MOs) are synthetically produced analogs of DNA or RNA that can be modified to bind with specific targeted regions in a genome. In this study the authors propose that in an animal model of JE, MOs specifically designed to bind with specific region of JE virus (JEV) genome, blocks virus production in cells of living organisms. This results in reduced mortality of infected animals. As the major target of JEV is the nerve cells, analysis of brain of experimental animals, post treatment with MOs, showed neuroprotection. Studies in cultured cells were also supportive of the antiviral role of the MOs. The potent anti-sense effect in animals and lack of obvious toxicity at the effective dosage make these MOs good research reagents with future therapeutic applications in JE.
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