Intracranial Administration of P Gene siRNA Protects Mice from Lethal Chandipura Virus Encephalitis
Intracranial Administration of P Gene siRNA Protects Mice from Lethal Chandipura Virus Encephalitis
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DOI：
10.1371/journal.pone.0008615
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发表时间：
2010-01-07
期刊：
影响因子：
3.7
通讯作者：
Arankalle, Vidya A.
中科院分区：
文献类型：
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作者：
Kumar, Satyendra;Arankalle, Vidya A.
背景：在印度的部分地区，Chandipura病毒（CHPV）已成为流行性和散发性脑炎的病原体。这种儿科疾病病程迅速，导致55-75%的死亡率。在没有特异性治疗的情况下，RNA干扰（RNAi）的有效性进行了评估。方法和发现：合成的短干扰RNA（siRNA）或短发夹RNA（shRNA）在保护小鼠免受CHPV感染的有效性进行了评估。靶基因是P基因和M基因，主要是因为前者在病毒复制中发挥重要作用，而后者具有致命性。采用真实的实时一步法RT-PCR和空斑试验检测基因沉默。利用pAcGFP 1 N1-CHPV-P，我们发现P-2 siRNA在体外抑制P基因表达的效果最好。两种定量测定都记录了当感染后2小时（PI）转染P-2、M-5或M-6 siRNA时，病毒滴度降低2log。组合使用这些siRNA没有导致效率提高。发现P-2 siRNA在中心容忍四个错配。与5种不同的shRNA相比，P-2 siRNA在抑制CHPV复制方面最有效。当用100 LD（50）CHPV颅内感染的小鼠同时用阳离子脂质复合物5 μ g P-2 siRNA治疗时，观察到存活时间延长。用10 LD（50）感染和用两个剂量的siRNA首先、同时和第二个24小时PI处理导致70%的存活。存活小鼠脑内CHPV滴度降低4log，无组织病理学变化或抗体应答。P-2 siRNA处理的小鼠的基因表达谱显示没有干扰素应答。第一次剂量的siRNA在2小时或4小时PI与第二次剂量在24小时分别导致40%和20%的生存率，提示潜在的应用在therapy.Conclusions：结果突出siRNA在治疗快速和致命的Chandipura脑炎的治疗潜力。
Background: In parts of India, Chandipura Virus (CHPV) has emerged as an encephalitis causing pathogen in both epidemic and sporadic forms. This pediatric disease follows rapid course leading to 55-75% mortality. In the absence of specific treatment, effectiveness of RNA interference (RNAi) was evaluated.Methods and Findings: Efficacy of synthetic short interfering RNA (siRNA) or short hairpin RNA (shRNA) in protecting mice from CHPV infection was assessed. The target genes were P and M genes primarily because important role of the former in viral replication and lethal nature of the latter. Real time one step RT-PCR and plaque assay were used for the assessment of gene silencing. Using pAcGFP1N1-CHPV-P, we showed that P-2 siRNA was most efficient in reducing the expression of P gene in-vitro. Both quantitative assays documented 2logs reduction in the virus titer when P-2, M-5 or M-6 siRNAs were transfected 2hr post infection (PI). Use of these siRNAs in combination did not result in enhanced efficiency. P-2 siRNA was found to tolerate four mismatches in the center. As compared to five different shRNAs, P-2 siRNA was most effective in inhibiting CHPV replication. An extended survival was noted when mice infected intracranially with 100 LD(50) CHPV were treated with cationic lipid complexed 5 mu g P-2 siRNA simultaneously. Infection with 10LD(50) and treatment with two doses of siRNA first, simultaneously and second 24 hr PI, resulted in 70% survival. Surviving mice showed 4logs less CHPV titers in brain without histopathological changes or antibody response. Gene expression profiles of P-2 siRNA treated mice showed no interferon response. First dose of siRNA at 2hr or 4hr PI with second dose at 24hr resulted in 40% and 20% survival respectively suggesting potential application in therapy.Conclusions: The results highlight therapeutic potential of siRNA in treating rapid and fatal Chandipura encephalitis.