Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes.
Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes.
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DOI：
10.3390/v8060163
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发表时间：
2016-06-10
期刊：
影响因子：
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通讯作者：
Fraser MJ
中科院分区：
文献类型：
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作者：
Mishra P;Furey C;Balaraman V;Fraser MJ
基孔肯雅病毒（CHIKV）是一种新兴的病原体，广泛分布在非洲，印度和亚洲地区，随着其主要媒介白纹伊蚊的引入，有可能传播到温带气候。CHIKV导致一种经常被误诊为登革热的疾病，具有潜在的危及生命的症状，可能导致长期的衰弱性关节炎。通过人类旅行和商业从流行地区传播的风险越来越大，加上目前缺乏疫苗，使世界上很大一部分人口面临感染这种疾病的风险。在这项研究中，我们设计并测试了锤头状核酶（hRzs）靶向CHIKV结构蛋白基因的RNA基因组作为潜在的抗病毒药物在细胞和体内水平。我们采用CHIKV毒株181/25，其在Vero细胞培养物和蚊子中表现出相似的感染率。对所有7种hRz的转化的Vero细胞克隆进行的病毒抑制测定证明，所有hRz都有效抑制Vero细胞中的CHIKV，其中hRz#9和#14最有效。piggyBac转化载体使用Ae.埃及伊蚊t-RNAval Pol III促进hRz#9和#14效应基因，以建立总共9个独特的转基因Higgs白色眼（HWE）Ae。埃及线。在通过实时聚合酶链反应（RT-PCR）确认转基因表达后，比较TCID 50-IFA分析、原位免疫荧光测定（IFA）和唾液CHIKV滴度分析证明，与对照HWE蚊子相比，在这些转基因系的每一个的杂合雌性中在7 dpi时有效抑制病毒复制。这份报告提供了一个证据，适当的工程hRzs是强大的抗病毒效应基因，适用于人口替代战略
The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs) targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNAval Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE) Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR), comparative TCID50-IFA analysis, in situ Immuno-fluorescent Assays (IFA) and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies