Maxizyme-mediated suppression of chikungunya virus replication and transmission in transgenic Aedes aegypti mosquitoes.
Maxizyme-mediated suppression of chikungunya virus replication and transmission in transgenic Aedes aegypti mosquitoes.
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DOI：
10.3389/fmicb.2023.1286519
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发表时间：
2023
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
--
作者：
基孔肯雅病毒(CHIKV)是一种新出现的蚊媒病原体，具有重要的公共卫生意义。目前尚无预防疫苗或治疗药物可用于控制CHIKV。已经提出的一种虫媒病毒控制方法是用那些对病毒感染不敏感的蚊子取代具有传播能力的蚊子。目前正在研究几种转基因效应器，认为它们可能对这种种群替代方法有用。我们曾成功地使用锤头状核酶(HRzs)作为抗病毒效应转基因来控制伊蚊对CHIKV的感染和传播。在这份报告中，我们研究了一种最大酶方法来增强催化活性并防止病毒突变体逃脱这些核酶。我们设计了一种包含两个hRZ的最小化(单体)版本的Maxizyme，我们之前证明了这两个hRZ在抑制CHIKV方面最有效。设计了三个版本的CHIKV MAXIZYME：活动的(Mz)、非活动的(ΔMz)和连接的CHIKV MAXIZYME(CMZ)。将Maxizyme及其表达单位(Ae-tRNA Val启动子及其终止信号)整合到带有选择标记和可视化标记的慢病毒载体中。在Vero细胞转化、筛选和单细胞分选后，用0.05和0.5moi的CHIKV感染克隆细胞群，用TCID50-IFA、RT-qPCR和caspase-3检测病毒抑制情况。获得了5个表达CMZ的转基因蚊株，并用夹板聚合酶链式反应(Splinkerette PCR)对转基因插入位点进行了鉴定。我们的结果表明，与其各自的非活性对照版本或两个亲本hRZ相比，表达Mz的Vero细胞克隆显示出对CHIKV复制的完全抑制。在口服携带CHIKV的转基因蚊子时，5个品系中有3个对CHIKV感染完全耐药，所有5个品系的唾液传播均为阴性。综上所述，这项研究表明，Maxizyme可以提供比hRzs更高的催化活性和病毒抑制。
Chikungunya virus (CHIKV) is an emerging mosquito-borne pathogen of significant public health importance. There are currently no prophylactic vaccines or therapeutics available to control CHIKV. One approach to arbovirus control that has been proposed is the replacement of transmission-competent mosquitoes with those that are refractory to virus infection. Several transgene effectors are being examined as potentially useful for this population replacement approach. We previously demonstrated the successful use of hammerhead ribozymes (hRzs) as an antiviral effector transgene to control CHIKV infection of, and transmission by, Aedes mosquitoes. In this report we examine a maxizyme approach to enhance the catalytic activity and prevent virus mutants from escaping these ribozymes. We designed a maxizyme containing minimized (monomer) versions of two hRzs we previously demonstrated to be the most effective in CHIKV suppression. Three versions of CHIKV maxizyme were designed: Active (Mz), inactive (ΔMz), and a connected CHIKV maxizyme (cMz). The maxizymes with their expression units (Ae-tRNA val promoter and its termination signal) were incorporated into lentivirus vectors with selection and visualization markers. Following transformation, selection, and single-cell sorting of Vero cells, clonal cell populations were infected with CHIKV at 0.05 and 0.5 MOI, and virus suppression was assessed using TCID50-IFA, RT-qPCR, and caspase-3 assays. Five transgenic mosquito lines expressing cMz were generated and transgene insertion sites were confirmed by splinkerette PCR. Our results demonstrate that Vero cell clones expressing Mz exhibited complete inhibition of CHIKV replication compared to their respective inactive control version or the two parent hRzs. Upon oral challenge of transgenic mosquitoes with CHIKV, three out of the five lines were completely refractory to CHIKV infection, and all five lines tested negative for salivary transmission. Altogether, this study demonstrates that maxizymes can provide a higher catalytic activity and viral suppression than hRzs.
影响因子：
46.9
作者：
Haasnoot J;Westerhout EM;Berkhout B
通讯作者：
Berkhout B
影响因子：
5.9
作者：
Yen PS;James A;Li JC;Chen CH;Failloux AB
通讯作者：
Failloux AB