State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; Department of Oral Pathology, College of Dentistry, Howard University, Washington, DC, 20059, USA; Department of Nephrology, Henan Provincial Key Laboratory of Kidney Disease and Immunology, Henan Provincial People's Hospital (Zhengzhou University People's Hospital), Zhengzhou, 450003, China.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
Virology. 2021 Jan 2;552:73-82. doi: 10.1016/j.virol.2020.09.001. Epub 2020 Oct 6.
Zika virus (ZIKV) is a mosquito-borne flavivirus that replicates in both vertebrate and insect cells, whereas insect-specific flaviviruses (ISF) replicate only in insect cells. We sought to convert ZIKV, from a dual-tropic flavivirus, into an insect-specific virus for the eventual development of a safe ZIKV vaccine. Reverse genetics was used to introduce specific mutations into the furin cleavage motif within the ZIKV pre-membrane protein (prM). Mutant clones were selected, which replicated well in C6/36 insect cells but exhibited reduced replication in non-human primate (Vero) cells. Further characterization of the furin cleavage site mutants indicated they replicated poorly in both human (HeLa, U251), and baby hamster kidney (BHK-21) cells. One clone with the induced mutation in the prM protein and at positions 291and 452 within the NS3 protein was totally and stably replication-defective in vertebrate cells (VSRD-ZIKV). Preliminary studies in ZIKV sensitive, immunodeficient mice demonstrated that VSRD-ZIKV-infected mice survived and were virus-negative. Our study indicates that a reverse genetic approach targeting the furin cleavage site in prM can be used to select an insect-specific ZIKV with the potential utility as a vaccine strain.
Zika 病毒（ZIKV）是一种通过蚊子传播的黄病毒，可在脊椎动物和昆虫细胞中复制，而昆虫特异性黄病毒（ISF）仅在昆虫细胞中复制。我们试图将 ZIKV 从双嗜性黄病毒转化为昆虫特异性病毒，最终开发出安全的 ZIKV 疫苗。反向遗传学用于在 ZIKV 前膜蛋白（prM）中的弗林裂解基序中引入特定突变。选择了复制良好的突变体克隆在 C6/36 昆虫细胞中，但在非人类灵长类动物（Vero）细胞中的复制能力降低。对弗林裂解位点突变体的进一步表征表明，它们在人类（HeLa、U251）和幼仓鼠肾（BHK-21）细胞中均复制不良。一个在 prM 蛋白中诱导突变的克隆，以及 NS3 蛋白中的位置 291 和 452 的突变，在脊椎动物细胞（VSRD-ZIKV）中完全且稳定地复制缺陷。在对 ZIKV 敏感的免疫缺陷小鼠中的初步研究表明，VSRD-ZIKV 感染的小鼠存活且呈病毒阴性。我们的研究表明，针对 prM 中的弗林裂解位点的反向遗传方法可用于选择具有作为疫苗株潜力的昆虫特异性 ZIKV。
