Nogales Aitor, Alonso Celia, Moreno Sandra, Lorenzo Gema, Borrego Belén, Martinez-Sobrido Luis, Brun Alejandro
Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas (INIA-CSIC), Madrid, Spain.
Texas Biomedical Research Institute, San Antonio, Texas, USA.
J Virol. 2025 Jan 31;99(1):e0178224. doi: 10.1128/jvi.01782-24. Epub 2024 Dec 12.
Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic disease that causes severe disease in both domestic and wild ungulates and humans, making it a significant threat to livestock and public health. The RVFV genome consists of three single-stranded, negative-sense RNA segments differing in size: small (S), medium (M), and large (L). Segment S encodes the virus nucleoprotein N and the virulence-associated factor non-structural (NSs) protein in opposite orientations, separated by an intergenic region (IGR). To overcome the current need to use secondary techniques to detect the presence of RVFV in infected cells, we used T7-driven polymerase plasmid-based reverse genetics to generate replication-competent recombinant (r)RVFV expressing Nanoluciferase (Nluc) or Venus fluorescent proteins. These reporter genes were used as valid surrogates to track the presence of RVFV in mammalian and insect cells. Notably, we explored the genome plasticity of RVFV and compared four different strategies by modifying the viral segment S to introduce the reporter gene foreign sequences. The reporter-expressing rRVFV were stable and able to replicate in cultured mammalian and insect cells, although to a lesser extent than the recombinant wild-type (WT) counterpart. Moreover, rRVFV-expressing reporter genes were validated to identify neutralizing antibodies or compounds with antiviral activity. , all mice infected with the reporter-expressing rRVFV displayed an attenuated phenotype, although at different levels. These rRVFV-expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of RVFV and represent an excellent biotechnological tool for developing new therapeutics against RVFV infections.
Rift Valley fever virus (RVFV) is a mosquito-borne virus and zoonotic agent threat that can be deadly to domestic or wild ungulates, and humans. In this work, we used reverse genetics approaches to explore the genome plasticity of RVFV by generating a set of recombinant (r)RVFV that express fluorescent or luminescent proteins to track viral infection. All the generated reporter-expressing rRVFVs were able to propagate in mammalian or insect cells and a mouse model of infection. Our studies may contribute to advances in research on RVFV and other bunyaviruses and pave the way for the development of novel vaccines and the identification of new antivirals for the prophylactic and therapeutic treatment, respectively, of RVFV infections.
裂谷热病毒（RVFV）是一种由蚊子传播的人畜共患病，可在家养和野生有蹄类动物以及人类中引发严重疾病，对家畜和公共卫生构成重大威胁。RVFV基因组由三个大小不同的单链负义RNA片段组成：小片段（S）、中片段（M）和大片段（L）。S片段以相反方向编码病毒核蛋白N和毒力相关因子非结构（NSs）蛋白，两者由基因间隔区（IGR）隔开。为满足当前检测感染细胞中RVFV存在时对二级技术的需求，我们利用基于T7驱动聚合酶质粒的反向遗传学方法，生成了表达纳米荧光素酶（Nluc）或维纳斯荧光蛋白的具有复制能力的重组（r）RVFV。这些报告基因被用作有效替代物，以追踪RVFV在哺乳动物细胞和昆虫细胞中的存在情况。值得注意的是，我们探究了RVFV的基因组可塑性，并通过修饰病毒S片段以引入报告基因外源序列，比较了四种不同策略。表达报告基因的rRVFV是稳定的，能够在培养的哺乳动物细胞和昆虫细胞中复制，尽管其复制程度低于重组野生型（WT）对应物。此外，表达报告基因的rRVFV经过验证，可用于鉴定中和抗体或具有抗病毒活性的化合物。所有感染表达报告基因的rRVFV的小鼠均表现出减弱的表型，尽管程度不同。这些表达报告基因的rRVFV提供了一种新方法，能更好地理解RVFV的生物学特性和发病机制，并且是开发针对RVFV感染的新疗法的优秀生物技术工具。
裂谷热病毒（RVFV）是一种由蚊子传播的病毒和人畜共患病原体，对家养或野生有蹄类动物以及人类可能具有致命性。在这项研究中，我们采用反向遗传学方法，通过生成一组表达荧光或发光蛋白以追踪病毒感染的重组（r）RVFV，来探究RVFV的基因组可塑性。所有生成的表达报告基因的rRVFV都能够在哺乳动物或昆虫细胞以及感染小鼠模型中繁殖。我们的研究可能有助于推动对RVFV和其他布尼亚病毒的研究进展，并分别为开发新型疫苗以及鉴定用于预防和治疗RVFV感染的新抗病毒药物铺平道路。
