Departments of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, TX 77555-1019, United States.
Departments of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, TX 77555-1019, United States; Institute of Human Infection and Immunity, The University of Texas Medical Branch, Galveston, TX 77555-1019, United States.
J Virol Methods. 2019 Oct;272:113701. doi: 10.1016/j.jviromet.2019.113701. Epub 2019 Jul 14.
Rift Valley Fever phlebovirus (RVFV), genus Phlebovirus, family Phenuiviridae, order Bunyavirales, has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. Here, we report the establishment of a strand-specific, quantitative reverse transcription (RT)-PCR assay system that can selectively distinguish between the genomic and antigenomic RNAs of each of the three viral RNA segments produced in RVFV-infected cells. To circumvent the obstacle of primer-independent cDNA synthesis during RT, we used a tagged, strand-specific RT primer, carrying a non-viral 'tag' sequence at the 5' end, which ensured the strand-specificity through the selective amplification of only the tagged cDNA in the real-time PCR assay. We used this assay system to examine the kinetics of intracellular accumulation of genomic and antigenomic viral RNAs in mammalian cells infected with the MP-12 strain of RVFV. The genomic RNA copy numbers, for all three viral RNA segments, were higher than that of their corresponding antigenomic RNAs throughout the time-course of infection, with a notable exception, wherein the M segment genomic and antigenomic RNAs exhibited similar copy numbers at specific times post-infection. Overall, this assay system could be a useful tool to gain an insight into the mechanisms of RNA replication and packaging in RVFV.
裂谷热病毒（RVFV），属布尼亚病毒科白蛉病毒属，为负链单股 RNA 病毒，基因组由 L、M 和 S 片段组成。本研究建立了一种可区分 RVFV 感染细胞中三种病毒 RNA 片段的基因组和抗原基因组 RNA 的特异性、定量逆转录（RT）-PCR 检测系统。为了避免 RT 过程中引物非依赖性 cDNA 合成的障碍，我们使用了带标记的、链特异性 RT 引物，其 5' 端带有非病毒“标记”序列，通过仅对实时 PCR 检测中的标记 cDNA 进行选择性扩增，确保了链特异性。我们使用该检测系统研究了 RVFV MP-12 株感染的哺乳动物细胞中细胞内积累的基因组和抗原基因组病毒 RNA 的动力学。在整个感染过程中，所有三个病毒 RNA 片段的基因组 RNA 拷贝数均高于其相应的抗原基因组 RNA，只有 M 片段的基因组和抗原基因组 RNA 在感染后特定时间具有相似的拷贝数，这是一个显著的例外。总的来说，该检测系统可能是深入了解 RVFV RNA 复制和包装机制的有用工具。
