Griffon Anne-Fleur, Rault Loeïza, Simon-Lorière Etienne, Dupont-Rouzeyrol Myrielle, Inizan Catherine
Dengue and Arboviroses - Research and Expertise Unit - Institut Pasteur in New Caledonia - Pasteur Network, Dumbéa-sur-Mer, New Caledonia.
Evolutionary genomics of RNA viruses, Institut Pasteur, Université Paris Cité, Paris, France.
bioRxiv. 2024 Sep 10:2024.09.10.611934. doi: 10.1101/2024.09.10.611934.
Comparing the fitness of dengue virus (DENV) isolates is a pivotal approach to assess the contribution of DENV strains' replicative fitness to epidemiological contexts, including serotype replacements. Competition assays are the gold standard to compare the replicative fitness of viral strains. Implementing competition assays between DENV serotypes requires an experimental setup and an appropriate read-out to quantify the viral progeny of strains belonging to different serotypes.
In the current study, we optimized an existing serotyping qRT-PCR by adapting primer/probe design and multiplexing the serotype-specific qRT-PCR reactions, allowing to accurately detect and quantify all four DENV serotypes. The qRT-PCR was specific, had a limit of detection of at least 5.08×10, 5.16×10, 7.14×10 and 1.36 ×10 genome copies/μL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R) of 0.99975, 0.99975, 0.9985, 0.99965 for DENV-1, -2, -3 and -4 respectively. Challenge of this multiplex serotype-specific qRT-PCR on mixes of viral supernatants containing known concentrations of strains from two serotypes evidenced an accurate quantification of the amount of genome copies of each serotype. We next developed an assay to compare the replicative fitness of two DENV serotypes in the human hepatic cell line HuH7: quantification of the viral progeny of each serotype in the inoculum and the supernatant using the serotype-specific multiplex qRT-PCR unveiled an enrichment of the supernatant in DENV-1 genome copies, uncovering the enhanced replicative fitness of this DENV-1 isolate.
This optimized qRT-PCR combined to a relevant cellular model allowed to accurately quantify the viral progeny of two DENV strains belonging to two different serotypes in a competition assay, allowing to determine which strain had a replicative advantage. This reliable experimental setup is adaptable to the comparative study of the replicative fitness of any DENV serotypes.
比较登革病毒（DENV）分离株的适应性是评估DENV毒株复制适应性对包括血清型替代在内的流行病学情况贡献的关键方法。竞争试验是比较病毒毒株复制适应性的金标准。在DENV血清型之间进行竞争试验需要一个实验设置和适当的读数来量化属于不同血清型的毒株的病毒后代。
在本研究中，我们通过调整引物/探针设计并对血清型特异性qRT-PCR反应进行多重化，优化了现有的血清型qRT-PCR，从而能够准确检测和量化所有四种DENV血清型。该qRT-PCR具有特异性，对DENV-1、-2、-3和-4的检测限分别至少为5.08×10、5.16×10、7.14×10和1.36×10个基因组拷贝/μL，效率分别为1.993、1.975、1.902、1.898，线性度（R）分别为0.99975、0.99975、0.9985、0.99965。对含有已知浓度的两种血清型毒株的病毒上清液混合物进行这种多重血清型特异性qRT-PCR检测，证明了对每种血清型基因组拷贝数量的准确量化。接下来，我们开发了一种试验来比较两种DENV血清型在人肝癌细胞系HuH7中的复制适应性：使用血清型特异性多重qRT-PCR对接种物和上清液中每种血清型的病毒后代进行量化，发现上清液中DENV-1基因组拷贝有所富集，揭示了该DENV-1分离株增强的复制适应性。
这种优化的qRT-PCR与相关的细胞模型相结合，能够在竞争试验中准确量化属于两种不同血清型的两种DENV毒株的病毒后代，从而确定哪种毒株具有复制优势。这种可靠的实验设置适用于任何DENV血清型复制适应性的比较研究。
