Sena Johnny, Karwal Lovkesh, Bell Callum, Devitt Nicholas, Schilkey Faye, Huang Claire, Livengood Jill, Das Subash, Dean Hansi J
National Center for Genome Resources, Santa Fe, NM, United States.
Vaccine Business Unit, Takeda Pharmaceuticals, Cambridge, MA, United States.
Biol Methods Protoc. 2024 Feb 3;9(1):bpae004. doi: 10.1093/biomethods/bpae004. eCollection 2024.
The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances >5 kb. As proof of concept, high-quality viral RNA of the Dengue 2 component of Takeda's tetravalent dengue vaccine candidate (TDV-2) was used to develop a reverse transcription-polymerase chain reaction protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long-read sequencing was validated with multiple clone-derived TDV-2 revertant variants and four complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in the differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single-genome molecule, which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.
该研究的目标是鉴定和表征含有分布在基因组距离大于5 kb的突变的RNA病毒变体。作为概念验证，使用武田四价登革热疫苗候选株（TDV-2）的登革热2组分的高质量病毒RNA来开发逆转录-聚合酶链反应方案，以扩增一个约5.3 kb的cDNA片段，该片段包含TDV-2减毒的三个遗传决定因素。将独特的分子标识符整合到每个病毒cDNA分子中用于PacBio文库制备，以提高在减毒位点观察到的变体的定量精度。经过检测优化后，PacBio长读长测序用多个克隆衍生的TDV-2回复变体和四种包含不同组成的TDV-2和回复病毒的复杂回复混合物进行了验证。PacBio测序分析正确鉴定并定量了所有测试样品中的变体组成，表明可以鉴定和表征TDV-2回复体，并支持使用该方法区分和定量其他RNA病毒的复杂变体。长读长测序可以鉴定在单基因组分子上含有多个突变的复杂RNA病毒变体，这对于减毒活病毒疫苗的深入遗传稳定性和回复体检测以及病毒进化研究以揭示免疫逃逸和宿主细胞适应机制很有用。
