Diaz Jennifer, Sears John, Chang Che-Kang, Burdick Jane, Law Isabella, Sanders Wes, Linnertz Colton, Sylvester Paul, Moorman Nathaniel, Ferris Martin T, Heise Mark T
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.
Viruses. 2024 Apr 19;16(4):636. doi: 10.3390/v16040636.
RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5' conserved sequence element that disrupt sequence but not structure did not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) did cause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent, primary sequence determinants in this loop play an important role in CHIKV biology. Our novel findings illustrate the utility of the U-CAN-seq competition assay.
RNA病毒会迅速发生细微的基因型变化，这些变化可能对病毒适应性和宿主范围产生重大影响，进而对人类健康造成潜在后果。因此，了解新型病毒变体相对于已深入研究的流行病毒基因型的进化适应性非常重要。竞争试验是评估病毒基因型相对适应性的一种有效且严格的系统。然而，快速且低成本地区分和量化非常相似的病毒基因型之间的适应性差异具有挑战性。在此，我们描述了一种将逆转录PCR与商业纳米孔测序服务相结合的方案，用于对未标记的RNA病毒进行竞争试验。我们的试验称为纳米孔测序通用竞争试验（U-CAN-seq），相对成本较低且灵敏度高。我们利用在基孔肯雅病毒（CHIKV）nsp3基因中一个已深入研究的N24A突变，证实我们能够通过U-CAN-seq检测到竞争劣势。我们还使用这种方法表明，CHIKV 5'保守序列元件发生的破坏序列但不破坏结构的突变不会影响CHIKV的适应性。然而，与相邻的CHIKV茎环（SL3）发生的类似突变相比野生型CHIKV确实导致了适应性劣势，这表明该环中与结构无关的一级序列决定因素在CHIKV生物学中起重要作用。我们的新发现说明了U-CAN-seq竞争试验的实用性。
