Quantitative Characterization of Defective Virus Emergence by Deep Sequencing
Quantitative Characterization of Defective Virus Emergence by Deep Sequencing
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DOI：
10.1128/jvi.02675-13
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发表时间：
2014-03-01
影响因子：
5.4
通讯作者：
Yin, John
中科院分区：
文献类型：
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作者：
Timm, Collin;Akpinar, Fulya;Yin, John
RNA病毒群体可以自发地产生基因组大小、序列和生物活性不同的变体。缺乏必需基因的缺陷变体仍然可以通过用活病毒共同感染细胞来繁殖，这一过程干扰了病毒的生长。这种缺陷干扰颗粒（DIP）在病毒群体中的丰度和生物活性如何变化尚不清楚。在此，将原型RNA病毒，水泡性口炎病毒（VSV）在BHK宿主细胞上培养三代，并对传代进行Illumina测序。当与全长病毒序列（11，161个核苷酸[nt]）比对时，来自初始群体的读数均匀分布在基因组中。然而，在传代过程中，读段计数的两个平台出现在负义病毒基因组的5'端。通过标准化和简单的滑动窗口方法进行的分析显示了平台边界，这表明至少有两种截短物种的出现和富集，这些截短物种具有中等（类似于5，900 nt）和短（类似于4，000 nt）基因组。通过定量逆转录-PCR（qRT-PCR）验证了基于读数计数的全长和截短物质的相对测量值。检测限分析表明，深度测序可能比用于检测和定量群体中缺陷颗粒的补充措施更灵敏。此外，来自透射电子显微镜的颗粒计数，加上感染性测定，将较小基因组的增加与截短颗粒和干扰活性的增加联系起来。总之，深度测序覆盖率的变化同时显示了截短基因组变异的大小，位置和相对水平，揭示了病毒基因组和粒子的其他措施所掩盖的群体异质性水平。重要的是，我们展示了如何深度测序可以用来表征病毒群体中截短病毒变异的出现，多样性和相对丰度。这种方法的适应自然分离株可能阐明影响自然界中病毒种群稳定性和持久性的因素。
Populations of RNA viruses can spontaneously produce variants that differ in genome size, sequence, and biological activity. Defective variants that lack essential genes can nevertheless reproduce by coinfecting cells with viable virus, a process that interferes with virus growth. How such defective interfering particles (DIPs) change in abundance and biological activity within a virus population is not known. Here, a prototype RNA virus, vesicular stomatitis virus (VSV), was cultured for three passages on BHK host cells, and passages were subjected to Illumina sequencing. Reads from the initial population, when aligned to the full-length viral sequence (11,161 nucleotides [nt]), distributed uniformly across the genome. However, during passages two plateaus in read counts appeared toward the 5' end of the negative-sense viral genome. Analysis by normalization and a simple sliding-window approach revealed plateau boundaries that suggested the emergence and enrichment of at least two truncated species having medium (similar to 5,900 nt) and short (similar to 4,000 nt) genomes. Relative measures of full-length and truncated species based on read counts were validated by quantitative reverse transcription-PCR (qRT-PCR). Limit-of-detection analysis suggests that deep sequencing can be more sensitive than complementary measures for detecting and quantifying defective particles in a population. Further, particle counts from transmission electron microscopy, coupled with infectivity assays, linked the rise in smaller genomes with an increase in truncated particles and interference activity. In summary, variation in deep sequencing coverage simultaneously shows the size, location, and relative level of truncated-genome variants, revealing a level of population heterogeneity that is masked by other measures of viral genomes and particles.IMPORTANCEWe show how deep sequencing can be used to characterize the emergence, diversity, and relative abundance of truncated virus variants in virus populations. Adaptation of this approach to natural isolates may elucidate factors that influence the stability and persistence of virus populations in nature.