Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies.
Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies.
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DOI：
10.1128/mbio.02020-16
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发表时间：
2017-03-14
期刊：
影响因子：
6.4
通讯作者：
Pfeiffer JK
中科院分区：
文献类型：
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作者：
Aguilera ER;Erickson AK;Jesudhasan PR;Robinson CM;Pfeiffer JK
空斑试验是用于测量病毒浓度的常用技术，其基于每个空斑代表单个感染单位的原理。因此，预期噬斑的数量与接种的病毒稀释度线性相关，并且每个噬斑应由单个创始病毒形成。在这里，我们检查了是否有一种以上的病毒会导致斑块形成。通过使用遗传标记脊髓灰质炎病毒的遗传和表型检测，我们发现即使在极低的感染复数下，5%至7%的斑块中也存在多个亲本病毒。我们通过视觉和生物物理测定证明，与许多病毒原液一样，我们的病毒原液含有单个颗粒和聚集体。这些数据表明，聚集的病毒粒子能够诱导共感染和嵌合空斑形成。事实上，在基于流式细胞术的测定中，通过暴露于低pH值诱导病毒体聚集增加了共感染。我们假设，高突变载量病毒产生的噬斑可能具有较高的合并感染频率，这是由于恢复适应性的过程，如互补和重组。事实上，我们发现合并感染频率与突变负荷相关，重度诱变病毒的嵌合空斑形成率为17%。重要的是，嵌合噬斑的频率可能被低估高达三倍，因为在我们的测定中不能对相同亲本病毒的共感染进行评分。这项工作表明，一种以上的病毒可以促进斑块的形成，并且在基因组损伤量高的情况下，合并感染可能有助于斑块的形成。定量病毒的最常用方法之一是空斑测定，其中通常假定每个空斑代表单个感染性病毒。使用遗传标记的脊髓灰质炎病毒，我们证明了一个空斑可以包含一个以上的亲本病毒，可能是由于病毒库内的聚集体诱导细胞的共感染。相对少量的空斑是我们标准病毒库的共感染产物。然而，具有增加的基因组损伤的诱变病毒原种产生更高量的嵌合噬斑。这些结果表明，合并感染可能有助于基因组损伤病毒的空斑形成，可能是由于互补和重组等过程。总体而言，我们的结果表明，病毒稀释度和空斑数之间的关系可能不是线性的，特别是对于诱变病毒群体。
The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high. One of the most common methods to quantify viruses is the plaque assay, where it is generally presumed that each plaque represents a single infectious virus. Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric. These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations.