Differences in Viral Disinfection Mechanisms as Revealed by Quantitative Transfection of Echovirus 11 Genomes
Differences in Viral Disinfection Mechanisms as Revealed by Quantitative Transfection of Echovirus 11 Genomes
复制标题
DOI：
10.1128/aem.00961-19
复制
发表时间：
2019-07-01
影响因子：
4.4
通讯作者：
Kohn, Tamar
中科院分区：
文献类型：
--
作者：
Torrey, Jason;von Gunten, Urs;Kohn, Tamar
病毒灭活机制可以通过测量特定病毒功能丧失（例如宿主附着、基因组内化和基因组复制）的方法来阐明。基因组功能经常通过基于 PCR 的方法进行评估，这种方法是间接的并且可能不准确；影响高保真 PCR 酶检测的基因组损伤可能不会对实际细胞酶产生功能性病毒的能力产生不利影响。因此，我们在这里开发了一种基于转染的检测方法，通过将病毒RNA直接插入宿主细胞来定量确定病毒基因组功能，以测量它们从受损病毒基因组中产生新功能病毒的能力。用臭氧、游离氯 (FC)、254 nm 紫外线 (UV254) 或热量处理埃可病毒 11，然后比较基因组功能和感染性的降低情况。臭氧按传染性成比例地降低基因组功能，表明基因组损伤是病毒失活的主要机制。相比之下，与感染性相比，FC 几乎没有或没有造成基因组功能损失，这表明蛋白质损伤的作用更大。对于 UV254，基因组功能丧失约占病毒失活的 60%，其余部分可能是由于蛋白质损伤所致。热处理不会导致基因组功能降低，这与热失活是由衣壳损伤引起的认识一致。我们的结果表明，先前研究中通过PCR酶测量的基因组完整性降低与消毒后的实际基因组功能（基因组是否可以产生病毒）之间存在根本差异。与 PCR 相比，定量转染测定可以更真实地了解消毒过程中的实际病毒基因组功能和整体灭活机制。 重要性 本研究通过直接测量病毒基因组在消毒后产生新病毒的能力，为评估病毒灭活机制提供了一种新工具。此外，我们还发现了 PCR 确定病毒基因组损伤的潜在缺陷，它不能反映基因组是否真正具有功能。这里提出的使用定量转染的结果证实了之前提出的一些病毒灭活方法（热）的病毒灭活机制，同时为其他方法（臭氧、FC 和 UV254）带来了更多见解。所开发的转染方法为评估常见水消毒剂的实际病毒灭活效果提供了一种更加机械化的方法。
Virus inactivation mechanisms can be elucidated by methods that measure the loss of specific virus functionality (e.g., host attachment, genome internalization, and genome replication). Genome functionality is frequently assessed by PCR-based methods, which are indirect and potentially inaccurate; genome damage that affects detection by high-fidelity PCR enzymes may not adversely affect the ability of actual cellular enzymes to produce functional virus. Therefore, we developed here a transfection-based assay to quantitatively determine viral genome functionality by inserting viral RNA into host cells directly to measure their ability to produce new functional viruses from damaged viral genomes. Echovirus 11 was treated with ozone, free chlorine (FC), UV light at 254 nm (UV254), or heat, and then the reductions in genome functionality and infectivity were compared. Ozone reduced genome functionality proportionally to infectivity, indicating that genome damage is the main mechanism of virus inactivation. In contrast, FC caused little or no loss of genome functionality compared to infectivity, indicating a larger role for protein damage. For UV254, genome functionality loss accounted for approximately 60% of virus inactivation, with the remainder presumably due to protein damage. Heat treatment resulted in no reduction in genome functionality, in agreement with the understanding that heat inactivation results from capsid damage. Our results indicate that there is a fundamental difference between genome integrity reductions measured by PCR enzymes in previous studies and actual genome functionality (whether the genome can produce virus) after disinfection. Compared to PCR, quantitative transfection assays provide a more realistic picture of actual viral genome functionality and overall inactivation mechanisms during disinfection.IMPORTANCE This study provides a new tool for assessing virus inactivation mechanisms by directly measuring a viral genome's ability to produce new viruses after disinfection. In addition, we identify a potential pitfall of PCR for determining virus genome damage, which does not reflect whether a genome is truly functional. The results presented here using quantitative transfection corroborate previously suggested virus inactivation mechanisms for some virus inactivation methods (heat) while bringing additional insights for others (ozone, FC, and UV254). The developed transfection method provides a more mechanistic approach for the assessment of actual virus inactivation by common water disinfectants.