Agarwal Shubhra, Baccam Prasith, Aggarwal Rakesh, Veerapu Naga Suresh
Virology Section, Department of Life Sciences, Shiv Nadar University, Gautam Buddha Nagar, Uttar Pradesh, India.
IEM, Inc., Bel Air, Maryland, USA.
J Virol. 2018 Jan 30;92(4). doi: 10.1128/JVI.01932-17. Print 2018 Feb 15.
Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 () or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses.
许多RNA病毒以一群基因多样、正在复制的群体形式存在，即所谓的突变云。这种突变云的遗传多样性（云大小）和组成可能会影响病毒的几个重要表型特征，包括其复制能力。我们采用了一种简单的无细菌方法，即使用易错PCR结合反向遗传学，从1型戊型肝炎病毒（HEV）毒株中产生具有不同遗传多样性水平的感染性突变RNA云。对所得云团中变体的包含开放阅读框1（ORF1）70%的基因组片段或全基因组进行克隆和测序，结果显示核苷酸突变的发生频率约为每复制一个核苷酸10次，并且存在显著的遗传多样性，具有较高的标准化香农熵值。突变云在细胞培养中表现出短暂复制，而野生型HEV则没有。这些细胞培养的横断面数据支持了不同大小和组成的云团对表型特征存在差异影响，例如（+）-RNA子代的复制水平、双链RNA（病毒复制速率的替代指标）和ORF1蛋白的量，以及干扰素刺激基因的表达。由于突变云的大小和组成会影响病毒的表型特性，更好地理解这种关系可能有助于进一步深入了解病毒进化和新兴病毒性疾病的预测。目前一些生物学或实际限制阻碍了对突变云表型行为的研究。我们开发了一种简单快速的方法来合成戊型肝炎病毒（HEV）——一种单链（+）-RNA [ss（+）RNA]病毒——具有不同且可控遗传多样性水平的突变云，然后可将其用于细胞培养系统中，以研究云大小和组成对病毒表型的影响。在横断面分析中，我们证明了一个具有极高遗传多样性的特定突变云的复制速率超过了野生型HEV。因此该方法应为理解ss（+）RNA病毒的表型行为提供一个有用的模型。
