Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan
J Virol. 2020 May 4;94(10). doi: 10.1128/JVI.01767-19.
Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures. The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.
流感 D 病毒（IDV）最初于 2011 年在美国分离。IDV 在全球范围内分布，是牛呼吸道疾病复合症（BRDC）的病原体之一，在育肥牛中引起高发病率和死亡率。IDV 致病的分子机制尚不清楚。反向遗传学系统不仅是研究病毒生物学的重要工具，而且还可用于重组疫苗病毒等应用。在这里，我们报告了用于 IDV 的基于质粒的反向遗传学系统的建立。我们首先验证了每个 7 节基因组 RNA 的 3'-末端核苷酸都含有尿嘧啶（U），这与之前的报道相反，然后我们能够通过共转染包含这些基因组 RNA 的 7 个质粒以及表达聚合酶蛋白和核蛋白的 4 个质粒将重组 IDV 成功生成到人直肠肿瘤 18G（HRT-18G）细胞中。与野生型病毒相比，重组病毒的生长受到限制，我们通过检查病毒粒子的基因组 RNA 含量来确定这种生长差异的原因。我们发现，与野生型病毒相比，重组病毒颗粒中病毒 RNA 节的比例不平衡，因此我们调整了转染中使用的每个质粒的量，以获得与野生型病毒具有相同复制能力的重组病毒。我们在这里建立的用于 IDV 的反向遗传学系统的工作将有广泛的应用，包括用于更好地研究 IDV 复制和致病性的研究，以及为开发 BRDC 对策做出贡献的研究。牛呼吸道疾病复合症（BRDC）在牛中引起高死亡率和发病率，在全球范围内造成经济损失。流感 D 病毒（IDV）被认为是 BRDC 的病原体之一。在这里，我们开发了一种反向遗传学系统，该系统允许从克隆 cDNA 生成 IDV 并在 IDV 基因组中引入突变。这种反向遗传学系统将成为用于研究理解病毒复制和致病性的分子机制的有力工具，并将导致针对 BRDC 的新对策的开发。
