Establishment of a Reverse Genetics System for Influenza D Virus
Establishment of a Reverse Genetics System for Influenza D Virus
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DOI：
10.1128/jvi.01767-19
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发表时间：
2020-05-01
影响因子：
5.4
通讯作者：
Horimoto, Taisuke
中科院分区：
文献类型：
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作者：
Ishida, Hiroho;Murakami, Shin;Horimoto, Taisuke
D型流感病毒(IDV)于2011年在美国首次分离。IDV广泛分布于世界各地，是引起牛呼吸道疾病复合体(BRDC)的病原之一，可导致饲养场牛的高发病率和高死亡率。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，也是用于重组疫苗病毒等应用的重要工具。在这里，我们报告了一个基于质粒的IDV反向遗传学系统的建立。我们首先验证了每个7段基因组RNA的3‘端核苷酸中含有尿嘧啶(U)，然后我们将含有这些基因组RNA的7个质粒与4个表达聚合酶蛋白和核蛋白的质粒共转染到人直肠肿瘤18G(HRT-18G)细胞中，成功地构建了重组IDV。与野生型病毒相比，重组病毒存在生长缺陷，我们通过检测病毒颗粒的基因组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，重组病毒在颗粒中掺入的病毒RNA片段比例不平衡，因此我们调整了每个质粒的转染量，以获得与野生型病毒具有相同复制能力的重组病毒。我们在建立IDV反向遗传学系统方面的工作将有广泛的应用，包括用于更好地了解IDV复制和致病性的研究，以及那些有助于开发BRDC对策的研究。D型流感病毒(IDV)被认为是BRDC的病原体。在这里，我们开发了一种反向遗传学系统，允许从克隆的cDNA中产生IDV，并将突变引入IDV基因组。这种反向遗传学系统将成为了解病毒复制和致病的分子机制相关研究的有力工具，并将导致开发针对BRDC的新对策。
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.IMPORTANCE 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.