Institute of Virology and Immunology, 3012 Bern, Switzerland.
Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland.
Viruses. 2021 Mar 18;13(3):502. doi: 10.3390/v13030502.
The ruminant-associated influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5' and 3' rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5' and 3' NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5' and 3' non-coding regions (NCR) highlighted that the terminal 3' end of all segments harbours an uracil instead of a cytosine nucleotide, similar to other influenza viruses. Subsequent analysis on the functionality of the 5' and 3' NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5' and 3' NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin, as well as by using an in vitro model recapitulating the natural replication site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.
反刍动物相关的 D 型流感病毒（IDV）宿主范围广泛，具有潜在的人畜共患病性。为了鉴定和分析影响 IDV 宿主范围的分子病毒决定因素，需要建立一个反向遗传系统。为此，我们首先对所有七个基因组片段进行了 5' 和 3' 快速扩增 cDNA 末端（RACE），然后评估了 5' 和 3' 非编码区（NCR）的活性，之后再将一株当代瑞士牛源 IDV 分离株（D/CN286）的病毒基因组片段构建到双向 pHW2000 载体中。将双向质粒转染到 HRT-18G 细胞中，然后在同一细胞类型上进行病毒拯救。对片段特异性 5' 和 3' NCR 的分析表明，所有片段的末端 3' 端都含有尿嘧啶而不是胞嘧啶核苷酸，与其他流感病毒相似。随后在 minireplicon 测定中对 5' 和 3' NCR 的功能进行分析，结果表明这些序列具有功能，并且 5' 和 3' NCR 的可变序列长度影响报告基因的表达。此后，我们评估了反向遗传克隆在人源、猪源和牛源常规细胞系以及体外模拟 IDV 在牛和猪中自然复制部位的模型中的复制效率。结果表明，反向遗传克隆 D/CN286 在所有细胞培养模型中都能有效地复制。综合这些结果，证明了从一株当代牛源 IDV 分离株成功建立了反向遗传系统，可用于未来鉴定和分析影响 IDV 广泛宿主范围的病毒决定因素。
