Introduction. Influenza is a global health issue causing substantial health and economic burdens on affected populations. Routine, annual vaccination for influenza virus is recommended for all persons older than 6 months of age. The propagation of the influenza virus for vaccine production is predominantly through embryonated chicken eggs. Hypothesis/Gap Statement. Many challenges face the propagation of the virus, including but not limited to low yields and lengthy production times. The development of a method to increase vaccine production in eggs or cell lines by suppressing cellular gene expression would be helpful to overcome some of the challenges facing influenza vaccine production. Aims. This study aimed to increase influenza virus titres by using a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an antisense molecule, to suppress protein expression of the host genes interferon alpha (IFN-α) and interferon beta (IFN-β) in chicken embryo fibroblast (DF-1) cells. Methods. The toxicity of PPMOs was evaluated by cytotoxicity assays, and their specificity to inhibit IFN-α and IFN-β proteins was measured by ELISA. We evaluated the potential of anti-IFN-α and anti-IFN-β PPMOs to reduce the antiviral proteins in influenza virus-infected DF-1 cells and compared the virus titres to untreated controls, nonsense-PPMO and JAK/STAT inhibitors. The effects of complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells were evaluated, and the virus titres were compared between treatment groups. Results. Suppression of IFN-α by PPMO resulted in significantly reduced levels of IFN-α protein in treated wells, as measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMOs increased the ability of the influenza virus to replicate in DF-1 cells. Over a 2-log10 increase in viral production was observed in anti-IFN-α and IFN-β PPMO-treated wells compared to those of untreated controls at the initial viral input of 0.1 multiplicity of infection. The data from complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells was about 82 and 97% compared to the combined PPMO-treated but uncomplemented group and untreated group, respectively. There was a 0.5-log10 increase in virus titre when treated with anti-IFN-α and IFN-β PPMO compared to virus titre when treated with JAK/STAT inhibitors. Conclusions. This study emphasizes the utility of PPMO in allowing cell cultures to produce increased levels of influenza for vaccine production or alternatively, as a screening tool to cheaply test targets prior to the development of permanent knockouts of host gene expression.

中文翻译：

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通过使用肽缀合的磷酸二酰胺吗啉寡聚物抑制干扰素 α 和 β 来增加鸡胚成纤维细胞 (DF-1) 细胞中流感病毒复制以生产疫苗的替代策略

介绍。流感是一个全球性健康问题，给受影响人群造成巨大的健康和经济负担。建议所有 6 个月以上的人每年定期接种流感病毒疫苗。用于疫苗生产的流感病毒主要通过含胚鸡蛋进行繁殖。假设/差距陈述。病毒的传播面临许多挑战，包括但不限于产量低和生产时间长。开发一种通过抑制细胞基因表达来增加鸡蛋或细胞系中疫苗产量的方法将有助于克服流感疫苗生产面临的一些挑战。目标。本研究旨在通过使用肽缀合的磷酸二酰胺吗啉寡聚物 (PPMO)（一种反义分子）来抑制鸡宿主基因干扰素 α (IFN-α) 和干扰素 β (IFN-β) 的蛋白质表达，从而提高流感病毒滴度胚胎成纤维细胞（DF-1）。方法。通过细胞毒性测定评估PPMO的毒性，并通过ELISA测定其抑制IFN-α和IFN-β蛋白的特异性。我们评估了抗 IFN-α 和抗 IFN-β PPMO 减少流感病毒感染的 DF-1 细胞中抗病毒蛋白的潜力，并将病毒滴度与未处理的对照、无义 PPMO 和 JAK/STAT 抑制剂进行了比较。评估 PPMO 处理的感染细胞中 IFN-α 和 IFN-β 蛋白的互补和重建效果，并比较处理组之间的病毒滴度。结果。通过 ELISA 测量，PPMO 对 IFN-α 的抑制导致处理孔中 IFN-α 蛋白的水平显着降低，并且显示在测试的有效浓度下对 DF-1 细胞没有任何细胞毒性。自我导向 PPMO 的处理增加了流感病毒在 DF-1 细胞中复制的能力。在初始病毒输入为 0.1 感染复数时，与未处理的对照相比，在抗 IFN-α 和 IFN-β PPMO 处理的孔中观察到病毒产量增加了2-log 10以上。与联合PPMO处理但未互补组和未处理组相比，PPMO处理的感染细胞中IFN-α和IFN-β蛋白的互补和重建数据分别约为82%和97%。与用 JAK/STAT 抑制剂处理时的病毒滴度相比，用抗 IFN-α 和 IFN-β PPMO 处理时，病毒滴度增加了0.5-log 10 。结论。这项研究强调了 PPMO 在允许细胞培养物产生更高水平的流感病毒以用于疫苗生产方面的效用，或者作为一种筛选工具，在开发宿主基因表达的永久敲除之前廉价地测试目标。