Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs. There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P < 0.05) of viral mRNA and DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses. In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.

中文翻译：

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多基因靶向 siRNA 下调 Immediate Early-2 (Ie2) 和 DNA 聚合酶基因介导的新型大鼠巨细胞病毒（All-03 株）抑制

巨细胞病毒 (CMV) 是一种机会性病原体，可在先天性感染的新生儿和无免疫能力的个体中引起严重的并发症。开发有效的疫苗是一项重大的公共卫生优先事项，目前的药物正面临着对接受者的耐药性和副作用。在本研究中，为了探索对抗 CMV 复制的新策略，对几种靶向 IE2 和 DNA 聚合酶基因区域的抗 CMV siRNA 进行了表征，并将其组合用于抗病毒治疗。在用 CMV 株 ALL-03 感染之前，用多 siRNA 转染大鼠胚胎成纤维细胞 (REF)。通过组织培养感染剂量 (TCID50)、细胞病变效应 (CPE) 和液滴数字 PCR (ddPCR) 测量病毒生长抑制，而通过实时 PCR 确定 IE2 和 DNA 聚合酶基因敲低。更昔洛韦被用作对照，以对各个 siRNA 的抗病毒活性的功效进行基准测试。对于通过 MTT 比色法分析的 REF 细胞上的所有 siRNA 组合，没有遇到显着的细胞毒性（P > 0.05）。与对照组相比，被 RCMV ALL-03 感染的细胞中的细胞病变效应 (CPE) 发展得明显更少，而且发展速度要慢得多。靶向基因的表达被成功下调导致病毒mRNA和DNA拷贝显着减少（P < 0.05）（dpb + dpc：79%、68%；dpb + ie2b：68%、60%；dpb + dpc + ie2b： 48%、42%）。流式细胞术分析显示，与对照组相比，经组合 siRNA 处理的细胞存活率和早期凋亡百分比更高。值得注意的是，对靶向基因区域的siRNA进行测序，未遇到突变，从而避免形成具有潜在抗性病毒的突变体。总之。该研究展示了创新方法的巨大前景，即同时部署针对多个基因的组合 siRNA，旨在控制宿主细胞中的 CMV 复制。