Linke Lyndsey M, Wilusz Jeffrey, Pabilonia Kristy L, Fruehauf Johannes, Magnuson Roberta, Olea-Popelka Francisco, Triantis Joni, Landolt Gabriele, Salman Mo
Animal Population Health Institute, Colorado State University, Fort Collins, CO, USA.
Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA.
AMB Express. 2016 Mar;6(1):16. doi: 10.1186/s13568-016-0187-y. Epub 2016 Feb 24.
Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.
甲型流感病毒对人类和动物构成重大的健康和经济威胁。禽流感病毒（AIV）的爆发对家禽业是一种负担，并增加了传播给人类的风险。在家禽中使用AIV疫苗存在局限性，这为控制疫情制造了障碍，因此需要其他有效的控制措施。RNA干扰（RNAi）技术的应用具有潜力；然而，RNAi介导剂的递送是限制其临床应用的一个众所周知的障碍。我们引入了一种新型抗病毒方法，使用靶向禽黏膜上皮细胞的细菌载体，并递送针对两种AIV基因（核蛋白（NP）和聚合酶酸性蛋白（PA））的小干扰RNA（siRNA）。使用红色荧光报告基因，我们首先证明了载体在禽上皮细胞中的递送和细胞内表达。随后，我们证明了预防性应用这些抗AIV载体时，AIV排泄物显著减少。这些抗病毒载体使排出的病毒滴度降低了多达10000倍，证明了使用这些新型抗AIV载体抑制AIV排泄物的体外概念验证。我们的结果表明，这种siRNA载体技术可能代表一种可扩展的、适用于禽类和人类流感的临床抗病毒技术，以及一种针对其他病毒的基于RNAi的载体原型。
