Specific interference shRNA-expressing plasmids inhibit Hantaan virus infection in vitro and in vivo

Liu, Yuan-yuan; Chen, Liang-jun; Zhong, Yan; Shen, Meng-xin; Ma, Nian; Liu, Bing-yu; Luo, Fan; Hou, Wei; Yang, Zhan-qiu; Xiong, Hai-rong

Since the discovery of RNAi, synthetic siRNA duplexes that generally consist of 21-23 nucleotides with 2-nt 3' overhangs have been used to target viral RNAs in a sequence-specific manner following transfection [21, 22] . However, the application of siRNAs has been limited by their short half-lives, the high costs of synthetic siRNAs and instability due to degradation by nucleases. Another method for inducing RNAi is via the transfection of plasmids that express antiviral short-hairpin RNAs (shRNAs), and this method is also an efficient means of eliciting RNAi in vivo [15, 21] . Our results indicated that the With the exception of ribavirin, no antiviral drugs for the treatment of hantavirus infection have been identified. Several members of the Bunyaviridae family, particularly HTNV, are sensitive to ribavirin [8] . We have reported that ribavirin can induce an up to 3.6-fold decrease in the vRNA level in HTNV infection at 4 dpi [20] , which is equivalent to the in vitro effects of pSilencer-S and -M observed in our experiments. With regard to the in vivo administration, the RNAi plasmid treatments increased the survival rate to 27.3% in a lethal HTNVinfected suckling mouse model. Zhou et al reported that the NP-specific siRNA expression plasmid pBabe-NP protected two of the eight mice (2/8) challenged with the lethal dose of avian influenza virus (H5N1) that killed all of the control mice [15] ; this result is similar to the antiviral effects of the other siRNA expression plasmids against HTNV observed in our in vivo experiments. However, we noticed that ribavirin has been reported to be capably of affording 100% protection against lethal Andes virus infections in hamsters [8] and also increases the survival rate to 81.8% in SEOV-infected suckling ICR mice [23] . The explanation of these phenomena may be related to the delivery of siRNA. The shRNA expression plasmid was distributed in the brain because the blood-brain barrier (BBB) of newborn mice is immature [24] . However, as a nonviral vector, the pSilencer shRNA expression vector does not readily cross the cellular membrane and is not stably introduced into the cells. Further studies are required to solve this problem, which is frequently considered a hurdle for the development of siRNA-based therapeutics [25] .