Date Published: December 13, 2018
Publisher: Public Library of Science
Author(s): Zicong Xie, Daxin Pang, Hongming Yuan, Huping Jiao, Chao Lu, Kankan Wang, Qiangbing Yang, Mengjing Li, Xue Chen, Tingting Yu, Xinrong Chen, Zhen Dai, Yani Peng, Xiaochun Tang, Zhanjun Li, Tiedong Wang, Huancheng Guo, Li Li, Changchun Tu, Liangxue Lai, Hongsheng Ouyang, Shou-Wei Ding.
Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.
Classical swine fever virus (CSFV) belongs to the genus Pestivirus within the family Flaviviridae . CSFV is an enveloped virus that possesses a single-strand positive-sense 12. 3kb RNA genome, which contains a long open reading frame that encodes a 3898-amino acid (aa) poly-protein . The co- and post-translational processing of the poly-protein by cellular and viral proteases results in cleavage of the poly-protein into individual proteins , including four structural proteins (C, Erns, E1 and E2) and eight non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) .
CSF is one of the most economically important infectious diseases affecting pigs worldwide. Due to the economic importance of this virus, intensive control strategies for CSFV have been implemented for several decades, but the disease is still listed by OIE (OIE 2017). There are several possible reasons for the failure to stamp out the disease: (a) the virulence of CSFV is a complex and multifactorial phenomenon that has not been completely characterized; (b) the acquired strategies of viral evasion of the host antiviral response require further in-depth research; (c) the impacts of geography, climate, national policy and people’s awareness of the elimination of disease must be considered; (d) there are limitations associated with current commercial vaccines; (e) the control of wild boar reservoirs is a significant challenge. Additionally, the singleness of control strategies based on vaccination may also be a contributing factor. Therefore, there remains a long way to before elimination of the virus. As an alternative, the breeding of anti-CSFV pigs via a genome editing-based strategy could be a direct and effective approach, which would facilitate the permanent introduction of novel disease resistance traits into the mass population of production pigs via conventional breeding techniques.