Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163
Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163
复制标题
DOI：
10.7150/ijbs.25862
复制
发表时间：
2019-01-01
影响因子：
9.2
通讯作者：
Li, Ning
中科院分区：
文献类型：
--
作者：
Chen, Jingyao;Wang, Haitao;Li, Ning
猪繁殖与呼吸综合征(PRRS)是一种高度传染性疾病，也是全球养猪业最重要的经济疾病。高致病性PRRS病毒(Hp-PRRSv)是PRRSV的变种，具有较高的发病率和死亡率。清道夫受体CD163含有9个富含半胱氨酸的清道夫受体(SRCR)结构域，是PRRSV的关键进入介质。先前的研究表明，SRCR5(SRCR5)是PRRSV体外感染所必需的，它由外显子7编码。在这里，我们使用CRISPR/Cas9系统与供体载体相结合，用人CD163样蛋白1(HCD163L1)的相应外显子替换猪CD163的外显子7。在CD163(Mut/Mut)猪中，修饰CD163基因对血红蛋白-结合珠蛋白(Hb-HP)复合体清除和红细胞生长没有不良影响。体外感染实验表明，CD163突变体通过抑制病毒脱壳和基因组释放而强烈抑制HP-PRRSV的复制。与体内野生型(WT)猪相比，Hp-PRRSV感染的CD163(Mut/Mut)猪血液中的病毒载量显著降低，并缓解了PRRSV诱导的发热。虽然WT猪全部死亡，但有4头CD163(Mut/Mut)猪存活并在实验结束时恢复。结果表明，修饰CD163能显著抑制PRRSV复制，保护猪免受HP-PRRSV感染，为利用基因编辑技术培育抗PRRSV猪奠定了良好的基础。
Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163(Mut/Mut) pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163(Mut/Mut) pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163(Mut/Mut) pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.