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Journal of Integrative Agriculture  2023, Vol. 22 Issue (7): 2188-2199    DOI: 10.1016/j.jia.2022.11.010
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

XU Kui1, 2*, ZHOU Yan-rong3*, SHANG Hai-tao2, 4*, XU Chang-jiang1, TAO Ran3, HAO Wan-jun3, LIU Sha-sha1, MU Yu-lian1#, XIAO Shao-bo3#, LI Kui1#

1State Key Laboratory of Animal Nutrition and Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

2Shenzhen Kingsino Technology Co., Ltd., Shenzhen 518106, P.R.China

3State Key Laboratory of Agricultural Microbiology and Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, P.R.China

4Precision Medicine Institute, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, P.R.China

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猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS)是猪传染性最强和致死性最高的病毒性疾病之一。尽管分化抗原簇163 (Cluster of differentiation 163, CD163) 蛋白被确定为介导PRRS病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)感染的必需受体,但CD163参与感染的重要氨基酸残基仍不清楚。鉴定这些关键残基对于研究PRRSV感染机制和制备抗PRRSV的基因编辑猪具有重要意义。对CD163结构分析表明,CD163 SRCR5结构域内的配体结合口袋(ligand-binding pocket, LBP)(位于CD163的第487位到499位氨基酸)loop 5–6(位于CD163的第544位到570位氨基酸可能参与了PRRSV的感染。CD163 LBP位点特异性编辑猪可以完全抵抗PRRSV感染,但loop 5-6的破坏或缺失是否能抑制PRRSV的感染尚未报道。R561(第561位的精氨酸(R))位于loop 5-6,R561A突变的PK-15细胞可以显著增强对PRRSV的抗感染能力,但定点编辑CD163 R561的猪肺泡巨噬细胞(porcine alveolar macrophages, PAMsPRRSV感染的靶细胞)或CD163 R561编辑猪能否抑制PRRSV的感染仍不清楚。这项研究中,我们首先利用CRISPR/Cas9介导的片段删除技术制备了CD163缺失40个氨基酸残基的永生化猪肺泡巨噬细胞(immortalized porcine alveolar macrophage, IPAM细胞系,这40个残基位于CD163的第523位到562位氨基酸,这其中包含了R561和部分loop 5–6结构,该细胞系被命名为IPAM-CD163523-562病毒感染实验表明,IPAM-CD163523-562可以完全抵抗 PRRSV的感染。同时,我们利用CRISPR/Cas9介导同源重组技术制备了携带CD163-R561ACD163561位精氨酸(R)被替换为丙氨酸(A))的基因编辑克隆猪,并分离得到原代CD163-R561A PAMs。PRRSV攻毒实验结果表明,与野生型的CD163-R561 PAMs相比,CD163-R561A PAMs PRRSV的易感性显著降低。以上研究结果表明,CD163的第523位到562位氨基酸中含有介导PRRSV感染的必需氨基酸残基,CD163 R561参与了PRRSV的感染过程,但它不是感染所必需的。这些位点可以作为了解PRRSV感染机制的新靶点CD163-R561A猪也可作为培育抗PRRSV猪群体的育种材料

Abstract  Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most infectious viral diseases of swine. Although Cluster of differentiation 163 (CD163) is identified as an essential receptor for mediating PRRS virus (PRRSV) infection, the important residues involved in infection on CD163 are still unclear. Therefore, it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs. In this study, we first generated immortalized porcine alveolar macrophage (IPAM) cell lines harboring 40-residues (residues 523–562, including R561 (arginine (R) at position 561)) deletion of CD163. PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection. We then generated cloned pigs carrying CD163- R561A (an arginine (R) to alanine (A) substitution at position 561 of CD163). PRRSV challenge experiments in porcine alveolar macrophages (PAMs) isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs. Through this study, we show that CD163 523–562 contains essential residues for mediating PRRSV infection, and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection. These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection. Furthermore, CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.
Keywords:  pigs        porcine alveolar macrophages        dual-sgRNA        homology-directed repair        PRRSV        CD163  
Received: 16 September 2022   Accepted: 10 November 2022

This work was supported by the Major Scientific Research Tasks for Scientific and Technological Innovation Projects of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202006), the National Transgenic Breeding Project (2018ZX08009-26B), the Shenzhen Science and Technology Plan Project (CJGJZD20210408092402006), and the Shenzhen Key Technology Projects (JSGG20180507182028625).

About author:  XU Kui, E-mail:; ZHOU Yan-rong, E-mail:; SHANG Hai-tao, E-mail:; #Correspondence MU Yu-lian, E-mail:; XIAO Shao-bo, E-mail: cn; LI Kui, E-mail: * These authors contributed equally to this study.

Cite this article: 

XU Kui, ZHOU Yan-rong, SHANG Hai-tao, XU Chang-jiang, TAO Ran, HAO Wan-jun, LIU Sha-sha, MU Yu-lian, XIAO Shao-bo, LI Kui. 2023. Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus. Journal of Integrative Agriculture, 22(7): 2188-2199.

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