Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function.
Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function.
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DOI：
10.1371/journal.ppat.1006206
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发表时间：
2017-02
期刊：
影响因子：
6.7
通讯作者：
Archibald AL
中科院分区：
文献类型：
--
作者：
Burkard C;Lillico SG;Reid E;Jackson B;Mileham AJ;Ait-Ali T;Whitelaw CB;Archibald AL
猪繁殖与呼吸综合征（Porcine Reproductive and Respiratory Syndrome，PRRS）是一种严重危害世界养猪业的传染病。PRRS在所有年龄的猪中表现不同，但主要导致母猪的晚期流产和死胎以及仔猪的呼吸道疾病。该疾病的病原体是正链RNA PRRS病毒（PRRSV）。PRRSV具有狭窄的宿主细胞嗜性，限于单核细胞/巨噬细胞谱系的细胞。CD 163已被描述为PRRSV的融合受体，其中清道夫受体富含半胱氨酸的结构域5（SRCR 5）区域被证明是病毒在体外的相互作用位点。CD 163在巨噬细胞表面以高水平表达，特别是在呼吸系统中。在这里，我们描述了CRISPR/Cas9对猪受精卵的应用，导致产生具有编码SRCR 5的CD 163基因的外显子7缺失的猪。SRCR 5的缺失显示在标准饲养条件下饲养的猪中没有不良影响，观察到正常的生长速率和全血细胞计数。从动物中分离肺泡巨噬细胞（PAM）和外周血单核细胞（PBMC），并在体外进行评估。通过CSF 1刺激从PBMC获得的PAM和巨噬细胞（PMM）均显示各自来源的巨噬细胞的特征性分化和细胞表面标志物表达。SRCR 5缺失CD 163在巨噬细胞表面的表达和正确折叠以及作为血红蛋白-触珠蛋白清除剂的蛋白质的生物活性被证实。用PRRSV基因型1、亚型1、2和3对PAM和PMM以及用PRRSV基因型2对PMM进行挑战，通过复制评估显示对病毒感染的完全抗性。共聚焦显微镜显示SRCR 5 CD 163缺失巨噬细胞中不存在复制结构，表明在基因表达之前，即在进入/融合或解包阶段，感染受到抑制。猪繁殖与呼吸综合征（Porcine Reproductive and Respiratory Syndrome，PRRS）是猪的一种地方性传染病，在不同年龄的猪中表现不同，但主要引起母猪的晚期流产和死胎以及仔猪的呼吸道疾病。该疾病的病原体是正链RNA PRRS病毒（PRRSV）。PRRSV仅感染单核细胞/巨噬细胞谱系的先天免疫系统的特定细胞亚群。前期研究发现，病毒需要一种特异性受体CD 163，才能使自身膜与宿主细胞膜融合在一个摄取囊泡中，将病毒遗传信息释放到胞质溶胶中，实现成功感染。CD 163具有珍珠串结构，其中发现“珍珠”/结构域编号5与病毒相互作用并允许其感染细胞。在这里，我们描述了我们如何在受精卵中使用所谓的CRISPR/Cas9基因编辑产生缺乏CD 163亚结构域5的猪。在正常饲养条件下，猪是健康的，并且发现由CD 163进行的其他生物学功能是完整的。我们从这些猪中分离出各种单核细胞和巨噬细胞，发现它们对PRRSV感染具有完全抗性。
Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages. Porcine Reproductive and Respiratory Syndrome is an endemic infectious disease of pigs, manifesting differently in pigs of different ages but primarily causing late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV only infects a specific subset of cells of the innate immune system of the monocyte/macrophage lineage. Previous research found that the virus needs a specific receptor, CD163, in order to make its own membrane fuse with the host cell membrane in an uptake vesicle to release the viral genetic information into the cytosol and achieve a successful infection. CD163 has a pearl-on-a-string structure, whereby the “pearl”/ domain number 5 was found to interact with the virus and allow it to infect a cell. Here we describe how we generated pigs lacking the CD163 subdomain 5 using so-called CRISPR/Cas9 gene editing in zygotes. The pigs were healthy under normal husbandry conditions and other biological functions conducted by the CD163 were found to be intact. We isolated a variety of monocyte and macrophage cells from these pigs and found them to be completely resistant to PRRSV infection.