PLoS Pathogens:英国学者利用基因编辑技术培育出可抵御致命病毒的

2017年2月23日，国际微生物学知名期刊《PLOS Pathogens》在线发表了英国爱丁堡大学罗斯林研究所Alan L. Archibald研究员的一篇研究论文，研究报告了研究组利用CRISPR/Cas9基因编辑技术培育出一批能抵御致命性猪蓝耳病病毒感染的“超级猪”。

猪蓝耳病又称猪繁殖与呼吸综合征(PRRS)，在猪之间接触传染性很高，且因快速进化，现有疫苗无法有效阻止该病毒传播，全世界养猪业都深受其害，仅欧洲每年因此病就导致15亿欧元以上的损失。技术一旦获得使用许可，全世界养猪产业将每年减少数十亿英镑损失。

研究已经证明，猪蓝耳病病毒靶向巨噬细胞这类免疫细胞，而这类细胞表面一种被称为“CD163”的分子在推动该病毒感染中扮演关键角色。之前也有团队培育出体内不含整个CD163分子的转基因猪，并能成功抵御PRRS病毒传播。

Alan L. Archibald团队与英国著名种畜公司Genus合作，使用CRISPR/Cas9工具剪切掉CD163基因中与猪蓝耳病病毒感染有关的小部分片段，培育出32头转基因猪。对这些猪细胞的实验室检测证明，其对两种能引起PRRS病的子病毒具有完全抵抗力，从而能阻止感染和传播。下一步他们会继续研究这些猪曝露在病毒环境下的抗感染能力。

Alan L. Archibald表示，部分切除而不是全部切除CD163分子，既能增强抵御猪蓝耳病的能力，又不会影响与CD163分子有关的其他功能。“CRISPR/Cas9用于种畜产业，不仅能通过减少疾病传播来提高食品安全，更能减少农业人员应对传染病的负担。”

原文链接：

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

原文摘要：

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.

doi:10.1371/journal.ppat.1006206

作者：Alan L. Archibald