Pigs Lacking the Scavenger Receptor Cysteine-Rich Domain 5 of CD163 Are Resistant to Porcine Reproductive and Respiratory Syndrome Virus 1 Infection.
Pigs Lacking the Scavenger Receptor Cysteine-Rich Domain 5 of CD163 Are Resistant to Porcine Reproductive and Respiratory Syndrome Virus 1 Infection.
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缺乏CD163的猪富含清道夫受体的富含半胱氨酸的结构域5具有对猪生殖和呼吸综合征病毒1感染的抗性。
DOI：
10.1128/jvi.00415-18
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发表时间：
2018-08-15
影响因子：
5.4
通讯作者：
Archibald AL
中科院分区：
文献类型：
--
作者：
Burkard C;Opriessnig T;Mileham AJ;Stadejek T;Ait-Ali T;Lillico SG;Whitelaw CBA;Archibald AL
猪繁殖与呼吸综合征（PRRS）病毒（PRRSV）是猪繁殖与呼吸综合征（PRRS）的病原体，可引起猪晚期流产、死产和呼吸道疾病，给全球养猪业造成重大经济损失。该病毒具有高度致突变性，可分为两个种，PRRSV-1和PRRSV-2，每个种含有几个亚型。目前的控制战略主要包括生物安全措施、人口减少和疫苗接种。疫苗充其量只能部分保护免受异源亚型和亚系的感染，并且经常有报道称经修饰的活疫苗会恢复毒力。在这里，我们证明了一个基因控制的方法，结果在体内对PRRSV感染的完全抗性。对CD 163进行编辑，以去除病毒相互作用结构域，同时保持蛋白质表达和生物学功能，避免与蛋白质敲除相关的任何潜在不良作用。这项研究证明了一种基因控制方法，对动物福利和猪肉行业都有潜在的好处。猪繁殖与呼吸综合征病毒（PRRSV）具有狭窄的宿主细胞嗜性，仅限于单核细胞/巨噬细胞谱系的细胞。CD 163蛋白在特定巨噬细胞类型的表面上以高水平表达，并且可溶性形式在血液中循环。CD 163已被描述为PRRSV的融合受体，其中清道夫受体富半胱氨酸结构域5（SRCR 5）区已被证明是病毒的相互作用位点。如前所述，我们已经产生了猪，其中在猪受精卵中使用CRISPR/Cas9编辑删除了CD 163基因的外显子7。这些猪表达缺乏SRCR 5的CD 163蛋白（Δ SRCR 5 CD 163），并且在标准饲养条件下饲养时未显示不良反应。不仅在巨噬细胞亚群的表面上检测到Δ SRCR 5 CD 163，而且还可以在编辑的猪的血清中检测到分泌的可溶性蛋白，如猪可溶性CD 163特异性酶联免疫吸附测定（ELISA）所示。先前的结果表明，来自Δ SRCR 5 CD 163动物的原代巨噬细胞在体外对PRRSV-1亚型1、2和3以及PRRSV-2感染具有抗性。在此，用高毒力的PRRSV-1亚型2毒株攻击Δ SRCR 5猪。与野生型对照组相比，Δ SRCR 5猪未显示感染体征，也未出现指示生产性感染的病毒血症或抗体应答。肺和淋巴结组织的组织学分析显示，在两种组织中均不存在病毒复制细胞。这表明Δ SRCR 5猪对病毒感染具有完全抗性。重要性猪繁殖与呼吸综合征（PRRS）病毒（PRRSV）是PRRS的病原体，可导致猪的晚期流产、死胎和呼吸道疾病，给全球养猪业造成重大经济损失。该病毒具有高度致突变性，可分为两个种，PRRSV-1和PRRSV-2，每个种含有几个亚型。目前的控制战略主要包括生物安全措施、人口减少和疫苗接种。疫苗充其量只能部分保护免受异源亚型和亚系的感染，并且经常有报道称经修饰的活疫苗会恢复毒力。在这里，我们证明了一个基因控制的方法，结果在体内对PRRSV感染的完全抗性。对CD 163进行编辑，以去除病毒相互作用结构域，同时保持蛋白质表达和生物学功能，避免与蛋白质敲除相关的任何潜在不良作用。这项研究证明了一种基因控制方法，对动物福利和猪肉行业都有潜在的好处。
Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiological agent of PRRS, causing late-term abortions, stillbirths, and respiratory disease in pigs, incurring major economic losses to the worldwide pig industry. The virus is highly mutagenic and can be divided into two species, PRRSV-1 and PRRSV-2, each containing several subtypes. Current control strategies mainly involve biosecurity measures, depopulation, and vaccination. Vaccines are at best only partially protective against infection with heterologous subtypes and sublineages, and modified live vaccines have frequently been reported to revert to virulence. Here, we demonstrate that a genetic-control approach results in complete resistance to PRRSV infection in vivo. CD163 is edited so as to remove the viral interaction domain while maintaining protein expression and biological function, averting any potential adverse effect associated with protein knockout. This research demonstrates a genetic-control approach with potential benefits in animal welfare as well as to the pork industry. Porcine reproductive and respiratory syndrome virus (PRRSV) has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 protein is expressed at high levels on the surface of specific macrophage types, and a soluble form is circulating in blood. CD163 has been described as a fusion receptor for PRRSV, with the scavenger receptor cysteine-rich domain 5 (SRCR5) region having been shown to be the interaction site for the virus. As reported previously, we have generated pigs in which exon 7 of the CD163 gene has been deleted using CRISPR/Cas9 editing in pig zygotes. These pigs express CD163 protein lacking SRCR5 (ΔSRCR5 CD163) and show no adverse effects when maintained under standard husbandry conditions. Not only was ΔSRCR5 CD163 detected on the surface of macrophage subsets, but the secreted, soluble protein can also be detected in the serum of the edited pigs, as shown here by a porcine soluble CD163-specific enzyme-linked immunosorbent assay (ELISA). Previous results showed that primary macrophage cells from ΔSRCR5 CD163 animals are resistant to PRRSV-1 subtype 1, 2, and 3 as well as PRRSV-2 infection in vitro. Here, ΔSRCR5 pigs were challenged with a highly virulent PRRSV-1 subtype 2 strain. In contrast to the wild-type control group, ΔSRCR5 pigs showed no signs of infection and no viremia or antibody response indicative of a productive infection. Histopathological analysis of lung and lymph node tissue showed no presence of virus-replicating cells in either tissue. This shows that ΔSRCR5 pigs are fully resistant to infection by the virus. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiological agent of PRRS, causing late-term abortions, stillbirths, and respiratory disease in pigs, incurring major economic losses to the worldwide pig industry. The virus is highly mutagenic and can be divided into two species, PRRSV-1 and PRRSV-2, each containing several subtypes. Current control strategies mainly involve biosecurity measures, depopulation, and vaccination. Vaccines are at best only partially protective against infection with heterologous subtypes and sublineages, and modified live vaccines have frequently been reported to revert to virulence. Here, we demonstrate that a genetic-control approach results in complete resistance to PRRSV infection in vivo. CD163 is edited so as to remove the viral interaction domain while maintaining protein expression and biological function, averting any potential adverse effect associated with protein knockout. This research demonstrates a genetic-control approach with potential benefits in animal welfare as well as to the pork industry.