Porcine reproductive and respiratory syndrome(PRRS)is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus(PRRSV),resulting in substantial economic losses in the swine industry.Modifyin...Porcine reproductive and respiratory syndrome(PRRS)is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus(PRRSV),resulting in substantial economic losses in the swine industry.Modifying the CD163 SRCR5 domain,either through deletion or substitution,can eff1ectively confer resistance to PRRSV infection in pigs.However,large fragment modifications in pigs inevitably raise concerns about potential adverse effects on growth performance.Reducing the impact of genetic modifications on normal physiological functions is a promising direction for developing PRRSV-resistant pigs.In the current study,we identified a specific functional amino acid in CD163 that influences PRRSV proliferation.Viral infection experiments conducted on Marc145 and PK-15CD163 cells illustrated that the mE535G or corresponding pE529G mutations markedly inhibited highly pathogenic PRRSV(HP-PRRSV)proliferation by preventing viral binding and entry.Furthermore,individual viral challenge tests revealed that pigs with the E529G mutation had viral loads two orders of magnitude lower than wild-type(WT)pigs,confirming effective resistance to HP-PRRSV.Examination of the physiological indicators and scavenger function of CD163 verified no significant differences between the WT and E529G pigs.These findings suggest that E529G pigs can be used for breeding PRRSV-resistant pigs,providing novel insights into controlling future PRRSV outbreaks.展开更多
【目的】猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS),俗称"蓝耳病",是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种高致死性传染病,对世...【目的】猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS),俗称"蓝耳病",是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种高致死性传染病,对世界养猪业造成了巨大的经济损失。由于PRRSV遗传变异性较大,因此国内外并未有理想疫苗能够对此病进行有效防制。Cluster of differentiation 163(CD163)是PRRSV感染猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)的受体之一,研究旨在利用CRISPR/Cas9技术结合体细胞核移植技术制备CD163基因编辑的大白猪。【方法】针对猪CD163基因的第7外显子设计构建CRISPR/Cas9基因编辑载体;转染大白猪胎儿成纤维细胞,获得基因编辑阳性细胞克隆;以基因编辑细胞为核供体、体外成熟的猪卵母细胞为核受体构建克隆胚胎;胚胎移植到受体母猪生产CD163基因编辑猪,并进行后续的扩繁试验。【结果】设计的g RNA能够高效的识别靶位点。对获得的127个细胞单克隆进行PCR和测序显示,共有21个克隆发生突变,其中14个克隆为单等位基因突变或双等位基因杂合突变,7个克隆为双等位基因纯合突变。通过体细胞核移植技术,成功获得了CD163双等位基因编辑的纯合大白猪;并获得首批F1代CD163基因编辑仔猪,目前健康状态良好。随后将有更多的F1代CD163基因编辑猪陆续出生。【结论】制备的无抗性筛选标记的CD163双等位基因编辑猪,能够安全并快速地为培育抗PRRSV新品种猪提供育种材料。展开更多
基金Major Scientific and Technological Projects in Agricultural Biological Breeding of China(2023ZD0404302)Youth Program of National Natural Science Foundation of China(32202754)。
文摘Porcine reproductive and respiratory syndrome(PRRS)is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus(PRRSV),resulting in substantial economic losses in the swine industry.Modifying the CD163 SRCR5 domain,either through deletion or substitution,can eff1ectively confer resistance to PRRSV infection in pigs.However,large fragment modifications in pigs inevitably raise concerns about potential adverse effects on growth performance.Reducing the impact of genetic modifications on normal physiological functions is a promising direction for developing PRRSV-resistant pigs.In the current study,we identified a specific functional amino acid in CD163 that influences PRRSV proliferation.Viral infection experiments conducted on Marc145 and PK-15CD163 cells illustrated that the mE535G or corresponding pE529G mutations markedly inhibited highly pathogenic PRRSV(HP-PRRSV)proliferation by preventing viral binding and entry.Furthermore,individual viral challenge tests revealed that pigs with the E529G mutation had viral loads two orders of magnitude lower than wild-type(WT)pigs,confirming effective resistance to HP-PRRSV.Examination of the physiological indicators and scavenger function of CD163 verified no significant differences between the WT and E529G pigs.These findings suggest that E529G pigs can be used for breeding PRRSV-resistant pigs,providing novel insights into controlling future PRRSV outbreaks.
