Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
J Virol. 2018 Oct 12;92(21). doi: 10.1128/JVI.01208-18. Print 2018 Nov 1.
Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain J knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain J knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain J knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the J knockout pigs, suggesting that the Ig heavy chain J knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and J knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected J knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems. According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain J knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel J knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and J knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected J knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.
戊型肝炎病毒（HEV）是导致戊型肝炎的病原体，是一种重要但尚未完全了解的病原体，可导致孕妇死亡率高，并导致免疫功能低下者发生慢性肝炎。导致肝损伤的潜在机制尚不清楚；然而，体液免疫反应被牵连在内。在这项研究中，使用 CRISPR/Cas9 技术生成了免疫球蛋白（Ig）重链 J 敲除无菌猪，以耗尽 B 淋巴细胞群，导致对基因型 3 HEV 感染无法产生体液免疫反应。与野生型无菌仔猪相比，Ig 重链 J 敲除仔猪的 B 淋巴细胞频率明显降低，尽管其他先天和适应性 T 淋巴细胞群体的水平相似。随后在重链 J 敲除和野生型无菌仔猪中确定了急性 HEV 感染的动态。数据表明，与 J 敲除仔猪相比，野生型仔猪粪便和血清中的病毒 RNA 载量更高，表明 Ig 重链 J 在猪中的敲除实际上降低了 HEV 复制水平。感染 HEV 的野生型和 J 敲除无菌仔猪比其他用常规猪进行的研究发展出更明显的淋巴浆细胞性肝炎和肝细胞坏死病变。与磷酸盐缓冲盐水接种组相比，感染 HEV 的 J 敲除仔猪的肝脏也明显肿大，肝脏/体重比也明显增大。这种新型无菌猪模型将有助于未来对 HEV 致病性的研究，这方面在现有动物模型系统中一直难以重现。根据世界卫生组织的数据，每年约有 2000 万人感染 HEV，导致 330 万例戊型肝炎和超过 4.4 万人死亡。缺乏能够模拟全谱感染结果的有效动物模型，阻碍了我们阐明 HEV 发病机制的能力。在这里，我们使用 CRISPR/Cas9 技术成功地生成了免疫球蛋白重链 J 敲除无菌猪，建立了新型 J 敲除和野生型无菌猪 HEV 模型，并系统地确定了无菌猪急性 HEV 感染的动态。结果表明，猪的 Ig 重链敲除降低了 HEV 的复制水平。感染的野生型和 J 敲除无菌仔猪比其他使用常规猪进行的研究发展出更明显的 HEV 特异性病变，感染的 J 敲除仔猪的肝脏明显肿大。这种新型模型的出现将有助于未来对 HEV 致病性的研究。
