Center for Food Animal Health, Ohio Agricultural Research and Development Center, Department of Animal Sciences, College of Food, Agricultural and Environmental Sciences, The Ohio State University, Wooster, OH 44691, USA.
Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Wooster, OH 44691, USA.
Viruses. 2022 Aug 20;14(8):1825. doi: 10.3390/v14081825.
Rotaviruses (RVs) are a significant cause of severe diarrheal illness in infants and young animals, including pigs. Group C rotavirus (RVC) is an emerging pathogen increasingly reported in pigs and humans worldwide, and is currently recognized as the major cause of gastroenteritis in neonatal piglets that results in substantial economic losses to the pork industry. However, little is known about RVC pathogenesis due to the lack of a robust cell culture system, with the exception of the RVC Cowden strain. Here, we evaluated the permissiveness of porcine crypt-derived 3D and 2D intestinal enteroid (PIE) culture systems for RVC infection. Differentiated 3D and 2D PIEs were infected with porcine RVC (PRVC) Cowden G1P[1], PRVC104 G3P[18], and PRVC143 G6P[5] virulent strains, and the virus replication was measured by qRT-PCR. Our results demonstrated that all RVC strains replicated in 2D-PIEs poorly, while 3D-PIEs supported a higher level of replication, suggesting that RVC selectively infects terminally differentiated enterocytes, which were less abundant in the 2D vs. 3D PIE cultures. While cellular receptors for RVC are unknown, target cell surface carbohydrates, including histo-blood-group antigens (HBGAs) and sialic acids (SAs), are believed to play a role in cell attachment/entry. The evaluation of the selective binding of RVCs to different HBGAs revealed that PRVC Cowden G1P[1] replicated to the highest titers in the HBGA-A PIEs, while PRVC104 or PRVC143 achieved the highest titers in the HBGA-H PIEs. Further, contrasting outcomes were observed following sialidase treatment (resulting in terminal SA removal), which significantly enhanced Cowden and RVC143 replication, but inhibited the growth of PRVC104. These observations suggest that different RVC strains may recognize terminal (PRVC104) as well as internal (Cowden and RVC143) SAs on gangliosides. Finally, several cell culture additives, such as diethylaminoethyl (DEAE)-dextran, cholesterol, and bile extract, were tested to establish if they could enhance RVC replication. We observed that only DEAE-dextran significantly enhanced RVC attachment, but it had no effect on RVC replication. Additionally, the depletion of cellular cholesterol by MβCD inhibited Cowden replication, while the restoration of the cellular cholesterol partially reversed the MβCD effects. These results suggest that cellular cholesterol plays an important role in the replication of the PRVC strain tested. Overall, our study has established a novel robust and physiologically relevant system to investigate RVC pathogenesis. We also generated novel, experimentally derived evidence regarding the role of host glycans, DEAE, and cholesterol in RVC replication, which is critical for the development of control strategies.
轮状病毒（RV）是导致婴幼儿和幼小动物（包括猪）严重腹泻的重要原因。C 组轮状病毒（RVC）是一种在全球范围内越来越多地在猪和人类中报告的新兴病原体，目前被认为是导致新生仔猪胃肠炎的主要病原体，给猪肉行业造成了重大经济损失。然而，由于缺乏强大的细胞培养系统，除了 RVC Cowden 株外，人们对 RVC 的发病机制知之甚少。在这里，我们评估了猪隐窝衍生的 3D 和 2D 肠类器官（PIE）培养系统对 RVC 感染的易感性。分化的 3D 和 2D PIE 被猪 RVC（PRVC）Cowden G1P[1]、PRVC104 G3P[18]和 PRVC143 G6P[5] 毒力株感染，并通过 qRT-PCR 测量病毒复制。我们的结果表明，所有 RVC 株在 2D-PIE 中复制能力较差，而 3D-PIE 支持更高水平的复制，这表明 RVC 选择性感染终末分化的肠细胞，而 2D-PIE 中的肠细胞比 3D-PIE 中的肠细胞少。虽然 RVC 的细胞受体尚不清楚，但细胞表面的靶碳水化合物，包括组织血型抗原（HBGAs）和唾液酸（SAs），被认为在细胞附着/进入中发挥作用。对 RVC 与不同 HBGAs 选择性结合的评估表明，PRVC Cowden G1P[1]在 HBGA-A PIE 中复制到最高滴度，而 PRVC104 或 PRVC143 在 HBGA-H PIE 中达到最高滴度。此外，在用唾液酸酶处理（导致末端 SA 去除）后观察到相反的结果，这显著增强了 Cowden 和 RVC143 的复制，但抑制了 PRVC104 的生长。这些观察结果表明，不同的 RVC 株可能识别神经节苷脂上的末端（PRVC104）和内部（Cowden 和 RVC143）SA。最后，测试了几种细胞培养添加剂，如二乙氨基乙基（DEAE）-葡聚糖、胆固醇和胆汁提取物，以确定它们是否可以增强 RVC 复制。我们观察到只有 DEAE-葡聚糖显著增强了 RVC 的附着，但对 RVC 的复制没有影响。此外，MβCD 耗尽细胞胆固醇抑制了 Cowden 的复制，而细胞胆固醇的恢复部分逆转了 MβCD 的作用。这些结果表明，细胞胆固醇在我们测试的 PRVC 株的复制中起着重要作用。总体而言，我们的研究建立了一种新的强大且生理相关的系统来研究 RVC 的发病机制。我们还生成了关于宿主糖、DEAE 和胆固醇在 RVC 复制中的作用的新的、实验衍生的证据，这对于控制策略的发展至关重要。
