Boshuizen Jos A, Reimerink Johan H J, Korteland-van Male Anita M, van Ham Vanessa J J, Koopmans Marion P G, Büller Hans A, Dekker Jan, Einerhand Alexandra W C
Laboratoryof Pediatrics, Pediatric Gastroenterology, and Nutrition, Erasmus MC/ Sophia, Rotterdam, The Netherlands.
J Virol. 2003 Dec;77(24):13005-16. doi: 10.1128/jvi.77.24.13005-13016.2003.
Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.
轮状病毒是婴幼儿肠胃炎的最重要病因。由于迄今为止尚未对轮状病毒感染的体内黏膜反应进行广泛研究，我们将小鼠小肠中的病毒复制与黏膜结构、上皮细胞稳态、细胞动力学及分化的改变联系起来。给7日龄的哺乳BALB/c小鼠接种2×10⁴个小鼠轮状病毒空斑形成单位，并与模拟感染的对照组进行比较。记录腹泻疾病和病毒排泄情况，并评估小肠组织中轮状病毒（NSP4和结构蛋白）及肠上皮细胞特异性（乳糖酶、SGLT1和L-FABP）的mRNA和蛋白表达。通过（免疫）组织化学评估形态学、凋亡、增殖和迁移情况。感染后第1至5天观察到腹泻，第1至10天观察到病毒排泄。感染后第1天和第4天观察到轮状病毒复制的两个高峰。组织学变化的特征为空泡化肠上皮细胞的积聚。令人惊讶的是，空泡化细胞的数量超过了可检测到病毒复制的细胞数量。感染后第1至7天凋亡和增殖增加，导致绒毛萎缩。上皮细胞更新明显快于对照组（<4天，对照组为7天）。由于上皮更新在4天内发生，病毒复制的第二个高峰很可能是由新合成细胞的感染引起的。早在感染后6小时，感染细胞中肠上皮细胞特异性基因的mRNA和蛋白水平表达就下调。总之，我们首次表明轮状病毒感染在体内诱导凋亡、上皮细胞更新增加以及显示病毒复制的肠上皮细胞中基因表达的关闭。肠上皮细胞特异性基因表达的关闭，连同成熟肠上皮细胞因凋亡而丧失以及这些细胞被分化程度较低的分裂细胞替代，可能导致肠上皮吸收功能缺陷，这有助于轮状病毒发病机制。
