Mukherjee Santanu, Zhou Xiaohong, Rajaiya Jaya, Chodosh James
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Howe Laboratory, Harvard Medical School, Boston, Massachusetts, United States.
Invest Ophthalmol Vis Sci. 2015 Jan 6;56(1):472-7. doi: 10.1167/iovs.14-15635.
We determined the ultrastructure of mouse adenovirus keratitis, a model for human adenovirus keratitis.
Adenovirus keratitis was induced in C57Bl/6j mice by intrastromal injection of human adenovirus species D type 37 (HAdV-D37) with a heat-pulled, glass, micropipette needle under compressed air. At select time points after infection, mice were euthanized and their corneas removed, fixed, and sectioned at 70-nm thickness for electron microscopy.
Injection of HAdV-D37 into the mouse corneal stroma placed virus predominantly in the pericellular corneal stromal matrix. Virus was seen bound to and entering stromal cells at 1 and 2 hours after infection, respectively. Cell membrane transit by virus was seen to involve two distinct structures resembling caveolae and macropinosomes. However, later during infection intracellular virus was not seen within membrane-bound organelles. By 8 hours after infection, intracellular virus had accumulated into densely packed, perinuclear arrays. Virus disassembly was not obvious at any time point after infection. Infiltrating neutrophils seen by one day after infection had engulfed degraded stromal cells by 4 days after infection.
By transmission electron microscopy, injected HAdV-D37 readily enters stromal cells in the C57Bl/6j mouse cornea and induces stromal inflammation, as was shown previously by light microscopy. However, electron microscopy also revealed dense, static arrays of intracytoplasmic virus, suggesting a block in viral capsid disassembly and viral DNA nuclear entry. These findings may explain why human adenoviruses do not replicate in the mouse corneal stroma.
我们确定了小鼠腺病毒角膜炎的超微结构，它是人类腺病毒角膜炎的一种模型。
在压缩空气下，用热拉制的玻璃微量移液器针将人腺病毒D种37型（HAdV-D37）基质内注射到C57Bl/6j小鼠体内，诱导腺病毒角膜炎。在感染后的特定时间点，对小鼠实施安乐死，取出其角膜，进行固定，并切成70纳米厚的切片用于电子显微镜检查。
将HAdV-D37注射到小鼠角膜基质中后，病毒主要位于角膜基质细胞周围的基质中。在感染后1小时和2小时分别观察到病毒与基质细胞结合并进入基质细胞。病毒通过细胞膜的过程涉及两种不同的结构，类似于小窝和巨吞饮小泡。然而，在感染后期，未在膜结合细胞器内观察到细胞内病毒。感染后8小时，细胞内病毒已积累成密集的核周排列。在感染后的任何时间点都未观察到明显的病毒解体。感染后1天可见浸润的中性粒细胞，到感染后4天已吞噬了降解的基质细胞。
通过透射电子显微镜观察，注射的HAdV-D37很容易进入C57Bl/6j小鼠角膜的基质细胞并诱导基质炎症，这与之前光学显微镜观察的结果一致。然而，电子显微镜还揭示了细胞质内病毒的密集、静态排列，提示病毒衣壳解体和病毒DNA进入细胞核存在障碍。这些发现可能解释了人类腺病毒为何不在小鼠角膜基质中复制。
