Serial Section Array Scanning Electron Microscopy Analysis of Cells from Lung Autopsy Specimens following Fatal A/H1N1 2009 Pandemic Influenza Virus Infection
Serial Section Array Scanning Electron Microscopy Analysis of Cells from Lung Autopsy Specimens following Fatal A/H1N1 2009 Pandemic Influenza Virus Infection
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DOI：
10.1128/jvi.00644-19
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发表时间：
2019-10-01
影响因子：
5.4
通讯作者：
Nakajima, Noriko
中科院分区：
文献类型：
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作者：
Kataoka, Michiyo;Ishida, Kinji;Nakajima, Noriko
2009年A/H1N1大流行性流感病毒（A/H1N1/pdm09）首次被确定为新型甲型流感大流行性病毒（IAV）。先前，我们报道了在a /H1N1/pdm09肺炎患者的尸检肺组织中检测到许多病毒抗原。确定病毒与宿主细胞之间的关系对于阐明IAV肺炎的发病机制具有重要意义。为了研究病毒颗粒的分布和宿主细胞的形态变化，我们使用透射电子显微镜（TEM）和一种新的扫描电子显微镜（SEM）方法对该患者的尸检肺标本进行了检查。我们将aec - ii作为病毒抗原阳性细胞，将单核/巨噬细胞（Ms/M phi s）和中性粒细胞（news）作为先天免疫细胞。我们发现了病毒颗粒和核内致密小管，它们与IAV的基质1 （M1）蛋白相关。大规模的二维观测是通过数字“拼接”在一起连续的扫描电镜图像。使用序列切片阵列(SSA)-SEM进行单个全细胞分析，鉴定出细胞质内和/或AEC-11s、Ms/M phi s和news表面周围的囊泡中的病毒颗粒；核内致密小管仅见于aec -11。计算机辅助处理来自每种细胞类型的SSA-SEM图像，实现了ACE-II、M/M phi和Neu中病毒颗粒分布的三维（3D）建模。一般来说，在季节性流感患者的死后样本中很难观察到禽流感病毒颗粒。事实上，在尸检肺切片的支气管上皮细胞中只检测到少数病毒抗原。以前，我们在肺aec -ll细胞中检测到许多病毒抗原。这是因为肺组织中的大多数A/H1N1/pdm09在血凝素蛋白的222位（D222G）上含有天冬氨酸到甘氨酸的取代。含有D222G取代的A/H1N1/pdm09对人aec上表达的α -2,3链唾液酸具有受体结合偏好，并以与H5N1和H7N9禽流感lav相同的方式感染它们。在这里，我们报告了首次成功观察病毒颗粒，不仅在AEC-11s，而且在Ms/M phi s和news中，使用电子显微镜。在囊泡内和细胞表面发现含有大量病毒颗粒的M/M phi，表明M/M phi参与了IAV原发性肺炎的发病机制。
A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/M phi s) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally "stitching" together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-11s, Ms/M phi s, and Neus; however, intranuclear dense tubules were found only in AEC-11s. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/M phi, and a Neu.IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-lls from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for alpha-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian lAVs. Here, we report the first successful observation of virus particles, not only in AEC-11s, but also in Ms/M phi s and Neus, using electron microscopy. The finding of a M/M phi) harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/M phi are involved in the pathogenesis of IAV primary pneumonia.