Infected and uninfected MDCK cells were embedded for standard electron microscopy or immunogold electron microscopy in a mixture of Epon-substitute and Araldite or in LR White resin (Ted Pella, Inc., Redding, CA, USA), respectively, as described (6
). The immunogold electron microscopy protocol used a goat antibody raised against the matrix protein of influenza A virus as a primary antibody and a donkey anti-goat antibody conjugated to 12-nm colloidal gold particles as a secondary antibody. In addition, lung tissues were obtained from the upper and lower respiratory tracts of 2 patients who died of pandemic (H1N1) 2009. Samples from each patient were negative for parainfluenza viruses and respiratory syncytial virus by PCR. Areas for examination were selected on the basis of strong immunohistochemical labeling for influenza virus. Sections for light microscopy sections were cut from the electron microscopy blocks, and areas were selected for either bronchus with submucosal glands or lung with alveoli.
Pandemic (H1N1) 2009 virus isolates grown in MDCK cells were morphologically similar to those of other influenza A viruses. In negative stain preparations, virions appeared mostly spherical (average diameter 104 nm) with some filamentous particles (up to 3.3 μm in length) and contained surface projections of the hemagglutinin and neuraminidase glycoproteins (, panel A). By thin section electron microscopy, infected cells showed virus particles being assembled mostly at the plasma membrane. Extracellular virions, averaging 86 nm in diameter, were mostly ovoid or filamentous (, panel B). The virions were surrounded by the glycoprotein spikes, and the individual nucleocapsids inside the virions were seen as thin threads (when cut longitudinally) or dark dot-like figures (when cut in cross-section) and measured ≈8 nm in diameter (, panels B and C).
Figure 1 Electron microscopy of pandemic (H1N1) 2009 virus. A) Negatively stained virions grown in MDCK cells showing spherical particles with distinct surface projections. Scale bar = 100 nm. B) Filamentous and ovoid particles assembling at the plasma membrane. (more ...)
The nuclei of some infected cells contained dense tubular structures, which had a rough outer edge and averaged 37 nm in width (, panel D). Immunogold electron microscopy labeling that used an anti–matrix protein goat antibody detected matrix (M) proteins on the dense tubular structures (, panel E) as well as on virions (C. Goldsmith et al., unpub. data).
Lung tissues from patients with fatal cases of pandemic (H1N1) 2009 were examined by electron microscopy. Infected cells and virions were observed in the alveolar spaces and in the submucosal glands (, panels A, B, and C). Spherical or ovoid extracellular viral particles, which may represent cross-sections through filamentous particles, were seen in respiratory tissues. Dense, amorphous material was associated with the virions. In addition, intranuclear dense tubules, similar to those seen in tissue culture–infected cells, were recognized in infected cells (, panel D).
Figure 2 Spherical and ovoid extracellular pandemic (H1N1) 2009 virus particles in human lung tissue found in the alveolar space (A) and in a submucosal gland (B). Nucleocapsids and surface projections are visible on some virions. Note the dense material (arrows) (more ...)