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Electron Tomography of Negatively Stained Complex Viruses: Application in Their Diagnosis

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Electron Tomography of Negatively Stained Complex Viruses: Application in Their Diagnosis

Jan Mast et al. Diagn Pathol.

Abstract

Background: Electron tomographic analysis can be combined with the simple and rapid negative staining technique used in electron microscopy based virus diagnosis.

Methods: Standard negative staining of representative examples of parapoxviruses and paramyxoviruses was combined with electron tomographic analysis.

Results: Digital sectioning of reconstructions of these viruses at a selected height demonstrated the viral ultrastructure in detail, including the characteristic diagnostic features like the surface threads on C-particles of a parapoxvirus and individual glycoproteins and the internal nucleoprotein strand of Newcastle disease virus. For both viruses, deformation and flattening were observed.

Conclusion: The combination of negative staining of complex viruses with electron tomographic analysis, allows visualizing and measuring artifacts typical for negative staining. This approach allows sharp visualisation of structures in a subnanometer-thick plane, avoiding blurring due to superposition which is inherent to TEM. In selected examples, such analyses can improve diagnosis of viral agents.

Figures

Figure 1
Figure 1
Negative staining and electron tomographic analysis of Orf parapoxvirus. Figure 1A and B represent micrographs of uranyl acetate-stained particles of M-type and C-type Orf parapoxvirus characterised by obvious and hardly visible surface threads, respectively. Figure 1C is a 44-nm thick digital section taken along the Z-axis of the tomogram of the C-particle shown in B. A, B and C are represented with the same scale. Bar: 100 nm.
Figure 2
Figure 2
Negative staining and electron tomographic analysis of Newcastle disease paramyxovirus. Figure 2A represents a micrograph of a PTA-stained virion. Figure 2B and C are 44-nm thick digital sections taken along the Z-axis of the tomogram of the virion shown in Figure 2A, taken in the central part (B) and at the level of the envelope (C). Figure 2D and Figure 2E represent the inserts in Figure 2B and Figure 2C, respectively, at higher magnification. Figure 2F and Figure 2G represent side and top views of the 3D ribbon-model of the density map of the F-glycoprotein of Newcastle disease virus. Bar A, B and C: 100 nm, Bar D: 10 nm, Bar E: 8 nm.
Figure 3
Figure 3
Negative staining and electron tomographic analysis of a suspected feature. Figure 3A represents a micrograph of a negatively stained feature that, based on its size and general aspect, can be mistaken for a virion of a paramyxovirus. Figure 3B is a central digital section of 44 nm thick along the Z-axis of the tomogram of the feature shown in Figure 3A. Bar A, B: 100 nm.

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