Physical Virology by Unknown

Author:Unknown
Language: eng
Format: epub
ISBN: 9783030147419
Publisher: Springer International Publishing

6.4 Negri Bodies

NBs are cytoplasmic inclusion bodies having a diameter up to a few μm. They have been discovered by Adelchi Negri in 1903 [16, 54] and are easily observed using Seller’s staining (a mixture of saturated solution of basic fuschin and methylene blue dissolved in methanol). These structures are typical of RABV infection of the brain and, as a consequence, have been used as a histological proof of the infection.

Similar structures, which are also referred as NBs, are observed in the cytoplasm of cell culture of both neuronal and non-neuronal origin [12, 13] (Fig. 6.4a). In electron microscopy, they appear as homogeneous dense spherical structures [12, 55] (Fig. 6.4b). At late stage of infections (16 h p.i. and after), they often wrap in a double membrane, most probably derived from rough endoplasmic reticulum [12], and their shape appears to be altered [12, 55]. At 24 h p.i., viral particles are observed budding from NBs into the compartment delimited by the associated double membrane [12, 56].

Fig. 6.4Negri bodies are liquid organelles. (a) BSR cells (a clone of BHK21 cells) were infected with CVS strain at a MOI of 0.5 and fixed at different times p.i. (8 h, 12 h, 16 h, 24 h). Confocal analysis was performed after staining with a mouse monoclonal anti-N antibody followed by incubation with Alexa-488 donkey anti-mouse IgG. DAPI was used to stain the nuclei. Scale bars correspond to 15 μm. (b) EM characterization of the ultrastructural aspects of NBs displaying an electron dense granular structure. Scale bar: 1 μm. (c) NBs can fuse together. BSR cells, infected by a recombinant RABV expressing a P protein fused to mCherry, were imaged at the indicated time. Images are shown at 2 min intervals. Scale bars: 3 μm. (d) Co-expression of RABV N and P proteins leads to the formation of inclusion bodies recapitulating NB properties. BSR cells constitutively expressing the T7 RNA polymerase (BSR-T7/5) were co-transfected by plasmids pTit-P and pTit-N [55]. N was revealed with a mouse monoclonal anti-N antibody followed by incubation with Alexa-488 donkey anti-mouse IgG, and P was revealed with a rabbit polyclonal anti-P antibody followed by incubation with Alexa-568 donkey anti-rabbit IgG. DAPI was used to stain the nuclei

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