Abstract
Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus–host cell interaction.
While there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus infected primary neurons, grown on plastic coverslips, to allow for sectioning of neurons and axons in their growth plane. This technique allows for TEM examination of cell bodies, axons, growth cones and varicosities, providing powerful insights into virus–cell interaction.
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References
Roingeard P (2008) Viral detection by electron microscopy: past, present and future. Biol Cell 100:491–501
Bozzola J, Russell LD (1999) Electron mircroscopy. Principles and techniques for biologist, 2nd edn. Jones and Bartlett Publishers, Burlington
Egerton RF (2008) Physical principles of ELectron microscopy. An introduction to TEM, SEM and AEM. Springer, New York
Griffiths G (1993) Fine structure immunocytochemistry. Springer-Verlag, Berlin Heidelberg
Saksena MM, Wakisaka H, Tijono B, Boadle RA, Rixon F, Takahashi H, Cunningham AL (2006) Herpes simplex virus type 1 accumulation, envelopment, and exit in growth cones and varicosities in mid-distal regions of axons. J Virol 80:3592–3606
Miranda-Saksena M, Boadle RA, Aggarwal A, Tijono B, Rixon FJ, Diefenbach RJ, Cunningham AL (2009) Herpes simplex virus utilizes the large secretory vesicle pathway for anterograde transport of tegument and envelope proteins and for viral exocytosis from growth cones of human fetal axons. J Virol 83:3187–3199
Aggarwal A, Miranda-Saksena M, Boadle RA, Kelly BJ, Diefenbach RJ, Alam W, Cunningham AL (2012) Ultrastructural visualization of individual tegument protein dissociation during entry of herpes simplex virus 1 into human and rat dorsal root ganglion neurons. J Virol 86:6123–6137
Miranda-Saksena M, Boadle RA, Diefenbach RJ, Cunningham AL (2015) Dual role of herpes simplex virus 1 pUS9 in virus anterograde axonal transport and final assembly in growth cones in distal axons. J Virol 90(5):2653–2663. https://doi.org/10.1128/JVI.03023-15
Tennyson VM (1970) The fine structure of the axon and growth cone of the dorsal root nerurobalst of the rabbit embryo. J Cell Biol 44:62–79
Nakata T, Terada S, Hirokawa N (1998) Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons. J Cell Biol 140:659–674
Goldberg JL (2003) How does an axon grow? Genes Dev 17:941–958
de Kort EJ, Gribnau AA, van Aanholt HT, Nieuwenhuys R (1985) On the development of the pyramidal tract in the rat. I. The morphology of the growth zone. Anat Embryol (Berl) 172(2):195–204
Zhou ZH, Dougherty M, Jakana J, He J, Rixon FJ, Chiu W (2000) Seeing the herpesvirus capsid at 8.5 A. Science 288:877–880
Holland DJ, Miranda-Saksena M, Boadle RA, Armati P, Cunningham AL (1999) Anterograde transport of herpes simplex virus proteins in axons of peripheral human fetal neurons: an immunoelectron microscopy study. J Virol 73:8503–8511
Grunewald K, Desai P, Winkler DC, Heymann JB, Belnap DM, Baumeister W, Steven AC (2003) Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science 302:1396–1398
Acknowledgments
This work was supported by the Australian National Health and Medical Research Grants (402457 and 570849), the Westmead Millennium Institute, and the Westmead Medical Research Foundation.
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Miranda-Saksena, M., Boadle, R.A., Cunningham, A.L. (2020). Preparation of Herpes Simplex Virus-Infected Primary Neurons for Transmission Electron Microscopy. In: Diefenbach, R., Fraefel, C. (eds) Herpes Simplex Virus . Methods in Molecular Biology, vol 2060. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9814-2_20
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DOI: https://doi.org/10.1007/978-1-4939-9814-2_20
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