High-resolution immuno-scanning electron microscopy using a non-coating method: study of herpes simplex virus glycoproteins on the surface of virus particles and infected cells.

The expression of two glycoproteins, i.e. glycoprotein C (gC) and glycoprotein D (gD), of herpes simplex virus type 1 (HSV-1) on the surface of extracellular particles of this virus was examined by immuno-scanning electron microscopy. Scanning electron microscopy specimens of infected cells immuno-labelled against the glycoproteins with colloidal gold particles were prepared by a conventional coating and a non-coating method. Surface ultrastructure of infected cells and gold particles were observed more clearly with specimens prepared by the non-coating method. The appearance of virus particles in association with glycoprotein expression on these particles and on the surface of infected cells was then studied. Progeny virus particles began to appear 6 h after infection, increased in number as the infection proceeded, and covered most of the cell surface by 16 h. Six to 24 h after the infection, the labelling density for each glycoprotein on virus particles remained constant. The labelling density for gD was always higher than that for gC. The patch-like distribution of gold-labelling against gD was often detected on infected cell monolayers at the exponential and late stage of one cycle of virus growth. The labelling density for gD on virus particles was the highest on these produced in Vero and L-929 cells, moderate in MRC-5, BHK-21 and FL cells, and the lowest in HEp-2 cells.