A Heparan-Dependent Herpesvirus Targets the Olfactory Neuroepithelium for Host Entry
Figure 3
Both olfactory neurons and sustentacular cells are direct infection targets.
a. The neuroepithelium of a naive BALB/c mouse was stained for cytokeratin-18 to reveal sustentacular cells (brown) and for olfactory marker protein to reveal neurons plus apical neuronal cilia (brown). Sections were counter-stained with Mayer's Hemalum. b. A scanning electron micrograph of the murine neuroepithelium showing the dense apical network of neuronal cilia. c. BALB/c mice were allowed to inhale eGFP+ MuHV-4 (106 p.f.u.) and 1 day later analysed for viral lytic antigen (anti-MuHV-4) and eGFP expression with polyclonal sera (brown). The sections were counter-stained with Mayer's Hemalum. Filled arrowheads show examples of virus+ nuclei in the sustentacular cell layer; open arrowheads show examples of eGFP+ nuclei in the neuronal layer. d. At day 3 after infection as in c, the neuroepithelium was stained for MuHV-4 lytic antigens, or for OMP to show the distribution of neurons. Most virus+ nuclei were in the sustentacular cell layer between the neuronal nuclei and neuronal cilia. e. At day 3 after infection as in c, adjacent neuroepithelial sections were stained for MuHV-4 lytic antigens or virus-expressed eGFP. The former again showed positive nuclei mainly in the sustentacular cell layer, while the latter also stained nuclei throughout the neuronal layer. f. At day 1 after infection with eGFP+ MuHV-4 as in c, adjacent sections were stained for virus-expressed eGFP or lytic antigens. Each zoomed image shows the corresponding boxed region. The number of eGFP+ cells consistently exceeded by at least 3-fold the number of lytic antigen+ cells. The arrows show examples of positive staining. g. BALB/c mice were infected as in c, either with wild-type eGFP+ MuHV-4 or with an equivalent dose of ORF50− (replication-deficient) eGFP+ MuHV-4. Samples taken 1 day later were stained for virus-expressed eGFP with a polyclonal serum. The arrows show examples of positive staining, which conformed mainly to the distribution of sustentacular cells. To the right, arrowheads show examples of sustentacular and neuronal staining at higher magnification.