A Heparan-Dependent Herpesvirus Targets the Olfactory Neuroepithelium for Host Entry
Figure 8
Virus binding to the olfactory neuroepithelium.
a. 1 week old C57BL/6 mice were allowed to inhale wild-type eGFP+ MuHV-4 (106 pf.u. in 2 µl). The next day neuroepithelial sections were analysed for viral lytic antigens and virus-expressed eGFP by immunofluorescent staining with polyclonal sera. Nuclei were counter-stained with DAPI. The zoomed image shows the boxed region of the merge. b. The same analysis was applied to ORF50−eGFP+ MuHV-4, which without complementation expresses eGFP but not new lytic antigens. c. 1 week old C57BL/6 mice were allowed to inhale MuHV-4 (106 pf.u. in 2 µl) and 1 min later the same volume of PBS. They were then analysed by immunofluorescent staining of neuroepithelial sections for viral antigens. Neuronal cilia were visualised by staining for α-tubulin. Nuclei were counter-stained with DAPI. An equivalent section from a naive mouse is shown for comparison. d. Mice were infected as in c and examined for virus binding to the neuroepithelium and respiratory epithelium after 5 min or 45 min. The arrows at 45 min show viral antigen+ neurons. e. In a higher power image of the neuroepithelium 45 min after virus inhalation (106 pf.u. in 2 µl), the dashed yellow lines outline neurons, as defined by α-tubulin staining, and the arrows show aggregated virion antigens along neuronal dendrites. f. After infection and staining as in d, virus binding was quantitated by counting MuHV-4+ pixels over a fixed area of apical epithelium and then normalizing by the α-tubulin signal of the same area. Each point shows the result for 3 sections from 1 mouse. The horizontal bars show medians. Comparison by Student's 2 tailed unpaired t test showed that neuroepithelial and respiratory epithelial binding were not significantly different at 1 min post-inoculation (p = 0.08) but at all subsequent time points neuroepithelial binding was significantly greater (p<0.05).