A Heparan-Dependent Herpesvirus Targets the Olfactory Neuroepithelium for Host Entry
Figure 4
Direct neuronal and sustentacular infections also occur in infant mice.
a. 1 week old C57BL/6 mice - BALB/c and C57BL/6 strains gave equivalent data in both adults and juveniles - were allowed to inhale luciferase+ MuHV-4 (104 p.f.u. in 2 µl), and imaged for light emission 5 and 14 days later. The scale shows maximum radiance (photons sec−1 cm−2 steradian−1). The images are representative of >30 infections. b. Mice were infected as in a, then monitored serially by luciferin infection and CCD camera scanning of emitted light. Each point shows the result for 1 mouse. SCLN = superficial cervical lymph nodes (neck signal). The dashed line shows the lower limit of assay sensitivity. c. 1 week old C57BL/6 mice were allowed to inhale wild-type eGFP+ MuHV-4 (106 p.f.u. in 2 µl) and 4 days later analysed by immunostaining for virus-expressed eGFP. The zoomed image shows the boxed region. The lower image shows a further magnified region of neuroepithelium with both sustentacular and neuronal eGFP expression. Equivalent results were obtained in 6 mice. d. 1 week old C57BL/6 mice were infected as in c but with replication-defective ORF50−eGFP+ MuHV-4 (106 p.f.u., assayed on complementing cells). Infection was identified 1 day later by immunostaining for virus-expressed eGFP. The zoomed image shows the boxed region, with arrowheads indicating examples of positive (sustentacular cell) staining. The lower image shows a further magnified region of neuroepithelium with eGFP+ neurons. Equivalent results were obtained in 6 mice. e. Immunofluorescent staining of naive 1 week old C57BL/6 mice shows the characteristic distributions of OMP (neuronal axons, dendrites and cell bodies), α-tubulin (broadly expressed, including neuronal cilia) and cytokeratin-18 (sustentacular cells). Nuclei were counterstained with DAPI. The merged image shows distinct layers of neuronal cell bodies, sustentacular cells and neuronal cilia. f. 1 week old C57BL/6 mice were allowed to inhale ORF50−eGFP+ (106 p.f.u. with complementation) and 1 day later analysed by immunofluorescent staining for eGFP, cytokeratin-18 and OMP. Nuclei were counterstained with DAPI. Co-localization appears yellow in the merged images. Equivalent results were obtained in 3 mice.