Receptor specificity of RGMal/01 and single and multi round replication of the virus in HAE cells. (A) Receptor specificity was measured by Glycan Array. Virus was analyzed at hemagglutination titers of 128 per 50 μl. The virus exhibited a strong preference for binding to mainly avian-type α 2–3 receptors, but not α 2–6 sialosides. 1–32 represent the glycans that contain α 2–3 SA (light gray), whereas 33–45 are the glycans with α 2–6 SA (dark gray). (B) HAE cultures were infected with RGMal/01 at an MOI of 1.0 and fixed at 7 hpi. The cilia (gray) and viral antigen (red) were visualized by double immunostaining. RGMal/01 is more efficient at infecting ciliated cells. (C) HAE cultures were inoculated via the apical side with the viruses at an MOI of 0.2. The progeny viruses released into the apical side were collected at the indicated time points and titrated in the MDCK cells by performing a TCID50 assay. Each bar represents the average for two independent experiments run with duplicate HAE cultures. (D) At end of the sampling (60 hpi) described from panel C, the cultures were fixed and stained for cilia (gray) and distribution of viral antigen (red).