2.5. Replication of APMVs in 1-day-old Chicks, 2-week-old Chickens, and 3-week-old Ducks Following Intranasal Inoculation Three 1-day-old chicks per group were inoculated with 100 ml of each virus (256 HA units/bird) via the intranasal route. On day 3 post-infection, tissue samples (lung, trachea, spleen, and brain) were collected for virus titration by limiting dilution and immunoperoxidase assay as described above. To evaluate the tropism of APMVs in older birds, groups of ten 2week-old SPF chickens were inoculated with 200 ml of each virus (256 HA units/bird) by the intranasal route. Three birds from each group were sacrificed at 4 dpi and tissues samples (lung, trachea, spleen, and brain) were collected and processed for immunohistochemistry as described later. Tissue samples also were homogenized for virus titration. Virus titers in DF1 or Vero cells were determined by limiting dilution as described above. To confirm the replication of viruses in infected birds, the tissue samples were also inoculated into 9-day-old embryonated chicken eggs. On 3 dpi, virus growth was determined by HA assay. The remaining birds were observed daily for 10 days for any clinical signs and then sacrificed for virus titration of various tissues as described above. To evaluate virus replication in different hosts, we further determined replication of APMVs in 3-week-old mallard ducks. Six birds each were infected with 500 ml of individual APMVs (256 HA units/bird) via the combined intranasal and intratracheal routes and sacrificed at 4 dpi for collection of tissue samples (lung, trachea, spleen, and brain). Homogenates were prepared for virus titration in cell cultures and replication in eggs as described above. The remaining birds were observed daily for 10 days for any clinical signs.