Birds that did not eat or drink on their own due to severe disease signs were euthanized, and their deaths were recorded on the following day of observation. Tracheal and cloacal swabs were collected from all ducks on days 3, 5, 7, and 10 p.i., and 0.5 ml of drinking water was sampled on days 1, 3, 5, 7, and 10 p.i. Influenza virus was detected by virus isolation in 10-day-old embryonated sellckchem chicken eggs as previously described (14, 47). The virus was titrated in positive samples by calculating the EID50, using the method of Reed and Muench (35); the lower limit of quantification was 0.75 log10 EID50/ml. Swab samples with detectable influenza virus but titers below the limit of quantification were reported as having a titer of <101 EID50/ml. All data shown were derived from two separate experiments.

All animal experiments were approved by the Animal Care and Use Committee of St. Jude Children’s Research Hospital (Memphis, TN) and were performed in compliance with relevant institutional policies, the Association for the Accreditation of Laboratory Animal Care guidelines, the National Institutes of Health regulations, and local, state, and federal laws. Transient expression of HA and NA proteins. Monolayers of Vero cells in 6-well dishes (85 to 95% confluence) were transiently transfected with 1 ��g of pCAGGS A/chicken/Vietnam/C58/04 HA DNA by using the Lipofectamine Plus expression system (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Transfected Vero cells were incubated for 4 h at 37��C.

DMEM (containing 10% fetal bovine serum and 1% glutamine) was then added to cells, and cells were incubated for 16 h at 37��C. Cells were then treated as indicated for each experiment. Neuraminidase (NA) protein was expressed by using 0.1 to 1.0 ��g of the pCAGGS A/chicken/Vietnam/C58/04 NA plasmid. Syncytium assay. Monolayers of Vero cells grown in 6-well plates were transfected with 1.0 ��g pCAGGS HA as described above or were infected with recombinant virus at an MOI of ~3 PFU per cell. At 16 h posttransfection or 6 h postinfection, cell monolayers were overlaid for 5 min with phosphate-buffered saline with magnesium and calcium (PBS+) that was adjusted to the reported pH with a 0.1 pH unit resolution using 0.1 M citric acid. Cells were neutralized by using DMEM (containing 10% fetal bovine serum and 1% glutamine) and were incubated at 37��C for 2 h. Samples were fixed and stained with a Hema 3 stat pack staining kit (Fisher) according to the manufacturer’s instructions. Representative Batimastat microscopic fields were captured with a Nikon D70 digital camera attached to a Nikon Eclipse TS100 inverted microscope (26). NA activity assay and NA inhibition.