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Experimental Infection of
North American Birds with the New York 1999 Strain of West Nile Virus
Nicholas Komar,* Stanley Langevin,* Steven Hinten,* Nicole Nemeth,*†
Eric Edwards,*† Danielle Hettler,*† Brent Davis,* Richard Bowen,† and
Michel Bunning*‡
*Centers for Disease Control and Prevention, Fort Collins, Colorado, USA;
†Colorado State University, Fort Collins, Colorado, USA; and ‡Office of
the Surgeon General, United States Air Force, Bolling Air Force Base,
Washington, D.C., USA
Appendix B (online only)
Venipuncture
We collected blood samples from each bird before infection to confirm
seronegative status for West Nile virus (WNV). Additional samples were
collected once a day for 7 days postinoculation (dpi) at 24-h intervals,
and a final sample was collected 1 week later to confirm seroconversion.
We collected blood with 1-mL syringes with attached sub-Q needles (27-gauge
needles for birds <60 g in mass or 26-gauge for all other birds.)
Initial and final blood samples ranged in volume from 0.2–0.6 mL, depending
on the size of the bird, and were collected in Microtainer serum separator
tubes (Becton, Dickinson and Co., Franklin Lakes, NJ). Specimens were
held at ambient temperature for at least 30 min and then centrifuged for
3 min at 7,500 rpm in a refrigerated Eppendorf centrifuge (Model 5417R;
Brinkmann Instruments, Inc., Westbury, NY). Once a day, blood samples
(0.1 mL for birds <60 g in mass or 0.2 mL for all other birds)
were diluted in 4.5 V of BA1 diluent (composed of Hank’s M-199 salts,
1% bovine serum albumin, 350 mg/L sodium bicarbonate, 100 U/mL penicillin,
100 mg/L streptomycin, and 1 mg/L Fungizone in 0.05 M Tris, pH 7.6) in
a 2-mL cryovial. After approximately 30 min at ambient temperature (to
permit coagulation), these cryovials were placed temporarily on wet ice
and then centrifuged cold at 3,750 rpm for 10 min in a refrigerated Beckman
centrifuge (Model GS-6R, Beckman Instruments, Inc., Palo Alto, CA), effectively
separating the clot from a 1:10 dilution of serum. Centrifuged cryovials
were then stored at –70°C until titrated by Vero plaque assay.
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