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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 C (online only)
Plaque Assay
Plaque assay for detecting and titrating infectious WNV particles in
blood samples, cloacal and oral swabs, and organ tissues was carried out
by using a double overlay system in Vero (a continuous line of African
Green Monkey kidney cells) culture. Most blood specimens were titrated
after a single freeze-thaw cycle. Organ samples were titrated after one
or two freeze-thaw cycles. Some high-titered specimens were titrated by
using serial 10-fold dilutions in BA1 after an additional freeze-thaw.
After inoculating cell monolayers with 0.1 mL (in duplicate) and incubating
the sample for 1 h at 37°C, 5% CO2, each well was overlaid
with 3 mL of 0.5% agarose in M-199 medium supplemented with 350 mg/L sodium
bicarbonate, 29.2 mg/L L-glutamine, and antibiotics as in BA1. After 48
h of additional incubation, a second 3-mL 0.5% agarose overlay containing
0.004 % neutral red dye was added for plaque visualization. Plaques were
counted on days 3 and 4 after infection of the Vero cells.
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