|
|
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 A (online only)
Plaque-Reduction Neutralization
Assay
Serum samples were assayed for West Nile virus (WNV)–specific antibodies
by using the plaque-reduction neutralization test as previously described
(1). Briefly, each serum sample was diluted 1:5 in BA1 and mixed with
an equal volume of BA1 containing a suspension of WNV NY99-4132 at a concentration
of approximately 200 PFU/0.1 mL, such that the final serum dilution was
1:10 and the final concentration of WNV was approximately 100 PFU/0.1
mL. For the postinoculation serum samples, we also tested serial twofold
dilutions of serum to determine endpoint 90%-neutralization titers. In
most cases, preinoculation serum samples were tested for neutralizing
antibodies to St. Louis encephalitis virus, a closely related flavivirus
that may cross-react serologically with WNV (2) and may partially protect
against WNV infection (3). After 1 h incubation at 37°C, 5% CO2,
virus-serum mixtures were assayed for virus content by plaque assay (see
above). Controls used included BA1 only (cell viability control), bird
serum-free virus mixture with BA1 only (to enumerate the number of PFU
in the challenge dose of virus), and WNV hyperimmune mouse ascitic fluid
(diluted 1:200) mixture with virus (positive control, to verify challenge
virus identity).
Appendix A References
- Beaty BJ, Calisher CH, Shope RE. Arboviruses. In: Schmidt NJ, Emmons
RW, editors. Diagnostic procedures for viral, rickettsial and chlamydial
infections, 6th ed. Washington: American Public Health Association;
1989. p. 797–855.
- Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS,
Westaway EG, et al. Antigenic relationships between flaviviruses as
determined by cross-neutralization tests with polyclonal antisera. J
Gen Virol 1989;70:37–43.
- Tesh RB, Travassos da Rosa AP, Guzman H, Araujo TP, Xiao SY. Immunization
with heterologous flaviviruses protective against fatal West Nile encephalitis.
Emerg Infect Dis 2002;8:245–51.
|
