In contrast, they mentioned that infection with WN virus after recovery from SLE virus produced very high antibody titers and a non-specific response that was highly variable among individual birds within this treatment group. wild birds for WN and JE virus. Twenty-one samples were positive for neutralizing antibodies to WN. These results suggest that WN virus is prevalent among wild birds in the Far Eastern region of Russia. Introduction West Nile (WN) virus belongs to the genus in the family = 4) were inoculated with 100 PFU of either JE or WN virus. After 3 weeks, the chicks (23 days old) were inoculated again, this time with 1,000 PFU of heterologous virus (WN virus in chicks previously inoculated with JE virus or JE virus in chicks previously inoculated with WN virus). Viremia titration. To confirm that the chicks were infected, the viremia titers of 2- to 9-day-old chicks were measured by plaque assay using baby hamster kidney cells (BHK-21, ATCC #CCL-10). The BHK cell monolayers were grown in 12-well plates and GSK690693 inoculated with serial dilutions of the viral solutions. After 60 min of viral adsorption, the viral solution was aspirated, and the cells were washed three times with PBS(?). A 1 mL volume of overlay consisting of Eagle’s minimal essential medium (EMEM; Nissui Pharmaceutical Co., Japan) containing 1.5% carboxymethyl cellulose (CMC; Wako, Japan) and 2% FCS (CMC-EMEM) was added to the cells, and the plates were incubated at 37C in a CO2 incubator. After 5 days of culture, the CMC-EMEM was aspirated, and the cells were fixed and stained with a solution of 0.1% crystal violet and 10% formalin in PBS(?). After staining for 2 h, the cells were washed with water and then dried, and the Tbp plaques were counted. The viral titer was expressed as the number of PFUs per mL. The minimum threshold for virus detection was 50 PFU/mL. Antibody determination. The sera of chicks and wild birds were tested for the presence of neutralizing antibody by the 80% FRNT using the fluorescent antibody technique used previously for tick-borne encephalitis virus.10 The test sera (15 L) were diluted serially in 2-fold steps from 1:20 to 1 1:2,560 in a 96-well plate. Each serum dilution was then combined with an equal volume of WN or JE virus, adjusted to give a final count of 50 focus-forming units per well. The serum-virus mixtures were incubated for 60 min at 37C in a CO2 incubator. After incubation, the mixtures were transferred to the wells of 96-well plates containing a monolayer of BHK cells. The plates were incubated for 60 min at 37C to allow for virus adsorption. After removing the mixture, the cells were covered with CMC-EMEM. After incubation for 24 h at 37C, the medium was removed and the cells were washed with PBS(?) three times and fixed with absolute methanol at room temperature for 20 min. Focus staining was performed by the fluorescent antibody technique. Fixed BHK cells were treated consecutively with anti-WN virus mouse hyperimmune ascitic fluid (1:500) or anti-JE virus mouse hyperimmune ascitic fluid (1:800) and Alexa Fluor 555 goat anti-mouse IgG (1:400, Invitrogen, Carlsbad, CA). Each incubation lasted 60 min and was followed by three washes with PBS with Tween 20(T) (PBS-T). The neutralizing antibody titer was expressed as the reciprocal of the highest dilution that reduced the number of foci to 80% of the control value. The cutoff titer was set at 1:20 and 1:160 for wild birds. We tested chick sera for WN virus using a plaque reduction neutralization test (PRFT). Briefly, a BHK cell monolayer was prepared on a 12-well plate and plaques were visualized by staining with crystal violet solution, as described previously for virus titration. Other procedures were the same as those used for the FRNT. The neutralizing test (NT) antibody titers by plaque GSK690693 reduction neutralizing test (PRNT) were similar to those obtained by FRNT (S. Totani and I. Takashima, unpublished data). Serological analysis of wild birds in Far Eastern Russia. To GSK690693 determine the prevalence of WN virus in the Far Eastern region of Russia, we analyzed the seroprevalence of WN virus among wild birds. A total of 152 wild birds were captured at Khanka Lake, Anyuy River, and GSK690693 Chor River in Far Eastern Russia in August 2005 and 2006, and blood and kidneys were collected. These areas are known to be resting points for migratory birds.11,12 The presence of WN virus RNA was determined.