Survival rate, viral burden, and B cell responses after WNV infection.

<p><b>A</b>. Adult or old mice were infected with 10² PFU of WNV (New York strain) via subcutaneous injection in the footpad. Survival was monitored for 30 days: <i>n</i> = 20 adult (16 weeks) and <i>n</i> = 34 old (18 months) mice from at least three independent experiments. The difference was statistically different (<i>P</i> < 0.01; log rank test). <b>B-D.</b> Viral burden after WNV infection of adult and old mice was measured by qRT-PCR from serum (<b>B</b>) and plaque assay from spleen and brain (<b>C-D</b>). The data is shown as the mean titer (log₁₀ PFU/ml) ± standard deviation (SD) and reflects 8 to 9 mice per time point from at least two independent experiments. The dashed lines represent the limit of sensitivity of the assay. Asterisks indicate statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; Mann-Whitney test). <b>E-H</b>. Serum was obtained from adult and old mice at days 5, 8, 15, and 90 after WNV infection. <b>E and H</b>. Neutralizing antibody titers were determined by a focus-reduction assay. Results are shown as the mean of the reciprocal log₁₀ titer ± SD that yields 50% inhibition of infection and reflects data from 5 to 11 mice per group from three independent experiments. <b>F-G</b>. Anti-WNV IgM (<b>F</b>) and IgG (<b>G</b>) levels were measured by ELISA. Data is plotted as the reciprocal log₁₀ titer, defined as three times greater than background. <b>I-J.</b> Ninety days after infection, WNV-specific antibody secreting long-lived plasma cells in the bone marrow (LLPC) were quantified. Shown is the LLPC frequency per 10⁶ bone marrow cells ± SD (<b>I</b>), as well as representative ELISPOT images from three adult and three old mice (<b>J</b>). Data is pooled from 6 to 9 mice per group from three independent experiments.