To evaluate the correlation between neutralization, epitope localization and in vivo protection, we assessed the therapeutic activity of different neutralizing monoclonal antibodies in an established mouse model 5 . Studies were performed with 5-week-old wild-type C57BL/6 mice, which have a ∼10% survival rate 5 . Mice were inoculated subcutaneously with 10 2 plaque-forming units (PFU) of WNV and administered a single dose of monoclonal antibody at day 2 after infection. Notably, 500 µg of the non-neutralizing monoclonal antibody E2 provided no protection (data not shown). In contrast, 100 µg of any of three different neutralizing monoclonal antibodies that map to K307 (E16, E24 or E34) protected greater than 90% of mice from lethal infection ( Fig. 3a-c) . Even a single 4 µg treatment of E16 or E34 on day 2 after infection prevented mortality. Given that humans can present with WNV infection of the CNS, we evaluated the therapeutic efficacy of monoclonal antibodies at later , or (c) E34 monoclonal antibodies. As controls, mice were independently administered saline (PBS) or a negative control monoclonal antibody (anti-SARS ORF7a, 500 µg). The survival curves were constructed using data from two independent experiments. The number of animals for each antibody dose ranged from 20 to 30. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown (P < 0.0001). (d) WNV burden in the brain of 5-week-old wild-type mice. At days 4, 5 and 6 after WNV infection, brains were harvested and viral burdens were determined by plaque assay. The following percentage of mice had viral burdens below detection (<20 PFU/g): day 4, 33%; day 5, 22%; day 6, 17%. (e,f) Efficacy of WNV-specific monoclonal antibody therapy at days 4 (e) and 5 (f) after infection. A single dose (0.5 mg at day 4 or 2 mg at day 5) of monoclonal antibody (E16, E24, E34 or anti-SARS ORF7a) was administered either 4 or 5 d after WNV infection. Data reflect approximately 20 mice per condition. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown at day 4 (P < 0.0001) and day 5 (E16, P = 0.0009; E24, P = 0.027). (g) Effect of E16 therapy on viral burden. Mice were treated with 2 mg of E16 or PBS on day 5 after WNV infection. On day 9, brains were recovered, homogenized and subjected to plaque assay. For a subset (6) that received PBS treatment, brains were harvested at days 7 and 8 from moribund mice. The data is expressed as PFU/g. Of 16 mice treated with E16 68% (11) had undetectable viral loads in the brain at day 9 whereas all mice (14 of 14) treated with PBS had detectable virus at the time of harvest. The dotted line represents the limit of sensitivity of the assay and the dark bars represent the mean of the log values. The differences were statistically different (P < 0.0001).