a, Schematic representation of study design. AG6 mice were infected with the JEV SA-14 strain. Subsequently, a 100 µl aliquot of a JEV NS1 antibody stock was intraperitoneally injected per mouse 12 h post-infection. Infected mice were inoculated with an equivalent quantity of untreated serum as a mock control. After waiting an additional 12 h for antibody dissemination, infected mice were subjected to daily mosquito biting from day 1 to day 3 post mouse infection. The mouse-blood-fed mosquitoes were reared for an additional 8 days for JEV detection. b, Production of murine polyclonal antibodies against JEV NS1. The JEV NS1 gene was cloned into a pET-28a (+) expression vector and expressed in E. coli BL21 DE3. Recombinant JEV NS1 in inclusion bodies was dissolved in 8 M urea and purified for antibody generation. The antibodies were validated by immunostaining with S2-expressed JEV NS1 protein. c, JEV infection in AG6 mice. Blood was collected from mouse tail veins from 0 to 3 days post JEV infection. The presence of infectious viral particles in the blood plasma was assessed using a plaque assay. Data are representative of at least five infected AG6 mice. Differences between untreated and JEV NS1 antibody-treated groups were assessed using a non-parametric Mann–Whitney test. d,e, Immunoblockade of JEV sNS1 in JEV-infected AG6 mice reduced JEV acquisition by C. pipiens pallens. The fed mosquitoes were reared for an additional 8 days for JEV detection by TaqMan qPCR. In d, the number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. In e, data are represented as the percentage of mosquito infection. Differences in mosquito infective ratios were assessed using Fisher's exact test. *P < 0.05, **P < 0.01, ***P < 0.001. In b–e, experiments were biologically reproduced at least three times. d.p.i., days post inoculation; NS, not significant.