a–c, The presence of recombinant DENV2 sNS1 in blood increased DENV2 acquisition by A. aegypti. Recombinant DENV2 sNS1 protein was expressed and purified from Drosophila S2 cells (a). A total of 10 µg of purified sNS1 was incubated with fresh human blood (500 µl) and supernatant from DENV2-infected Vero cells (500 µl) to feed A. aegypti via an in vitro membrane blood meal. In b and c, 1 × 105 p.f.u. ml–1 DENV2 was used for mosquito oral infection. Mosquitoes fed the same amount of BSA served as negative controls. d–f, Immunoblockade of DENV sNS1 in supernatant from infected Vero cells reduced DENV acquisition by A. aegypti. Serially diluted anti-DENV2 NS1 antisera were mixed with supernatant from DENV2-infected Vero cells (500 µl) and fresh human blood (500 µl) for in vitro membrane feeding of A. aegypti (d). In e and f, 1 × 106 p.f.u. ml–1 DENV2 was used for oral infection. As a mock control, mosquitoes were fed the same dilution of pre-immune sera. g–i, The presence of purified native sNS1 (nNS1) in viremic human blood increased DENV2 acquisition by A. aegypti. Infectious DENV2 particles and native sNS1 protein were purified from the supernatant of DENV2-infected Vero cells. Following this, native sNS1 and 1 × 106 p.f.u. purified DENV2 virions were incubated with human blood (1 ml) for mosquito oral feeding. The equivalent amount of BSA was used as a negative control. In a–i, the DENV2 NGC strain was used for mosquito oral infection. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. In b, e and h, the number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. In c, f and i, data are represented as the percentage of mosquito infection. Differences in mosquito infective ratio were compared using Fisher's exact test. In i, P values were adjusted using the Bonferroni correction to account for multiple comparisons. Differences were considered significant if P < 0.017. In a–i, experiments were biologically repeated at least three times with similar results. NS, not significant.