Intrathoracic injection of DENV2 strains and midgut escape isolates into HWE mosquitoes. (A) Intrathoracic injection of DENV2-PR159 (American genotype DENV2 from Puerto-Rico), EM41 (midgut escape mutant derived from the PR159 strain), and DENV2-Jam1409 (American-Asian genotype DENV2 from Jamaica as control) into females of the laboratory adapted Higgs White Eye (HWE) strain. Viruses had been propagated for 12–14 days in C6/36 cells. Prior to injection, virus-cell suspensions had titers ranging from 5x105 pfu/ml to 7x106 pfu/ml. (B) Intrathoracic injection of DENV2-QR94, (American genotype DENV2 from Quintana Roo, Mexico), EM33 (midgut escape mutant derived from the QR94 strain), and DENV2-Jam1409 into HWE females. Virus-cell suspensions had titers of ~106 pfu/ml. Virus titers of individual midguts and carcasses were assayed by plaque assays in LLC-MK2 (monkey kidney) cells at 7 days post-bloodmeal (pbm). Bars in the diagram represent mean values. * indicate significant difference between mean virus titers as analyzed on Y = Log(Y) transformed data by ANOVA and Bonferroni’s Multiple Comparison Test.