Membrane feeding of dengue patient’s blood as a substitute for direct skin feeding in studying Aedes-dengue virus interaction

Cheong-Huat Tan, Pei-Sze Jeslyn Wong, Mei-Zhi Irene Li, Hui-Ting Yang, Chee-Seng Chong, Linda K. Lee, Shi Yuan, Yee-Sin Leo, Lee-Ching Ng, David C. Lye

Understanding the interaction between Aedes vectors and dengue viruses (DENV) has significant implications in determining the transmission dynamics of dengue. The absence of an animal model and ethical concerns regarding direct feeding of mosquitoes on patients has resulted in most infection studies using blood meals spiked with laboratory-cultured DENV. Data obtained from such studies may not reflect the natural human-mosquito transmission scenario. This study explored the potential of using membrane feeding of dengue patient's blood as a substitute for direct skin feeding. Four to six-day old female Ae. aegypti were provided the opportunity to feed via direct exposure to a patient's forearm for 15 min or via exposure to EDTA-treated blood from the same patient through an artificial membrane for 30 min. Mosquitoes from both feeding methods were incubated inside environmental chambers. Mosquitoes were sampled at day 13 post-feeding. Midgut and salivary glands of each mosquito were dissected to determine DENV infection by RT-qPCR and viral titration, respectively. Feeding rates: Direct skin feeding assay (DSFA) consistently showed higher mosquito feeding rates (93.3-100 %) when compared with the membrane feeding assay (MFA) (48-98.2 %). Midgut infection: Pair-wise comparison between methods showed no significant difference in midgut infection rates between mosquitoes exposed via each method and a strong correlation was observed in midgut infection rates for both feeding methods (r = 0.89, P < 0.0001). Overall midgut viral titers (n = 20) obtained by both methods were comparable (P ≥ 0.06). Salivary gland infection: Pair-wise comparison between both methods revealed no significant difference in salivary gland infection rate. Strong correlation in salivary gland infection was observed between DSFA and MFA (r = 0.81, P < 0.0001). In general, mosquitoes fed directly on dengue patients and those on patients' blood (n = 11) had comparable virus titer (P ≥ 0.09). DENV midgut and salivary gland infection rates showed good concordance between DSFA and MFA blood meal exposure methods. Freshly-obtained venous blood in EDTA from dengue patients for MFA can be used as a substitute to DSFA, especially in circumstances where bioethics approval or patient recruitment is difficult to obtain for vector competence studies. Nevertheless, mosquito numbers will need to be increased to compensate for lower feeding rate in MFA.