title: Membrane feeding of dengue patient's blood as a substitute for direct skin feeding in studying Aedes-dengue virus interaction.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore, Singapore. Tan_Cheong_Huat@nea.gov.sg.|Faculty of Science, Monash University, Melbourne, Australia. Tan_Cheong_Huat@nea.gov.sg.
authors.forename: Cheong-Huat
authors.initials: CH
authors.lastname: Tan
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore, Singapore.
authors.forename: Pei-Sze Jeslyn
authors.initials: PS
authors.lastname: Wong
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore, Singapore.
authors.forename: Mei-Zhi Irene
authors.initials: MZ
authors.lastname: Li
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore, Singapore.
authors.forename: Hui-Ting
authors.initials: HT
authors.lastname: Yang
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore, Singapore.
authors.forename: Chee-Seng
authors.initials: CS
authors.lastname: Chong
authors.affiliation: Communicable Disease Center, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Linda K
authors.initials: LK
authors.lastname: Lee
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore, Singapore.
authors.forename: Shi
authors.initials: S
authors.lastname: Yuan
authors.affiliation: Communicable Disease Center, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Yee-Sin
authors.initials: YS
authors.lastname: Leo
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore, Singapore.|School of Biological Sciences (SBS), Nanyang Technological University, Singapore, Singapore.
authors.forename: Lee-Ching
authors.initials: LC
authors.lastname: Ng
authors.affiliation: Communicable Disease Center, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: David C
authors.initials: DC
authors.lastname: Lye
mesh_terms: D000330:Aedes / Q000502:physiology* / Q000821:virology; D000818:Animals; D003715:Dengue / Q000097:blood* / Q000635:transmission / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D005247:Feeding Behavior; D005260:Female; D006801:Humans; D007303:Insect Vectors / Q000502:physiology* / Q000821:virology; D008297:Male; D012867:Skin / Q000469:parasitology*
keywords: Aedes aegypti; Dengue; Direct Skin Feeding Assay (DFSA); EDTA blood; Membrane Feeding Assay (MFA)