Feasibility of Using the Mosquito Blood Meal for Rapid and Efficient Human and Animal Virus Surveillance and Discovery

Abstract

Mosquito blood meals taken from humans and animals potentially represent a useful source of blood for the detection of blood-borne pathogens. In this feasibility study, Anopheles stephensi mosquitoes were fed with blood meals spiked with dengue virus type 2 (DENV-2) and harvested at serial time points. These mosquitoes are not competent vectors, and the virus is not expected to replicate. Ingested blood was spotted on Whatman FTA cards and stored at room temperature. Mosquito abdomens were removed and stored at -80°C. Control blood meal aliquots were stored in vials or applied onto FTA cards. After 4 weeks of storage, the samples were extracted using beadbeating and QIAamp Viral RNA kit (Qiagen Sciences, Germantown, MD). Recovered viral RNA was analyzed by DENV-2 TaqMan RT-PCR assay and next-generation sequencing (NGS). Overall viral RNA recovery efficiency was 15% from the directly applied dried blood spots and approximately 20% or higher for dried blood spots made by blotting mosquito midgut on FTA cards. Viral RNA in mosquito-ingested blood decreases over time, but remains detectable 24 hours after blood feeding. The viral sequences in FTA-stored specimens can be maintained at room temperature. The strategy has the potential utility in expedited zoonotic virus discovery and blood-borne pathogen surveillance.

Figure 1.

Experiment design for the feasibility study of using blood meal in mosquito abdomens for blood-borne virus surveillance. Blood spiked with dengue virus type 2 (DENV-2) was used to feed Anopheles stephensi mosquitoes. Controls and mosquito blood meal specimens, kept in vials at −80°C or on FTA cards at room temperature, were subjected to storage, nucleic acid extraction, and analyses by real-time reverse-transcription polymerase chain reaction (qRT-PCR) or next-generation sequencing.

Figure 2.

Recovery of nucleic acids from Whatman FTA cards. Aliquots of artificial whole blood with dengue virus type 2 (DENV-2) were collected before and after feeding mosquitoes, kept in vial (empty bars) or spotted on FTA cards (gray bars), and subjected to storage at −80°C and room temperature for 4 weeks, respectively, nucleic acids extraction, and quantitation by real-time reverse-transcription polymerase chain reaction (qRT-PCR). The error bars are standard deviations of data for eight samples.

Figure 3.

Dengue virus type 2 (DENV-2) in mosquito abdomens after blood feeding. Mosquito abdomen specimens, collected at 0–24 hours after blood feeding, were cut and kept in vial (empty bars) and subjected to storage at −80°C, nucleic acids extraction, and quantitation by real-time reverse-transcription polymerase chain reaction (qRT-PCR). The error bars are standard deviations of data for eight samples.

Figure 4.

Dengue virus type 2 (DENV-2) in FTA dried blood meal spots for mosquito abdomens after blood feeding. Mosquito abdomen specimens, collected at 0–24 hours after blood feeding, were blotted on FTA cards (gray bars) and subjected to storage at room temperature, nucleic acids extraction, and quantitation by real-time reverse-transcription polymerase chain reaction (qRT-PCR). The error bars are standard deviations of data for eight samples.