Xenosurveillance: a novel mosquito-based approach for examining the human-pathogen landscape

Abstract

Background: Globally, regions at the highest risk for emerging infectious diseases are often the ones with the fewest resources. As a result, implementing sustainable infectious disease surveillance systems in these regions is challenging. The cost of these programs and difficulties associated with collecting, storing and transporting relevant samples have hindered them in the regions where they are most needed. Therefore, we tested the sensitivity and feasibility of a novel surveillance technique called xenosurveillance. This approach utilizes the host feeding preferences and behaviors of Anopheles gambiae, which are highly anthropophilic and rest indoors after feeding, to sample viruses in human beings. We hypothesized that mosquito bloodmeals could be used to detect vertebrate viral pathogens within realistic field collection timeframes and clinically relevant concentrations.

Methodology/principal findings: To validate this approach, we examined variables influencing virus detection such as the duration between mosquito blood feeding and mosquito processing, the pathogen nucleic acid stability in the mosquito gut and the pathogen load present in the host's blood at the time of bloodmeal ingestion using our laboratory model. Our findings revealed that viral nucleic acids, at clinically relevant concentrations, could be detected from engorged mosquitoes for up to 24 hours post feeding by qRT-PCR. Subsequently, we tested this approach in the field by examining blood from engorged mosquitoes from two field sites in Liberia. Using next-generation sequencing and PCR we were able to detect the genetic signatures of multiple viral pathogens including Epstein-Barr virus and canine distemper virus.

Conclusions/significance: Together, these data demonstrate the feasibility of xenosurveillance and in doing so validated a simple and non-invasive surveillance tool that could be used to complement current biosurveillance efforts.

Fig 1. Laboratory preparation of mosquito bloodmeals for detection of viral RNA.

Mosquitoes were offered bloodmeals from artificial membrane feeders containing virus-spiked sheep’s blood or virus-infected hamsters. The bloodmeals were applied to FTA cards 12 h post blood feeding, unless otherwise indicated. The FTA cards were left to air dry for 2 hours and stored at room temperature for 2 weeks. The mosquito dried blood spots (M-DBS) were removed from the FTA cards and RNA was extracted.

Fig 2. Virus-specific differences between the efficiency and duration of viral RNA detection from M-DBS.

WNV (red) or HIV-1 (blue) were mixed with blood at 10⁶ viral RNA copies/ml. (A) The means (shown with SEM) of virus-specific RNA detected directly from 2 μl of blood-virus mix (not applied to FTA cards), 2 μl of the blood-virus mix applied to FTA cards and mosquito bloodmeals applied to FTA cards 12 h post feeding (hpf) were compared using a one way ANOVA with Tukey’s multiple comparisons adjustment. Application of the blood-virus mix (P = 0.0012) and mosquito bloodmeals (P = 0.0008) to FTA cards resulted in significant losses in WNV RNA detection compared to the original blood-virus mix, but not between the treatments applied to FTA cards. Comparisons between HIV-1 treatments were not significant. (B) WNV or (C) HIV-1 mixed with blood were offered to mosquitoes and the bloodmeals were applied to FTA cards at the indicated times post blood feeding. The blood-virus mixes (2 μl each) were applied to FTA cards before blood feeding (pre-feed) to compare to viral RNA detection from mosquito dried blood spots (M-DBS). The number of blood spots positive (>10⁴ RNA copies/ml) for virus RNA per dried blood spot tested (n = 5) are shown above each time point. The means (shown with SEM) of the post blood feeding time points were compared to the pre-feed using a one way ANOVA with Dunnet’s multiple comparisons adjustment. The comparisons were not significant.

Fig 3. Detection of viral RNA from infected hamsters is not significantly different than from artificial bloodmeals.

Hamsters were infected with (A) WNV or (B) PIRV and blood was collected on days 2, 4 and 6 post infection to determine viral RNA copies/ml (n = 3 represented by different colors shown in graphs). Prior to blood collection, the hamsters were offered to mosquitoes as bloodmeals. The tables show the number of mosquito dried blood spots (M-DBS) that were positive for virus-specific RNA, determined by qRT-PCR titers greater than the lower limit of quantification (LLOQ), per M-DBS tested for each hamster and time point. Table text colors represent different hamsters offered as bloodmeals and correspond to the colors in the graphs. (C) Ratios of viral RNA titers (means with min to max whiskers) post blood feeding (M-DBS) to pre blood feeding (2 μl blood applied to FTA cards) were used were used to determine the recovery of viral RNA from mosquito bloodfeeding. The post/pre blood feeding ratios between virus-spiked artificial and infected hamster bloodmeal sources were compared using unpaired t tests and were not significant.

Fig 4. Sequencing reads from mosquito bloodmeals collected in Liberia aligning to Epstein-Barr virus (EBV) and canine distemper virus (CDV) genomes.

Bloodmeals from indoor resting mosquitoes were collected from two villages in northern Liberia and sequenced. (A) All pooled M-DBSs contained 100bp reads aligning to EBV and (B) one pool of M-DBSs (Village A d7) contained 100bp reads aligning to CDV. N, nucleoprotein; P/V/C, phospho- and nonstructural V and C proteins; M, matrix protein; H, hemagglutinin protein.