Xenosurveillance: A Novel Mosquito-Based Approach for Examining the Human-Pathogen Landscape

<div><p>Background</p><p>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 <i>Anopheles gambiae</i>, 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.</p><p>Methodology/Principal Findings</p><p>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.</p><p>Conclusions/Significance</p><p>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.</p></div

GBV-C sequences from H and M-DBS cluster phylogenetically with GBV-C strains from Sierra Leone and Liberia.

<p>The longest contig assembled to GBV-C, a 491 n.t. segment, was used as input to create a phylogenetic tree using a neighbor-joining method. The red line corresponds to input sequence generated from NGS data. See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006348#pntd.0006348.s002" target="_blank">S1 Fig</a> for accession numbers corresponding to each GBV-C strain used in the analysis.</p

PathoScope reads alignment summaries.

<p>EBV, Epstein-Barr virus; CDV, canine distemper virus.</p><p>^aControl pool generated from laboratory-raised <i>An</i>. <i>gambiae</i> mosquitoes that fed upod sheep’s blood.</p><p>^bDenotes percentage after PathoQC.</p><p>^cDenotes reads aligning to the sheep reference library.</p><p>^dDenotes reads aligned to EBV strain B95–8 (GenBank V01555.2)</p><p>^eDenotes reads aligned to CDV strain Uy251 (GenBank KM280689.1)</p><p>PathoScope reads alignment summaries.</p

NGS reads aligning to human viruses.

<p>NGS reads aligning to human viruses.</p

HBV sequences from H and M-DBS cluster phylogenetically with HBV strains from West Africa.

<p>The longest contig assembled to HBV was a 542 n.t. segment. This was used as input to create a phylogenetic tree using a neighbor-joining method. Letters correspond to HBV genotype. See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006348#pntd.0006348.s003" target="_blank">S2 Fig</a> for accession numbers corresponding to each HBV used in the analysis.</p

Laboratory preparation of mosquito bloodmeals for detection of viral RNA.

<p>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.</p

The majority of reads from M-DBS and H-DBS, for both RNA and DNA NGS pools, align to host nucleic acid.

<p>The taxonomic makeup of each sequencing pool was determined using the Blastn -Megablast tool with individual sequencing reads as input. Blue circles show taxonomic make up at the level of kingdom for all reads in each pool. Red circles show taxonomic makeup from a subset of reads aligning to eukaryotes. While the most reads in H-DBS aligned to human nucleic acid, reads from M-DBS aligned to both human and mosquito nucleic acid. Reads to human pathogens were detected in both H/M-DBS in the remaining reads.</p

Summary of enrollment and sampling data.

<p>Summary of enrollment and sampling data.</p

Phylogenetic tree of 29 Zika virus isolates.

<p>The tree was generated by Neighbor-Joining methods using the Tamura-Nei genetic distance model (bootstrapped 1,000 times). Similar results were obtained using maximum parsimony or maximum likelihood algorithms. Strains used in this study are shown in bold; distinct lineages are East African (MR766), West African (41525), and Asian (PRVABC59).</p

Select strains of East and West African ZIKV have higher fitness <i>in vitro</i> than the American strain PRVABC59.

<p>Equal amounts of virus (MOI 0.01) was used to infect the indicated cell line. Supernatant was harvested and proportion of each virus was determined by sequencing and analyzing chromatograms. Data are presented as proportion of the first virus listed present at the indicated time point in (A) Vero, (B) Huh7, C6/36 (C), and (D) Aag2 cells.</p