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

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

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

Mosquito bloodmeal limits of detection and comparisons to clinical ranges.

<p>^aData from blood donors.</p><p>^bClinical data for Lassa fever virus. WNV, West Nile virus; HIV-1, human immunodeficiency virus-1; PIRV, pirital virus; CHIKV, chikungunya virus; n.t., not tested.</p><p>Mosquito bloodmeal limits of detection and comparisons to clinical ranges.</p

Liberia mosquito collection summary^a and RNA-sequencing pools.

<p>NA, not applicable. M-DBS, mosquito dried blood spots.</p><p>^aAll of the aspirated mosquitoes were identified by PCR as <i>An</i>. <i>gambiae</i> sensu stricto.</p><p>^bControl pool generated from laboratory-raised <i>An</i>. <i>gambiae</i> mosquitoes that fed upon sheep’s blood.</p><p>^cUp to four M-DBS from each house aspirated were pooled and used for RNA-sequencing.</p><p>Liberia mosquito collection summary<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003628#t002fn002" target="_blank">^a</a> and RNA-sequencing pools.</p