The trade-off hypothesis is not supported by results from <i>in vivo</i> competitions.

<p>After serial or alternate passage WNV strains were competed against a reference virus in chicks, <i>Cx. pipiens</i> mosquitoes (Pip.) and <i>Cx. quinquefasciatus</i> mosquitoes (Quinq.). Each of four treatments (serial passage in chicks, serial passage in mosquitoes, final chick passage of the alternate series, final mosquito passage of the alternate series) was performed in triplicate (represented by light, medium and dark shades of each color). Inocula (squares) contained approximately equal parts passed test virus and unpassed reference virus and were identical across cohorts except for 5 Pip. cohorts for which comparable inocula had to be re-created (points with white centers). Each cohort comprised 7–10 chicks or 9–11 mosquitoes with each animal represented by a circle. Mean proportions of test WNV for each cohort were compared with the inocula means in unpaired t-tests where P≤0.05 was considered significant (astrices). Bars indicate cohort mean and standard error of the mean. Dashed lines at 0.1 and 0.9 indicate the range of high accuracy for the quantitative sequencing assay used as determined by Fitzpatrick et al <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002335#ppat.1002335-Fitzpatrick1" target="_blank">[21]</a>.</p

Intrahost genetic diversity is associated with decreased fitness in chickens but not mosquitoes.

<p>Fitness was computed as the difference between the test:REF ratio at input and after competition, such that numbers greater than zero indicate fitness increases and numbers less than zero indicate fitness declines. Sequence diversity was computed as the proportion of nucleotides in the test virus population with mutation, as described by Jerzak et al <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002335#ppat.1002335-Jerzak3" target="_blank">[18]</a>. Fitness was measured in <i>Cx. pipiens</i> (blue circles) <i>Cx. quinquefasciatus</i> (red squares) and chickens (green triangles). Open symbols indicate passage history in mosquitoes, closed symbols indicate passage history in chickens. Sequence diversity was significantly negatively correlated with fitness in chickens (Spearman r = −0.9856, P = 0.0028).</p

Combined average proportions of total WNV RNA comprised of competitor RNA after competition against a marked reference virus in chicks or mosquitoes.

1<p> = input is the inoculum.</p>2<p> = output is either day 2 chick serum or day 7 whole mosquito homogenate.</p>3<p> = p-value was determined in an unpaired t-test between input and output for each cohort (significance is defined as p≤0.05 and is noted in bold).</p

Serial and alternate passage experimental design.

<p>Virus derived from a WNV infectious clone was passed 20 times through chicks, 20 times through <i>Cx. pipiens</i> mosquitoes or 20 times alternating between the two (10 cycles). Each passage series was performed in triplicate and the final virus stocks were then used in <i>in vivo</i> competition assays to assess gains or losses in replicative fitness.</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

Size and abundance of small RNA reads Mmapping to the viral genomes.

<p>The abundance of 19–30-mer sRNA reads mapping to the WNV (A), SINV (B) and LACV (C) genomes based on size. Abundance is represented as a percentage of the total viRNAs from each sample. The black bars correspond with samples collected from S2 cells and white bars from C6/36 cells.</p

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

Small RNA profiles from C6/36 and S2 infected cells.

<p>Small RNA profiles from C6/36 and S2 infected cells.</p

viRNA coverage of the LACV/Human/1960 strain genome in C6/36 and S2 cells.

<p>Complete genome of LACV/Human/1960 strain showing intensity at each nucleotide of the genome in C6/36 (A,C,E) and S2 (B,D,F) cells. A and B correspond with the L gene segment (6,980 nt), C and D the M gene segment (4,526 nt), and E and F to the S gene segment (984 nt). Plotted are the 19–30-mer viRNA reads across the length of each segment represented by the x-axis. Reads originating from the genomic, negative strand are represented in red below the x-axis and those originating from the positive strand are represented in blue above the x-axis.</p