Production of piRNA-like viral small RNAs in the mosquito soma.

<p>Size distribution, density plots, and nucleotide analysis of virus-derived small RNAs in <i>A. aegypti</i> (<b>A</b>), <i>A. albopictus</i> (<b>B</b>) and head and thorax of <i>A. albopictus</i> (<b>C</b>) infected with CHIKV. Example weblogos are shown for the predominant size classes. Arrows denote approximate start of 26S mRNA.</p

Model for RNA-based immune pathways modulating alphavirus pathogenesis in the mosquito soma.

<p>Following entry and uncoating, the genomic (+) strand RNA of an alphavirus serves both as mRNA and as a template for the synthesis of complementary (−) strand RNA. The viral (−) strands then serve as templates for the synthesis of new genomic-length (+) strand RNAs, as well as for shorter subgenomic (+) strand RNAs (26S mRNA) that encode the virus' structural genes. Alphaviruses are thought to synthesize (−) strand RNAs for a limited duration of time early in the infection, establishing an upper limit on the number of dsRNA RIs present in the cell. However, production of the (+) single-stranded genomic (49S) and subgenomic (26S) RNAs continues much longer, ultimately becoming the predominant virus-specific RNAs present in the cell. In this model antiviral siRNA and piRNA-like viral small RNA biogenesis pathways compete for a limited number of precursor dsRNA RIs in the infected cell. While recognition of dsRNA activates both pathways, secondary piRNA-like viral small RNAs are preferentially generated from viral mRNAs. Efficient processing of dsRNA RIs by Dcr-2 may restrict the amount of precursor substrate available to enter the piRNA-like viral small RNA biogenesis pathway. The B2 protein binds both siRNA duplexes and long dsRNAs preventing the protein components of antiviral pathways access to dsRNAs, but inhibition is not absolute. Elevated levels of viral replication may increase amplification of secondary piRNA-like viral small RNAs from 49S and 26S mRNA substrates.</p

Identification of <i>dcr-2</i> null mutant mosquito cell lines.

<p>Size distribution and nucleotide analysis of virus-derived small RNAs in u4.4 cells (<b>A</b>), <i>dcr-2^FS−1</i> (C6/36) cells (<b>B</b>) and <i>dcr-2^{del 33}</i> (C7-10) cells (<b>C</b>) infected with CHIKV. Schematics indicating Dcr-2 domains (<b>D</b>). The <i>A. albopictus</i> Dcr-2 contains a DExH/D protein family domain (DEAD) and helicase conserved C-terminal domain (H); a domain of unknown function (DUF); a PAZ domain; and tandem RNase III domains. Asterisks indicate locations of deletions in <i>dcr-2</i> sequences.</p

B2-mediated suppression of piRNA-like viral small RNAs in <i>dcr-2</i> null mutant cells.

<p>Size distribution and nucleotide analysis of virus-derived small RNAs in <i>dcr-2^FS−1</i> cells infected with CHIKV-B2 (FHV) (<b>A</b>) or CHIKV-B2 (C44A) (<b>B</b>). Single representative TruSeq libraries are shown (replicate #2 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002470#ppat.1002470.s004" target="_blank">Table S1</a>). CHIKV (+) strands per virus-derived small RNA in 1 ug of total RNA (calculated from normalized 25–29 nt reads identified in replicate TruSeq libraries) (<b>C</b>). Error bars indicate the standard deviation among three biological replicates. Modulation of alphavirus infection by an antiviral piwi-like RNA pathway in <i>dcr-2^FS−1</i> (C6/36) cells (<b>D</b>). Time course of cytopathology in <i>dcr-2^FS−1</i> (C6/36) cells infected with recombinant CHIK viruses (20X magnification).</p

Sources of error per sample preparation method.

<p>(A) Intra-host single nucleotide variants (iSNVs) that were present in multiple samples at greater than 1% of population are shown in a heat map format to visualize patterned diversity acquired during sample preparation. The total number of samples containing iSNVs in greater than 1% of population are summarized in the column “#> 1% of Pop” in green. “Codon” column (second column from right) provides both the nucleotide and protein translation of the site. (B) The detected mean error rates of iSNVs greater than 0. 2% of population (error/site/copy) are stratified by presence in the plasmid (Origin), detection after transcription/reverse transcription (Transc) or preparation, and preparation/sequencer error (Prep). (C) Error profiles are expressed as the number of iSNVs per percent of population obtained from each of the 5 sample preparation methods.</p

Study sample diagram.

<p>Study sample diagram.</p

Cooler Temperatures Destabilize RNA Interference and Increase Susceptibility of Disease Vector Mosquitoes to Viral Infection

<div><p>Background</p><p>The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi) pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.</p><p>Methodology/Principal Findings</p><p>We utilized transgenic “sensor” strains of <i>Aedes aegypti</i> to examine the role of temperature on RNA silencing. These “sensor” strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2) or Argonaute-2 (AGO-2). We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.</p><p>Conclusions/Significance</p><p> This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.</p></div

Percentage of errors by type of acquired diversity determined during sample preparation.

<p>(A) Percentage of error attributed to synonymous vs. non-synonymous variants. (B) Percentage of errors attributed to transitions vs. transversions. (C) Percentage of errors attributed to insertions or deletions vs. Intra-host single nucleotide variants (iSNV).</p

Total error by sample preparation method.

<p>(A) The mean read depth per position from two DNA controls (PLASMID and P_AMP) and five RNA sample preparation methods (n = 2 of replicate experiments). (B) Errors calculated as error/site/copy (plasmid or transcript) are presented as the average of duplicate experiments as in A. Intra-host single nucleotide variants present in the original plasmid were removed from the calculation of the error (reference positions: 4,697 and 4,725). Student t-tests were performed to demonstrate differences between the mean error per site per copy in the control plasmid (* = <i>p</i><0.05) and each sample preparation method. (C) The error calculated in B is converted to fold change over the control (“PLASMID”). Error bars in all panels represent standard deviation of the mean.</p

Low-temperature activation of RNAi sensor mosquitoes is reversible and can be induced in adults.

<p>Photographs taken at 7 or 14 days post emergence (7 d or 14 d) of typical individuals from <i>Ae. aegypti</i> RNAi sensor strain #2 following rearing at 18°C (<b>A</b>) or 28°C (<b>B</b>). Adult females were transferred to the indicated temperature at 1 day post-emergence; photographs are EGFP (top panel) or DsRED (bottom panel). Real-time qPCR of EGFP mRNA levels in 3×P3-sensor mosquito heads following rearing at 18°C (<b>C</b>) or 28°C (<b>D</b>), with newly emerged adults held at the alternate temperature for the indicated number of days. Error bars indicate one standard deviation corresponding to technical variation for a representative biological replicate. (<b>E</b>) Real-time qPCR of EGFP mRNA levels in transgenic RNAi sensor heads following rearing at 18°C or 28°C, with mosquitoes remaining at the same temperature as adults. Error bars indicate the standard deviation among three biological replicates; *** indicates significance at the p<0.001 level as determined by two-tailed Student's t-test.</p