Trait means, genetic variance (V_G), total phenotypic variance (V_P), heritability (H²), and coefficient of genetic variation (CV_G) in titre and mortality following KV infection in the DGRP.

<p>Trait means, genetic variance (V_G), total phenotypic variance (V_P), heritability (H²), and coefficient of genetic variation (CV_G) in titre and mortality following KV infection in the DGRP.</p

KV causes male-biased mortality, increased lethargy, and decreased fecundity.

<p>(A) Injection of KV virus into <i>OreR</i> flies led to sex-specific mortality. Infected females (red dotted line) experienced a small but significant increase in mortality, but males (blue dotted line) experienced a significantly larger rate of mortality after day 10. Flies injected with control gradient solution were unaffected (solid lines). Each point is the mean and standard error for the proportion of flies alive in each vial (10 vials of 10 flies). (B) Although females remained alive for longer, they were more lethargic. We assessed daily movement of flies injected with either chloroform-inactivated KV (green) or active KV (purple). KV-infected flies moved less from days 3–7 post-infection. (C) Females also displayed altered egg laying behaviour. Thirty pairs of flies were injected with inactive chloroform treated KV (green) or active KV (purple). KV-infected flies laid a slightly, but not significantly, higher number of eggs during early infection (1 and 2 DPI) but laid significantly fewer eggs in late infection (7 and 8 DPI). This reduction in egg laying is due to a shutdown of oogenesis before vitellogenesis (D, E), and ovaries from KV-infected flies house a lower proportion of ovarioles that include late-stage and mature egg chambers (F) and a higher proportion which contain apoptotic nurse cells (G). Ovaries were analysed 10 DPI, and error bars (F,G) show the standard error.</p

Confirmation of antiviral genes identified in GWAS.

<p>KV titre was measured in flies expressing a foldback hairpin targeting 18 genes identified in the GWAS, using GAL4 lines that knock each down in either the whole fly or specifically in the gut. (A) The data were used to estimate random effects associated with each gene knock down, plotted with 95% highest posterior density intervals. (B) Knock-down of the most confident association in the GWAS, <i>Cip4</i>, caused reduced <i>Cip4</i> RNA levels and (C) increased viral titre. (D) The associated variant (3L_4363810_SNP), was polymorphic (G/A), representing a nonsynonymous polymorphism in some splice variants, and survival following KV infection was significantly increased in fly lines with the “A” genotype, especially in females. Each point in comparison of survival in the two genotypes is a line mean. (*MCMCp < 0.05).</p

Genome-wide association of polymorphism in the DGRP with KV-induced titre and mortality.

<p>Manhattan plots showing the p-value for the effect of each polymorphism on viral titre (purple) and mortality (green). The top SNPs for each phenotype are shown in expanded inset panels, including surrounding genes. For clarity “CG” is omitted from gene identifiers. Horizontal lines show significance thresholds obtained through randomisation (p_rand = 0.05 in blue; p_rand = 0.01 in red).</p

Isolation of KV and growth in flies.

<p>(A) Density gradient and virus titre: Kallithea virus (purple) was effectively separated from DAV (green) at 1.18 g/mL (dotted line) in fractions 15 and 16 of an iodixanol gradient. (B) Transmission electron micrograph of KV-positive fractions showed KV to be a rod-shaped enveloped particle, as has been described previously for other nudiviruses [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007050#ppat.1007050.ref055" target="_blank">55</a>]. We did not observe unenveloped KV particles, bacteria, or RNA viruses in the isolate. (C) Relative viral titres normalised by the number of fly genomic copies and virus levels at time zero in each sex. Each point represents a vial of 10 flies. Viral titres peaked at 10 days post-infection, and were generally higher in females (red) than males (blue) late in infection. (D) We were able to infect adult flies orally by applying the viral isolate to <i>Drosophila</i> medium, although relative copy number of the virus was very low and infection was inefficient, with only 2 of 16 vials (each of 10 flies) having increased titre after one week, indicating an infectious rate lower bound of ~1% at 5x10³ ID50.</p

Genetic variation in resistance to KV.

<p>(A) We measured LT50 in both sexes, and titre in females, following KV injection in the DGRP. For titre, each bar represents the mean (and standard error) titre relative to fly genome copy-number, as assessed by qPCR for 5 vials of 10 flies for each of 125 DGRP lines. For LT50, each bar represents the mean time until half the flies (in a vial of 10) were dead, for three vials per line, per sex. (B, C) We used a multi-response linear mixed model to calculate genetic correlation between the traits. Shown are the raw data (left), and the estimated line effects (right) after accounting for any injection date and qPCR plate effects, and for the estimated variance among lines. Each point is a DGRP line measured for both phenotypes. We find a strong positive correlation between male and female LT50 values (B). We also observe a weak positive correlation between titre and LT50 (C).</p

KV induces differential regulation of chorion, virus defense, and serine endopeptidase genes.

<p>(A) Volcano plots showing fold changes and p-values from Wald tests for differential expression of <i>D</i>. <i>melanogaster</i> genes following KV infection for females (left), males (center), those different between the sexes (right), with DAV read count fit as a covariate and nominal significance threshold of p < 0.001. In each panel, the genes with the smallest p-values are labelled. All of the genes that were significantly differentially regulated between the sexes are highly significant in females. (B) Highly induced genes are mostly functionally unannotated, but include some with known roles in viral pathogenesis. (C) The male response is correlated with females, but muted, with few genes identified as significantly differentially expressed in males. Genes with weak evidence of differential expression in either sex (p < 0.05) are plotted, where the dotted line represents a perfect correlation, and red points are genes identified as significantly differentially expressed. (D) The top GO enrichment terms for each GO class (Molecular Function, Biological Process, Cellular Component) were genes involved the chorion, virus defense, and serine peptidase activity. For each plot, estimated fold changes and their associated standard errors are plotted for every gene matching the GO term, regardless of the significance of the Wald test. Generally, chorion genes were downregulated, virus defense genes were upregulated, and serine peptidases were downregulated.</p

The rapid VACV-induced reduction of modified and unmodified forms of miR-27b-3p is inhibited by arrest of viral replication.

<p>Northern blot analysis of RNA extracted from HeLa or CHO cells infected with VACV or MVA at an MOI of 10, or mock infected, in the absence or presence of AraC as indicated. RNA was extracted at 0 or 6 hpi. The probe was a DNA oligonucleotide perfectly complementary to miR-27b-3p.</p

Individual miRNAs show varying susceptibility to VACV-induced polyadenylation and reduction in abundance.

<p>The top row shows the relative abundance of unmodified or templated (blue) and modified (red) (a) miR-16-5p, (b) miR-29a-3p, and (c) and miR-27b-3p in uninfected and infected cells at 6 and 24 hpi, with and without AraC treatment. Data shown are the average number of reads for three replicates; error bars represent SD. The middle row shows northern blot analyses of RNA extracted from HeLa cells under the same conditions as in a, d, g, using probes complementary to (b) miR-16-5p, (e) miR-29a-3p, and (h) miR-27b-3p. The arrows in 5e and 5h indicate likely pre-miRNA. The bottom row compares the modifications to the miRNA read for (c) miR-16-5p, (f) miR-29a-3p and (i) miR-27b-3p in uninfected and infected cells at 6 and 24 hpi, with and without AraC treatment.</p

Unmodified miRNAs are shortened in VACV infected cells.

<p>(a) Logo images displaying the length of reads mapping to the indicated miRNAs in uninfected cells (left column) and VACV-infected cells (right column). For comparison, a dotted black line has been placed on the images at the same position for uninfected and infected samples in order to visualize the difference between 3’ end composition in uninfected and infected cells, (b) The average proportion (n = 3) of miRNA reads of different lengths in uninfected and infected samples. Statistically significant differences (p<0.05, t-test) are indicated with an asterisk.</p