Correlation between Nora virus infection and impaired defense against P. aeruginosa intestinal infection as well as lowered fitness

The Oregon-R sub-strain infected with Nora virus is referred to as Ore-R(SM), while the non-infected sub-strain is referred to as Ore-R(SC). (A) Survival of Ore-R(SM) (Nora(+)) and Ore-R(SC) (Nora(-)) flies following oral exposure to P. aeruginosa PA14 (OD 0.25) at 25 °C. NI: not infected. (B) Lifespan analysis of Nora-infected and non-infected flies maintained under standard laboratory conditions at 25 °C. (C) Nora virus RNA levels in Ore-R(SM) and Ore-R(SC) stocks quantified by RT-qPCR. (D) Quantification of phospho-histone H3 (PHH3)-positive nuclei in the midgut of Ore-R(SM) (Nora(+)) and Ore-R(SC) (Nora(-)) flies at 2 days following oral PA14 infection or in non-infected (NI) controls. PHH3-positive nuclei counts were assessed as a measure of epithelial cell proliferation. (E) Nora virus RNA levels measured by RT-qPCR in the original Ore-R(SM) stock and in stocks cured of Nora virus by embryo bleaching. (F) Survival of uncured and cured Ore-R(SM) stocks to the ingestion of PA14. Each graphic is representative of three independent experiments. Survival panels are presented as mean ± SEM with each curve representing a triplicate of 20 flies. Other panels are presented as box and whiskers where the middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Each dot in Nora virus load panels (C, E) represent a sample of 5 flies. Each dot in the PHH3 quantification panel (D) represents one single posterior midgut. Survival data were analyzed using a log-rank (Mantel-Cox) test. Nora virus load in (C) was analyzed using a Mann-Whitney nonparametric test. PHH3 quantification (D) and Nora load (E) were analyzed using one-way ANOVA with Tukey’s post-hoc tests. Statistical significance is indicated as **p < 0.01, ***p < 0.001, ****p < 0.0001.

Infection of Nora(-) flies with the purified Nora virus recapitulates the fitness properties of Nora naturally infected flies

Flies used in these experiments were derived from Ore-R(SM) stocks cured of Nora virus by egg bleaching. A subset of cured flies was experimentally re-infected with a purified Nora virus preparation (Nora(+) re-inf virus), while control flies remained uninfected (Nora(-)). PA14: infection with P. aeruginosa PA14; NI: noninfected. (A) Transmission electron microscopy image of the purified Nora virus preparation used for re-infection. (B) Nora virus RNA levels measured by RT-qPCR in Nora(+) and Nora(-) flies across successive generations following experimental re-infection: G0 to G2. (C) Survival of re-infected Nora(+) and Nora(-) flies following oral exposure to PA14 at 25 °C. (D) Quantification of phospho-histone H3 (PHH3)-positive nuclei in the midgut of re-infected Nora(+) and Nora(-) flies following two days of PA14 ingestion or in non-infected (NI) controls. (E) Lifespan analysis of Nora(+) and Nora(-) flies maintained under standard laboratory conditions at 29 °C. (F) Lifespan analysis of re-infected Nora(+) and Nora(-) flies maintained on a sucrose-only diet at 25 °C. Each graphic is representative of three independent experiments. Survival panels are presented as mean ± SEM with each curve representing a triplicate of 20 flies. Other panels are presented as box and whiskers where the middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Each dot in the Nora virus load panel (B) represents a sample of 5 flies. Each dot in the PHH3 quantification panel (D) represents one single posterior midgut. Survival data were analyzed using a log-rank (Mantel-Cox) test. Nora virus load was analyzed using a Mann-Whitney nonparametric test (B). PHH3 quantification (D) was analyzed using one-way ANOVA with Tukey’s post-hoc test. Statistical significance is indicated as ***p < 0.001, ****p < 0.0001.

Influence of age, diet, and microbiota on Nora virus load and fly fitness

(A) Nora virus RNA levels measured by RT-qPCR at 3 days following oral exposure to P. aeruginosa PA14 in Nora-infected and non-infected flies maintained on either standard diet or rich diet (standard medium supplemented with 5x yeast). (B) Nora virus RNA levels measured by RT-qPCR in young (3-day-old) and aged (30-day-old) Nora-infected flies. (C) Quantification of total culturable bacterial microbiota by colony-forming unit (CFU) counts in young Nora(+) and Nora(-) flies maintained for 8 days on a sucrose-only diet, and in aged flies maintained on standard fly food. CFU values are presented on a logarithmic scale. (D) Lifespan analysis of Nora-infected (Nora(+) and non-infected (Nora(-) flies maintained on standard diet in the presence or absence of antibiotics (ABX). (E) Lifespan analysis of Nora-infected (+) and non-infected (-) flies maintained on rich diet in the presence or absence of antibiotics (ABX). Each graphic is representative of three independent experiments. Survival panels are presented as mean ± SEM with each curve representing a triplicate of 20 flies. Other panels are presented as box and whiskers where the middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Relative unit was used for Nora virus load panels. Each dot in Nora virus load panels (A, B) represent a sample of 5 flies. Each dot in the CFU quantification panel (C) represents one single posterior midgut microbiota. Survival data were analyzed using a log-rank (Mantel-Cox) test. Nora virus load was analyzed using a Mann-Whitney nonparametric test (A, B). CFU quantification was analyzed using one-way ANOVA with Tukey’s post-hoc test (C). Statistical significance is indicated as *p < 0.05, **p < 0.01, ****p < 0.0001.

Alterations in intestinal barrier integrity and systemic bacterial dissemination in Nora virus-infected flies

(A) Representative images from the SMURF assay in 30-day-old non-infected fly (top) and Nora-infected fly (bottom) fed on a sucrose solution containing a blue dye. (B) Quantification of SMURF-positive Nora(-) or Nora (+) flies at 30 days of age maintained on rich diet (standard medium supplemented with 5x yeast) (C) Representative LB agar plate showing bacterial growth from serial dilutions of hemolymph collected from non-infected flies and from Nora-infected flies exhibiting either SMURF-negative or SMURF-positive phenotypes. Age of flies corresponding to the LT50 observed in the survival assay shown in Fig. 3E (Nora(-): 65 days; Nora(+): 30 days). (D) Quantification of bacterial titers in the hemolymph at 3 days following oral exposure to P. aeruginosa PA14 in Nora-infected and non-infected flies. (E) Relative expression of the antimicrobial peptide gene Diptericin in dissected midguts of Nora(+) flies measured by RT-qPCR following 2 days of PA14 intestinal infection or after feeding with 1% SDS for 4-6 hours. Each graphic is representative of three independent experiments. All panels are presented as box and whiskers where the middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Each dot in the SMURF quantification panel (A) represents a single SMURF-positive fly. Each dot in the CFU quantification panel (C) represents one single fly hemolymph bacterial titer. Each dot in the Diptericin gene expression panel represents a sample of 5 midguts. The percentage of SMURF-positive flies was analyzed using a Mann-Whitney nonparametric test (B). CFU counts (D) and Diptericin gene expression levels (E) were analyzed using one-way ANOVA with Tukey’s post-hoc test. Statistical significance is indicated as *p < 0.05, ****p < 0.0001.

Nora virus is initially found in intestinal progenitor cells and is also located in enterocytes upon infectious or chemical stresses

(A) Nora virus RNA levels measured by RT-qPCR in whole flies or in dissected intestines. (B) Representative confocal images of intestines from 5-day-old non-infected flies. Tissues were fixed and stained for DNA (DAPI, blue), actin (FITC, green), and Nora virus (Cy3, red). The Cy3 secondary antibody (goat anti-mouse) shows background signal in intestinal muscles. Scale bar, 5 µm. (C) Representative confocal images of intestines from 5-day-old Nora-infected flies stained for DNA (DAPI, blue), actin (FITC, green), and Nora virus (Cy3, red). Scale bars, 3 µm (left) and 5 µm (right). (D) Colocalization analysis of Nora virus signal with progenitor cells marked using the esg-Gal4, Gal80ts driver crossed to UAS-GFP. Intestines were fixed and stained for DNA (DAPI, blue) and Nora virus (Cy3, red) Scale bars, 2 µm (left) and 5 µm (right). (E) Representative confocal images of intestines from Nora-infected GFP::Dicer-2 flies stained for DNA (DAPI, blue), actin (RFP, red), Dicer-2 (GFP, green), and Nora virus (Cy5, purple). Scale bars, 10 µm (left) and 5 µm (right). (F) Survival analysis of Nora-infected (+) and non-infected flies (-) following intestinal infection with P. aeruginosa PA14 (OD 0.25) or S. marcescens Db11 (OD 0.25), or exposure to 1 mM paraquat. (G) Nora virus RNA levels measured by RT-qPCR in flies from the experimental conditions shown in (F). (H) Representative confocal images of intestines from Nora-infected flies following PA14 infection, Db11 infection, or paraquat exposure. Tissues were fixed and stained for DNA (DAPI, blue), actin (FITC, green), and Nora virus (Cy3, red). Scale bar, 3 µm. (I) Quantification of (H). EC: enterocyte; ISC: progenitor cells. Each graphic is representative of three independent experiments. Survival panels are presented as mean ± SEM with each curve representing a triplicate of 20 flies. Other panels are presented as box and whiskers where the middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Relative unit was used for Nora virus load panels. Each dot in Nora virus load panels (A, G) represent a sample of 5 flies. Survival data were analyzed using a log-rank (Mantel-Cox) test. Nora virus load was analyzed using a Mann-Whitney nonparametric test (A). Comparisons of Nora virus load under pathogen infection or paraquat exposure (G) and of quantification of Nora (+) ECs (I) were analyzed using one-way ANOVA with Tukey’s post-hoc test. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Inhibition of apoptosis prevents epithelial turnover and the proliferation of Nora virus

(A) Quantification of apoptotic nuclei in the posterior midgut (region R4 & R5 [see Fig. S5J]) using ApopTag staining following P. aeruginosa PA14 infection or 1 mM paraquat exposure and controls. (B) Quantification of phospho-histone H3 (PHH3)-positive nuclei in the posterior midgut of Nora-infected flies 24h after PA14 infection or paraquat exposure. (C) Survival analysis of non-infected flies (Nora(-)) following PA14 intestinal infection (+ PA14) in control genotypes (NPG4G80 driver: [NP1-Gal4, Gal80ts] and UAS-p35 alone) and in flies with enterocyte expression of the apoptosis inhibitor gene p35 (NP>p35). (D) Survival analysis of Nora-infected flies (Nora(+)) following PA14 intestinal infection in control genotypes and in flies expressing the p35 gene in the intestine. (E) Quantification of PHH3-positive nuclei in the posterior midgut at 5 days following PA14 infection in flies from panels (C) and (D). PHH3-positive nuclei were quantified in control and apoptosis-inhibited intestines. (F) Nora virus RNA levels measured by RT-qPCR at 3 days following PA14 infection in intestines from flies shown in (D). (G) Representative confocal images of old intestines (30-day-old) from Nora-infected flies in control genotypes (NP driver (NPG4G80) and UAS-p35 alone) and in flies expressing p35 in the intestine (NP>p35). Tissues were fixed and stained for DNA (DAPI, blue), actin (FITC, green), and Nora virus (Cy3, red). Scale bar, 5 µm. (H) Quantification of Nora virus-positive cells in the intestine from the experimental conditions shown in (G). Each graphic is representative of three independent experiments. Survival panel is presented as mean ± SEM with each curve representing a triplicate of 20 flies. Other panels are presented as box and whiskers where the middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Relative unit was used for Nora virus load panels. Each dot in Nora virus load panel (F) represents a sample of 5 flies. Each dot in the PHH3 quantification panels (B, E) represent one single posterior midgut. Each dot in the ApopTag quantification panel (A) represents one single posterior midgut. Each dot in the Nora virus quantification panel (H) represents a region of one single posterior midgut. Survival data were analyzed using a log-rank (Mantel-Cox) test. ApopTag, PHH3, and Nora virus quantifications were analyzed using one-way ANOVA with Tukey’s post-hoc test (A, B, E, F, H). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Inhibition of the JAK-STAT pathway prevents epithelial turnover and the proliferation of Nora virus

(A-B) Survival analysis of non-infected flies (A) and infected flies (B) following P. aeruginosa PA14 intestinal infection of flies in which RNAi-mediated silencing of JAK-STAT pathway component genes (domeless and Stat92E) in intestinal stem cells was achieved using the Delta driver. Control genotypes and non-infected (NI) survival assays are shown in the lower right insets. (C) Relative expression of JAK-STAT pathway-related genes (upd3, dome, STAT92E, SOCS36E, and PIAS) measured by RT-qPCR at 3 days following PA14 infection in Nora-infected (+) and non-infected (-) flies’ midguts. (D) Quantification of upd3-GFP reporter signal in the posterior midgut at 2 days following PA14 infection in Nora-infected (+) and non-infected (-) flies. (E) Quantification of phospho-histone H3 (PHH3)-positive nuclei in the posterior midgut at 3 days following PA14 infection in flies with RNAi-mediated silencing of domeless or Stat92E in intestinal stem cells using the Delta-Gal4 driver. PHH3-positive nuclei were used as a measure of epithelial proliferation. (F) Nora virus RNA levels measured by RT-qPCR in intestines from flies shown in (E). (G) Representative confocal images of intestines from Nora-infected flies in control genotypes (Delta>control RNAi) and UAS-p35 alone) and in flies expressing p35 in the intestine (NP>p35). Tissues were fixed and stained for DNA (DAPI, blue), actin (FITC, green), and Nora virus (Cy3, red). Scale bar, 5 µm. (H) Quantification of Nora-positive cells of the intestinal epithelium displayed in (G). EC: enterocyte; ISC: progenitor cells. Each graphic is representative of three independent experiments. Survival panels are presented as mean ± SEM with each curve representing a triplicate of 20 flies. Other panels are presented as box and whiskers where the middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Relative unit was used for Nora virus load panels. Each dot in Nora virus load panel (F) represents a sample of 5 flies. Each dot in the PHH3 quantification panels (E) represent one single posterior midgut. Each dot in the gene expression panel (C) represents 5 posterior midguts. Each dot in the upd3-GFP quantification panel (D) represents one single posterior midgut. Survival data (A-B) were analyzed using a log-rank (Mantel-Cox) test. PHH3 quantification (E), gene expression analysis (C), upd3-GFP quantification (D), Nora virus load (F), and the number of Nora (+) enterocytes (H) were analyzed using one-way ANOVA with Tukey’s post-hoc test. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Model of Nora virus propagation in Drosophila intestine

Schematic representation of Nora virus propagation in the posterior midgut from intestinal stem cells to enterocytes. The Nora virus initially invades ISC in the midgut and remains rather quiescent. External factors such as age, infections or exposure to xenobiotics stress the host intestinal epithelium and induce ISC compensatory proliferation that ensures a degree of homeostasis by replacing damaged enterocytes. ISC cell division appears to stimulate the proliferation of the Nora virus that ultimately contaminates enterocytes. This is likely to further damage the epithelium and lead to a loss of the integrity of this intestinal barrier, allowing the enhanced passage of gut bacteria to the hemocoel.