Infested Nora flies have a weaker fitness and are more sensitive to various stress conditions

Flies infested with Nora virus are noted as Ore-R(SM) and non-infested flies as Ore-R(SC).

(A) Ore-R(SM) (Nora(+)) were more susceptible to an intestinal infection with PA14 in survival experiments at 25°C. Infected flies: PA14; noninfected control flies: NI.

(B) Nora infested flies presented a reduced lifespan compared to uninfested flies.

(C) Nora titer of Ore-R(SM) and Ore-R(SC) stocks measured by qRT-PCR.

(D) A significant difference of the number PHH3 positive nuclei in Drosophila midgut at 2 days after an intestinal infection with PA14 (PA14) or noninfected controls (NI) was observed between Ore-R(SM) (Nora(+)) and Ore-R(SC) (Nora(-)) flies.

(E) Nora virus titer of the original and cured stocks as measured by qRT-PCR.

(F) Flies used for these experiments were all originally OreR(SM) Drosophila infested with the Nora virus. Some flies have been cured from Nora virus (Nora(-)) by bleaching the eggs and kept separately from the Nora virus infested flies (Nora(+)).

All data represent the mean of biological triplicates ± SEM of one representative experiment out of three.

LogRank test was performed for survival test.

Mann-Whitney nonparametric t-test was performed for Nora load.

One-Way ANOVA with multiple comparison was perform for PHH3 quantification.

*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.

Nora virus causes an enhanced susceptibility to PA14 intestinal infections

Flies used for these experiments were from Ore-R(SM) stocks cured from Nora infestation by egg bleaching. Some of these flies were re-infected with a pure suspension of Nora virus (Nora(+) re-inf virus) or not (Nora(-)). (PA14) were the flies infected with PA14 and (NI) the uninfected control flies.

(A) Electronic microscopic picture of the pure Nora virus preparation. Black arrows indicate Nora virus particles.

(B) A significant difference in the Nora virus titer measured by qRT-PCR was observed between Nora(+) and Nora(-) flies in the first (G1) and second (G2) generation after re-infection with virus. The observed difference between the initially re-infected G0 generation flies (G0 Nora(+) re-inf virus) or not (G0 Nora(-)) was not significant.

(C) Nora(+) flies were more susceptible than Nora(-) flies to an intestinal infection with PA14 at 25°C.

(D) A difference in the number of PHH3 positive nuclei in midguts was observed between Nora(+) and Nora(-) flies whether the flies had ingested PA14 for two days or not. A significant increase of PHH3 positive nuclei was detected after a PA14 intestinal infection in both Nora(+) and Nora(-) flies.

(E) Nora(+) flies displayed a shorter lifespan than Nora(-) flies.

(F) A marked increase of the death rate on a sucrose diet was observed in Nora(+) flies compared to Nora(-) flies.

All data represent the mean of biological triplicates ± SEM of one representative experiment out of three.

LogRank test was performed for survival test.

Mann-Whitney nonparametric t-test was performed for Nora load.

One-Way ANOVA with multiple comparison was perform for PHH3 quantification.

*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.

Aging, nutrient, and microbiota increase susceptibility of Nora infested flies to PA14 infections

(A) Quantification of Nora virus by RT-qPCR at 3d post-infection from flies infested or not from standard food or rich food (= standard medium plus 5x extra yeast) with PA14 co-infection.

(B) A significant difference in the Nora virus titer measured by qRT-PCR was observed between Nora(+) young (3d old) versus old (30d old) flies.

(C) A strong increase of four orders of magnitude (Log scale) of the microbiotal titer was observed between young Nora(+) and Nora(-) flies kept for eight days on a sucrose diet. A significant increase was detected in old flies kept on standard fly food.

(D) Nora infested flies presented a reduced lifespan compared to uninfested flies on standard food with or without the addition of antibiotics (antiB).

(E) Nora infested flies presented a reduced lifespan compared to uninfested flies on rich food (= standard medium plus 5x extra yeast) with or without the addition of antibiotics (antiB).

All data represent the mean of biological triplicates ± SEM of one representative experiment out of three.

LogRank test was performed for survival test.

Mann-Whitney nonparametric t-test was performed for Nora load.

One-Way ANOVA with multiple comparison was perform for PHH3 and CFU quantification.

*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.

Flies infested with Nora virus show a defect in intestinal barrier integrity

(A) Comparison for SMURF assay of 30d old non infected fly (top) and Nora infected fly (bottom).

(B) Percentage of SMURF positive flies at 30d on rich food (= LT50 on the survival test in Fig. 2E)

(C) Picture of LB plate with drop of hemolymph in dilution series from non infected flies, infected flies with SMURF negative or SMURF positive phenotype.

(D) A significant difference of the PA14 titer in the hemolymph was measured at day three of the infection between Nora(-) and Nora(+) flies.

(E) A marked increase of Diptericin expression in midguts of Nora(+) flies was detected by qRT-PCR after a PA14 intestinal infection at two days or when fed on SDS 1% for 4-6 hours.

All data represent the mean of biological triplicates ± SEM of one representative experiment out of three.

Mann-Whitney nonparametric t-test was performed for percentage of SMURF flies.

One-Way ANOVA with multiple comparison was perform for CFU and Dipt expression quantification.

*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.

The Nora virus is located in the intestinal stem cells (ISCc)

(A) Quantification of Nora virus by RT-qPCR in the whole fly or the intestine only.

(B) Confocal pictures of non-infested 5d old intestine. Intestine fixed and stained for DNA (DAPI, in blue), actin (FITC, in green) and Nora (Cy3, in red). The Cy3 antibody (goat anti-mouse) have a background signal in the intestinal muscles. Scale bar = 5 µm.

(C) Confocal pictures of infested 5d old intestine. Intestine fixed and stained for DNA (DAPI, in blue), actin (FITC, in green), and Nora (Cy3, in red). Scale bar = 3 µm (left) and 5 µm (right).

(D) Colocalization of Nora virus in intestinal stem cells (ISCs) using the reporter esg-Gal4Gal80ts crossed with UAS::GFP. Intestine fixed and stained for DNA (DAPI, in blue), and Nora (Cy3, in red). Scale bar = 2 µm (left) and 5 µm (right).

(E) Confocal picture of infected GFP::Dicer-2 intestine. Intestine fixed and stained for DNA (DAPI, in blue), actin (RFP, in red), Dicer (GFP, in green), and Nora (Cy5, in purple). Scale bar = 10 µm (left) and 5 µm (right).

(F) Survival test of flies infected or not by Nora with PA14 or Db11 co-infection or exposure to paraquat. As previously shown, Db11 co-infection with Nora virus has no survival effect. In opposition to PA14 and paraquat where Nora(+) flies are more sensitive.

(G) Quantification of Nora virus by RT-qPCR from (F).

(H) Confocal picture of Nora infested intestine (Nora(+)) with PA14 co-infection (Nora(+) / PA14), Db11 co-infection (Nora(+) / Db11), and paraquat exposure (Nora(+) / Paraquat). More Nora virus is detected in ECs when combined with PA14 infection or paraquat exposure as shown in (G). Intestine fixed and stained for DNA (DAPI, in blue), actin (FITC, in green) and Nora (Cy3, in red). Scale bar = 3 µm.

All data represent the mean of biological triplicates ± SEM of one representative experiment out of three.

LogRank test was performed for survival test.

Mann-Whitney nonparametric t-test was performed for Nora load.

One-Way ANOVA with multiple comparison was perform for Nora load plus pathogen co-infection or paraquat exposure.

*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.

Apoptosis inhibition via p35 expression do not allow Nora virus to spread through the intestine

(A) Increased quantification of ApopTag positive nucleus in posterior midgut with PA14 co-infection or paraquat exposure.

(B) A significant increase of PHH3 positive nucleus in posterior midgut of Nora infested flies co-infected with PA14. There is a correlation with Nora load increase (Fig. 5G) and PHH3 positive nucleus increase.

(C) Survival test of non infested flies with PA14 co-infection in controls (NP driver (NPGal4G80ts) and UAS-p35 alone) and apoptosis inhibition (NP>p35).

(D) Survival test of infested flies with PA14 co-infection in controls (NP driver (NPGal4G80ts) and UAS-p35 alone) and apoptosis inhibition (NP>p35). Inhibition of apoptosis in reducing the lethality induce by co-infection of Nora plus PA14.

(E) Significant reduction of PHH3 positive nucleus in posterior midgut from (C) and (D) at 5d post-infection in flies expressing the apoptosis inhibitor p35 in intestine.

(F) Quantification of Nora virus by RT-qPCR from (E) at 3d post-infection with low level of Nora virus detected in intestines deficient for apoptotic activity.

(G) Confocal picture of infested intestine in control NP driver (NPG4G80) and UAS-p35 (UAS-p35), or without apoptosis (NP>p35). The loss of intestinal apoptosis in induce this anarchic structure with enterocytes layers. Intestine fixed and stained for DNA (DAPI, in blue), actin (FITC, in green) and Nora (Cy3, in red). Scale bar = 5 µm.

(H) Quantification of Nora positive cells in the intestine.

All data represent the mean of biological triplicates ± SEM of one representative experiment out of three.

LogRank test was performed for survival test.

One-Way ANOVA with multiple comparison was perform for ApopTag, PHH3, and Nora load quantification.

*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.

JAK-STAT pathway inhibition does not allow Nora virus to spread through the intestine

(A) Survival test of flies non-infested plus PA14 infection with RNA inhibition of JAK-STAT pathway genes showing small survival resistance with dome and Stat92E RNAi inhibition in the intestinal stem cells using delta driver. Non infected control survival test on the right bottom.

(B) Survival test of flies infested plus PA14 infection with RNA inhibition of JAK-STAT pathway genes showing significant survival resistance with dome and Stat92E RNAi inhibition in the intestinal stem cells using delta driver. Non infected control survival test on the right bottom.

(C) Expression of JAK-STAT genes by RT-qPCR from flies infested or not with PA14 co-infection at 3d post-infection. The upd3 cytokine is more expressed under PA14 infection.

(D) Quantification of upd3::GFP in the posterior midgut of flies infested or not with PA14 co-infection at 2d post-infection. As observe in (C), upd3::GFP is more detected in presence of PA14 co-infection.

(E) Significant loss of PHH3 positive nucleus in posterior midgut at 3d post-infection when the JAK-STAT pathway is altered bu RNAi inhibition of dome and Stat92E in intestinal stem cells using delta driver.

(F) Quantification of Nora virus by RT-qPCR from (E) with significant loss of Nora proliferation in intestines with JAK-STAT pathway inhibition in their stem cells.

All data represent the mean of biological triplicates ± SEM of one representative experiment out of three.

LogRank test was performed for survival test.

One-Way ANOVA with multiple comparison was perform for PHH3, gene expression, upd3::GFP, and Nora load quantification.

*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 intially 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.