We noted that two Oregon-R (Ore-R) wild-type stocks kept by different investigators in the laboratory, hereafter referred to as (SM) and (SC), displayed a remarkably distinct survival pattern in one out of two models of intestinal infections (Limmer et al., 2011; Nehme et al., 2007) in which flies respectively continuously feed either on Pseudomonas aeruginosa PA14 (Fig. 1A) or Serratia marcescens Db11 (Fig. S1A). Namely, the Ore-R (SM) was more sensitive to PA14 and there was a slight trend toward higher susceptibility to Db11. We also noted that Ore-R (SM) displayed a shorter lifespan under non-infected conditions either on our standard food or on sucrose solution (Fig. 1B, Fig. S1B, and see below). We wondered whether the difference might be due to an infection and checked the stocks for the presence of common microbes known to affect Drosophila stocks (Haller et al., 2014; Lestradet et al., 2014). The only notable difference we identified between the two stocks was the presence of the Nora enteric virus in the infection-sensitive Ore-R(SM) (Fig. 1C).

We therefore examined the guts of flies from the two Ore-R stocks and did not detect any major morphological differences. However, when we measured basal ISC proliferation by counting phospho-histone H3-positive (PHH3) cells in the intestine, we found an enhanced rate of ISC proliferation in the Nora-positive Ore-R(SM) stock (Fig. 1D). When we challenged the Nora-negative Ore-R(SC) stock with P. aeruginosa, we found a small but nevertheless significant increase in ISC proliferation. However, this P. aeruginosa-induced increase was three-times larger in the Nora-positive Ore-R(SM) stock (Fig. 1D).

Overall, these observations suggest that flies infected with Nora have a lower fitness and are more susceptible to infections than non-infected flies.

As expected, bleaching the eggs laid by the Nora-infected Ore-R(SM) stock appeared to eradicate the virus ((Habayeb et al., 2009b), Fig. 1E and Fig. S2A). This treatment did not have a noticeable adverse impact as bleached stocks displayed a normal survival for at least ten days on sucrose (Fig. S1E-I). Furthermore, cured Ore-R(SM) flies fed with P. aeruginosa were less susceptible to this intestinal challenge than the uncured stock (Fig. 1F).

We placed our cured flies in a vial that had hosted infected males for several days for fecal-oral transmission (Habayeb et al., 2009a) and found that they became again Nora-positive over several generations (Fig. S2A). The reinfected stock was more sensitive to the ingestion of P. aeruginosa than the cured stock (Fig. S2B). This correlated with an enhanced ISC proliferation in the Nora virus reinfected flies, whether challenged with P. aeruginosa or not (Fig. S2C).

The drawback of the fecal transmission route is that other enteric pathogens may be transferred along with the Nora virus. To exclude this possibility, we used gradient centrifugation to purify and concentrate Nora virus from an infected fly extract. This preparation was homogenous with particles of the expected size when observed by cryo-electron microscopy (Fig. 2A). Using RT-PCR, we confirmed the identity of the virus and ruled out the presence of other Drosophila viruses (DAV, DCV, DBV, DTV, DXV, CrPV, FHV…; see Table 1 Primer Sequence). Cured flies were fed on this pure viral preparation for 24 hours. This was sufficient to stably reinfect the stock over several generations (Fig. 2B). As with the fecal contamination route, we observed that the flies reinfected with the pure viral preparation were more sensitive to the ingestion of P. aeruginosa (Fig. 2C) and displayed an enhanced rate of ISC proliferation in the gut (Fig. 2D). In terms of lifespan on standard food or on sucrose solution, the re-infected stocks displayed a largely decreased fitness (Fig. 2E-F), in contrast to the findings of (Habayeb et al., 2009a; Nigg et al., 2024) who reported only a mild effect upon the injection of the Nora virus. Altogether, these results demonstrate that Nora virus is responsible for the enhanced sensitivity to P. aeruginosa intestinal infection.

We suspected that a polymorphism in the gene pastrel might be the cause of the different susceptibility to Nora virus between Ore-R(SM) and Ore-R(SC) (Magwire et al., 2012). To test this hypothesis, we determined by RT-PCR the presence of a pastrel sensitive or resistant alleles (pst^S, pst^R) in several stocks commonly used in the laboratory. Strikingly, the sensitive allele was found in homozygous condition only in the Ore-R(SM) stock, while it was heterozygous in our w (A5001) stock (Fig. S1D). Interestingly, the w (A5001) (Thibault et al., 2004) and the DD1 cn bw (Jung et al., 2001) stocks were found to be also harboring the Nora virus, the latter one being pst^R/ pst^R (Fig. S1C-D). The w (A5001) and DD1 cn bw cured stocks as well as the Canton-S stocks were all more susceptible to P. aeruginosa ingestion after having been converted to a Nora-positive status by the prior ingestion of the purified virus preparation (Fig. S1E-G). Since even stocks harboring two copies of the resistant pastrel allele became more sensitive to ingested P. aeruginosa (Fig. S1D, S1F-G), we can rule out that distinct pastrel alleles were also the cause of the enhanced sensitivity to bacterial intestinal infections. Unexpectedly, the Ore-R(SC) strain appeared to be resistant to the ingestion of Nora since it did not display an enhanced sensitivity to oral P. aeruginosa infection (Fig.S1I). Thus, this line may harbor in addition to the homozygous pst^R allele a polymorphism at an unidentified locus that restricts the Nora virus.

In the following, we will use the Ore-R(SM) cured stock as a Nora-negative control. We will further characterize the impact of Nora virus on infected flies using the cured Ore-R(SM) stock reinfected with the pure Nora preparation, which will be referred to as Nora-positive flies.

We confirmed that Nora re-infected flies succumbed much earlier than Nora-negative control flies when orally challenged with P. aeruginosa in a sucrose solution after five days of prior feeding on our standard food or a rich food corresponding to our standard food containing five times more yeast (Fig. S3A-B). Strikingly, we observed that an oral infection with PA14 increased the Nora burden (thrice on standard food and four-times on rich food). Actually, both the Nora virus load and the P. aeruginosa titer was higher for flies fed on rich food (Fig. 3A, Fig. S3C). Of note, flies fed on rich food succumbed earlier to the challenge, independently of their Nora status (compare S3B to S3A). We noted a correlation between the Nora load in the absence or presence of PA14 and the measured proliferation of ISCs, which held also on rich food (compare Fig. 3A and Fig. S3D).

As aging flies exhibit a dysbiosis that correlates with an impaired homeostasis of the intestinal epithelium (Biteau et al., 2008; Buchon et al., 2009a; Choi et al., 2008), we also compared the Nora burden in young (3-5 day-old) and old (30-35 day-old) flies and found an about four-fold increase (Fig. 3B), which correlated with an enhanced rate of ISC proliferation (Fig. S3E), in keeping with a previously published study (Hanson and Lemaitre, 2023). While checking for an increase of the microbiota load in old flies (Iatsenko et al., 2018), we noted that the microbiota titer was much higher in Nora-positive than in Nora-negative flies (Fig. 3C). This observation also held when flies were kept feeding only on a sucrose solution (Fig. S3F). To determine whether the microbiota influenced the fitness of Nora-negative or -positive flies, we first monitored their survival when kept on sucrose solution. An antibiotic mix treatment substantially decreased the lifespan of Nora-negative flies (Fig. S3G), possibly reflecting a positive impact of the microbiota on this amino-acid depleted food solution even though the microbiota is minimal under these sucrose conditions (Fig. S3F). In contrast, antibiotics treatment had no effect on Nora-positive flies feeding on sucrose solution (Fig. S3G). Interestingly, the proliferation of ISCs was increased in antibiotics-treated Nora-positive flies kept under these amino-acid depleted conditions (Fig. S3H), which inversely correlates with the life span of those flies. We next monitored the fitness of Nora-negative or -positive flies on our standard or on rich food. The antibiotic treatment had a limited effect on Nora-negative flies with a somewhat enhanced fitness that failed to reach statistical significance (Fig. 3D-E). In contrast, it significantly extended the lifespan of Nora-positive flies on either standard or rich food (note that the detrimental effect of the Nora virus is attenuated on rich food, at least for about two-thirds of their lives, until they rapidly succumb). However, the antibiotics treatment failed by far to protect the flies to the level of Nora-negative flies. We conclude that under these different feeding conditions, the microbiota contributes at best only partially to the decreased fitness of Nora-positive flies.

As an increased rate of ISC division may mirror a phenomenon of compensatory proliferation driven by damage to enterocytes, we assessed the permeability of the gut of Nora-positive flies, first using the SMURF assay on 30-day old flies; the SMURF assay monitors the permeability of the gut barrier upon ingestion of a dye (Rera et al., 2011). The percentage of SMURF-positive flies was much higher in 30-day old Nora-positive flies fed on rich food (a time point corresponding to the death of 50% (LT50) of the flies in fitness experiments shown in Fig. 3E) (Fig. 4A-B). In keeping with this result, when we plated hemolymph collected from flies, we observed a strong microbial growth for that retrieved from SMURF-positive flies, but not from SMURF-negative flies, whether Nora-positive or Nora-negative (Fig. 4C). Thus, SMURF-positive flies do not only display an enhanced permeability to the dye but also likely to the midgut microbiota, an indication of severe disruption of the barrier function of the intestinal epithelium. These results were further confirmed after P. aeruginosa ingestion: already after three days, the passage of PA14 through the intestinal epithelium was much higher in Nora-positive than in Nora-negative flies (Fig. 4D). Of note, at Day 3, some 220 bacterial colonies on average were also retrieved from the hemolymph of Nora-positive control flies that had not ingested PA14, possibly reflecting an early intestinal barrier dysfunction in Nora-positive flies. Indeed, there was a detectable induction of a local immune response in the midgut induced by PA14 ingestion only in Nora-positive flies at two days instead of around five days for Nora-negative flies (Limmer et al., 2011), as monitored by measuring Diptericin expression levels (Fig. 4E). This immune response is triggered by the proliferation of P. aeruginosa in the hemolymph (Chen et al., 2024; Limmer et al., 2011). We conclude that the Nora virus affects the integrity of the intestinal epithelium and its barrier function.

The experiments above show that the presence of the Nora virus in a stock increases its susceptibility to ingested P. aeruginosa. Since P. aeruginosa ultimately causes a systemic infection, we also tested whether Nora-positive flies would be more sensitive to other types of systemic infections. As shown in Fig. S4A, we did not observe any difference in the survival curves between Nora-positive or -negative flies exposed to spores of the entomopathogenic fungus Beauveria bassiana, an infection model in which no macroscopic wounds are inflicted. In contrast, Nora-positive flies succumbed significantly earlier than Nora-negative control flies when challenged with the Gram-positive Enterococcus faecalis or the Gram-negative Enterobacter cloacae bacterial pathogens in a septic injury model (Fig. S4B-C). However, we found in control experiments that Nora-positive flies pricked with a needle dipped into a sterile PBS solution succumbed at a rate that was similar to that observed in flies submitted to a septic injury, whereas control Nora-negative flies were more resistant to aseptic injury (compare Fig. S4D to Fig. S4B-C). Unexpectedly, MyD88 flies displayed an enhanced apparent sensitivity to a clean injury, a phenomenon we have observed before and that depends on the endogenous microbiota of MyD88 flies (Xu et al., 2023). To understand why Nora-infected flies were more susceptible to a near-sterile wound, we measured the proliferation rate of ISCs as it has been reported that injury triggers a ROS-response, in the hypodermis, hemocytes, and in the gut that leads to enterocyte apoptosis and an increased compensatory proliferation of ISCs (Chakrabarti and Visweswariah, 2020; Takeishi et al., 2013). As reported, we observed a modest, statistically not significant, increase of ISC proliferation in Nora-negative flies after injury of the cuticle (Fig. S4E). Nora-positive flies displayed a higher basal rate of ISC proliferation that was however not altered by injury, suggesting that gut damage is unlikely to account for the enhanced sensitivity of Nora-positive flies to wounding of the cuticle. We finally compared the survival rates of flies pricked with a clean needle to that of unchallenged controls and did not find any significant difference (Fig. S4F). We conclude that the apparent sensitivity of Nora-positive flies to clean injuries is actually due to the shortened lifespan of Nora-positive flies. This effect only becomes apparent in systemic infections with mild pathogens that kill the flies slowly (Fig. S4B-C).

Since the Nora virus is mostly detected in the gut by RT-qPCR (Habayeb et al., 2009a) and plating assays (Ekstrom and Hultmark, 2016) (Fig. 5A) and since it affects the barrier function of the intestinal epithelium, we raised an antibody against the virus, which is essentially specific except for a cross reaction of the secondary antibodies with intestinal muscles (compare Figs. 5B & S5A to 5C), which precludes drawing any conclusion as regards a possible localization of the Nora virus also in these muscles, as has been previously described for DCV oral infection (Ferreira et al., 2014). In contrast, a positive signal was detected in basally located small triangular cells of the intestinal epithelium of flies that had ingested the Nora virus five days earlier and not in non-infected controls (Figs. 5C-D, Fig. 5B & Fig. S5A). The right panel of Fig. 5C displays a dividing stem cell that yields a basally located ISC and a differentiating progenitor cell (enteroblast or enteroendocrine cell). Indeed, a similar non-basally located small cell is shown in Fig. S5B. We confirmed the localization of the Nora virus to progenitor cells of the intestinal epithelium by staining the Nora virus in esg-Gal4Gal80^ts >UAS-GFP flies (Fig. 5D). The Nora-positive signal was always found in the GFP-expressing progenitor cells. Using transgenic fly lines that express Dicer2-fluorescent protein fusions (Donelick et al., 2020; Girardi et al., 2015), we noted that even though the construct was expressed under the direct control of the poly-ubiquitin promoter (ubi-p63E), we failed to detect the fusion protein in ISCs (Figs. 5E & S5C), even though GFP is known to be stable in progenitor cells (Fig. 5D). If Dicer-2 were to be unstable specifically in ISCs, this might account for the initial localization of the Nora virus to ISCs, a proposition that would require further experimental confirmation.

We have shown above that young Nora-positive flies are more susceptible to P. aeruginosa ingestion. We found that the Nora-positive flies were also more susceptible to the strongly oxidizing agent paraquat in survival experiment (Fig. 5F). As for P. aeruginosa oral infection, the ingestion of paraquat led to similarly enhanced levels of the viral load as monitored by RTqPCR (Fig. 5G) and by staining with the Nora antibody (Figs. 5H & S5D). Immunohistochemistry further revealed that the virus starts also proliferating within enterocytes under pathogenic or chemical stress (Fig. 5H-I). Interestingly, aged flies raised on our standard food or young flies raised on rich food also exhibited a growth of the virus in enterocytes (Fig. S5E-I), in keeping with the increased viral burden found in these 30-day-old flies (Fig. 3A-B). In addition, the detection of the Nora virus in enterocytes (Fig. 5I & Fig. S5I) also correlated with an enhanced proliferation of ISCs (Figs. 3C & S3D).

In conclusion, the Nora virus is initially restricted to progenitor cells in basal conditions but appears to start proliferating also inside enterocytes upon stress and/or increased proliferation of ISCs. Importantly, Nora-positive cells were detected only in the posterior midgut (scheme in Fig. S5J). These observations open the possibility that Nora virus is transmitted to differentiating cells after ISC division (Figs. 5C & S5B) and then ultimately to enterocytes.

The above conclusion implies that ISC proliferation is likely needed to promote the proliferation of the virus. To test this hypothesis, we used two complementary approaches that rely on the fact that enterocyte damage triggers the compensatory proliferation of ISCs.

First, we measured the degree of apoptosis in epithelial cells through ApopTag/TUNEL staining. We noted that Nora-positive bacteria do not display enhanced levels of apoptosis in this tissue under basal conditions (Fig. 6A, Fig. S6A). However, the ingestion of P. aeruginosa led to enhanced levels of apoptosis, significantly more so in Nora-positive flies (Fig. 6A, Fig. S6B). In contrast, paraquat induced much higher levels of positive signals, independently of the Nora status of the exposed flies (Fig. 6A, Fig. S6C). This situation was roughly mirrored in the number of mitotic ISCs (Fig. 6B), although the degree of proliferation induced by paraquat was similar to that induced by P. aeruginosa infection, possibly mirroring an adverse effect of paraquat exposure to the division of ISCs (Chatterjee and Ip, 2009).

Next, we attempted to block the induction of apoptosis in adult enterocytes by expressing there the baculovirus p35 protein that inhibits executioner caspases (Hay et al., 1994). p35 did partially protect the flies from the noxious effects of P. aeruginosa ingestion, with a relatively stronger effect on Nora-positive flies, which correlates with the higher number of apoptotic cells measured in those flies (Fig. 6C-D, Fig. 6A). As expected, the expression of p35 in enterocytes blocked the compensatory proliferation of ISCs (Fig. 6E). Strikingly, Nora virus was obliterated in both control or P. aeruginosa-infected, originally Nora-positive, flies, as monitored by RTqPCR (Fig. 6F) and by immunohistochemistry in 30-day old flies (Fig. 6G-H) for which less than ten Nora-positive progenitor cells per midgut were detected at best. The second approach was to interfere with the signals that drive ISC proliferation upon enterocyte damage. JAK-STAT pathway activation in ISCs promotes their division (Buchon et al., 2009b; Cronin et al., 2009; Jiang et al., 2009). Silencing in ISCs the genes that encode either the JAK-STAT pathway receptor Dome or the STAT92E transcription factor also provided protection against ingested P. aeruginosa, in both Nora-positive and Nora-negative cells (Fig. 7A-B). Indeed, the upd3 gene encoding one Dome ligand and the gene encoding the JAK-STAT-regulated inhibitor SOCS36E were induced in dissected midguts (crop and Malpighian tubules not included) by P. aeruginosa ingestion, again more strongly so in Nora-positive flies (Fig. 7C). This result was further corroborated using a transgenic fly line that expresses an UPD3-GFP reporter protein (Fig. 7D). As expected, silencing Dome or STAT abolished the compensatory proliferation of ISCs (Fig. 7E). Again, as for the ectopic expression of p35 in enterocytes, blocking JAK-STAT pathway signaling in ISCs prevented any proliferation of the Nora virus in midguts, whether infected orally with P. aeruginosa or not (Fig. 7F). Microscopic analysis of immunostained posterior midguts confirmed that when the JAK-STAT signaling pathway was blocked specifically in ISCs using a Dome-Gal4 driver line, Nora virus remained confined to progenitor cells and was not detected in enterocytes (Fig. 7G-H).

Wild-type Ore-R(SM) Nora(+) and Ore-R(SC) Nora(-) fly stocks were found in our laboratory. Both stocks of wild-type Oregon-R flies tested negative for Wolbachia infection. Stock used for the septic injury and natural infections are the following: wild-type w^A5001 (Thibault et al., 2004), mutant MyD88c03881 (Tauszig-Delamasure et al., 2002), mutant DD1 cn bw (Rutschmann et al., 2000), key^cO2831(Ferrandon D., unpublished), NP1-Gal4 (Cronin et al., 2009), esg-Gal4Gal80^ts(Micchelli and Perrimon, 2006), delta-Gal4 (Zeng et al., 2010), VDRC GD control line (#6000 VDRC), UAS-dome^RNAi (#36355 GD VDRC) (Borensztejn et al., 2013), Bloomington control line UAS-mCherry^RNAi (#35787 Bloomington Drosophila Stock Center) (Borensztejn et al., 2013), UAS-p35 (#5073 Bloomington Drosophila Stock Center) (Hay et al., 1994; Rahmatika et al., 2019), UAS-Stat92E^RNAi(#26899 Bloomington Drosophila Stock Center) (Nagai et al., 2023), and upd3::GFP (Zhou et al., 2013).

All stocks have been checked for the presence of known pathogens and symbiont besides Nora virus (Haller et al., 2014; Lestradet et al., 2014; Niehus et al., 2012). Fly stocks were kept at 25°C and nearly 60% humidity, on a standard semi-solid cormeal medium (6.4% (w/v) cornmeal (Moulin des moines, France), 4.8% (w/v) granulated sugar (Erstein, France), 1.2% (w/v) yeast brewer’s dry powder (VWR, Belgium), 0.5% (w/v) agar (Sobigel, France), 0.004% (w/v) 4-hydroxybenzoate sodium salt (Merck, Germany)). For lifespan analysis, 3 x 20 female flies were kept at 25°C with 60% humidity, on standard fly food. Flies were transferred without anesthesia on fresh food every 4 days. For survival test on a sucrose only diet, 3x 20 female flies were fed on 2 mL on a 50 mM sucrose solution and kept at 25°C with 60% humidity.

Microbes were grown in these conditions: Pseudomonas aeruginosa PA14 in Brain-Heart-Infusion Broth (BHB), overnight, at 37°C (Rahme et al., 1995); Serratia marcescens Db11 (Nehme et al., 2007) in Luria Bertoni Broth (LB), overnight, at 37°C; Enterobacter cloacae (Lemaitre et al., 1997), in LB, overnight, at 30°C; Enterococcus faecalis (TX0016) in BHB overnight, at 37°C; Beauvaria bassiana (Lemaitre et al., 1997) on malt agar plates at 25°C. Intestinal infections were performed as described previously with PA14 (Haller et al., 2014) and Db11 (Lee et al., 2016). Briefly, flies were continuously feeding on a sucrose solution containing the bacteria at the indicated OD and deposited on filter pads in the absence of standard food. In the case of S. marcescens 100 µL of 100 mM sucrose solution was added to the cage every day. Infected and control flies were kept at 25°C. For PA14 infection, 3x 20 female flies were used per experiment. For E. cloacae septic injury, 50 mL of an overnight culture of bacteria was centrifuged 30 min at 3 000 x g. The supernatant was removed and pellet used to infect the flies. For E. faecalis septic injury, overnight culture was diluted to 1/50 in fresh BHB and allowed to growth for 3 more hours at 37°C. The final culture was centrifuged 10 min at 3000 x g and the pellet washed one time with sterile PBS. The bacteria were resuspended to a final OD = 0.5 that was used to infect female flies with a septic injury. 3x 20 females were infected with each bacterium. The septic injury and the PBS clean injury were performed as described (Haller et al., 2014): an electrolysis-sharpened tungsten wire dipped or not in a microbial solution was used to prick flies in the thorax at a position corresponding to alary muscles. 20 females were injured. For B. bassiana natural infection, flies were anesthetized, deposited and shaken on a sporulating plate containing the fungus. 3 x 20 females were infected. Flies were kept at 29°C with 60% humidity and transferred, without anesthesia, to fresh food every 2-3 days.

Paraquat solution was prepared by dissolving Paraquat dichloride (Sigma-Aldrich # 50636) in 50 mM sucrose solution to obtain a final paraquat concentration at 1 mM. For survival test on a paraquat supplemented sucrose only diet, 3x 20 female flies were fed on 2 mL on a 50 mM sucrose plus 1 mM paraquat solution and kept at 25°C with 60% humidity.

Nora(+) flies were allowed to lay eggs overnight at 25°C in a cage on apple juice agar plate with yeast paste in the center of the plate. Eggs were collected, washed with water, and dechorionated with a 50% bleach solution for 3 min with constant up and down pipetting of the solution. Eggs were abundantly rinsed with water, aligned under the microscope on a piece of agar medium and transferred by capillarity on a coverslip. One drop of mineral oil was applied to cover the eggs, and the coverslip was deposited on a petri dish with normal Drosophila food. After 2 days, larvae were transferred to normal food vial (single bleaching) or treated again with bleach solution as previously and then transferred to normal food vial (double bleaching or even triple bleaching if needed). Once flies emerged, they were tested for Nora virus infection. Usually, there was no difference in the results obtained with the single or double bleaching procedures although this observation applies only to short term observations.

Nora(+) flies (around 5 000 flies) were crashed in ice cold PBS using a potter homogenizer. The homogenate (around 35 mL) was transferred in a 50 mL Falcon tube and centrifuged at 1 500 rpm for 10 min, at 4°C in an Eppendorf 5810R centrifuge to remove wings, legs and other fly debris. The resulting supernatant was sequentially filtrated through 0.8, 0.45 and 0.22 μm filter units and stored at −80°C. The homogenate (around 26 mL) was then layered in two 15 ml Beckmann Ultra-Clear centrifuge tubes over 1.5 mL of a 30 % (wt/wt) sucrose solution (50 mM Hepes pH 7.0, 0.1 % BSA) and centrifuged at 24,000 rpm for 6.5 hours at 11°C using a JS-24.15 rotor (Beckmann). The pellets were resuspended in a total volume of 500 μL Hepes solution (Hepes 50 mM, pH 7.0) and transferred into a 1.5 mL Eppendorf tube. The solution was then centrifuged at 10,000 rpm for 10 min in a 5424 Eppendorf centrifuge to discard insoluble material. The resulting supernatant containing virus particles was layered over a 40-10 % (wt/wt) sucrose gradient (50 mM Hepes, pH 7.0) prepared in a 15 mL Ultra-Clear centrifuge tube (Beckmann) and centrifuged at 11°C for 4h at 24,000 rpm in a JS-24.15 rotor (Beckmann). An opalescent band, that migrated near the middle of the tube, containing virus particles was then collected by puncturing the tube with a 25-gauge needle mounted on a 1 ml syringe. This solution was then layered onto a 30 % (wt/wt) sucrose solution contained in a 15 mL Beckmann Ultra-Clear centrifuge tubes (50 mM Hepes pH 7.0, 0.1 % BSA) and centrifuged at 24,000 rpm for 6.5 hours at 11°C using a JS-24.15 rotor (Beckmann). The pellet was resuspended in 500 μl of Tris solution (Tris-HCl 10 mM, pH 7.5), aliquoted, and stored at −80°C.

The sample was prepared by plunge-freezing of 2.5 μL of the sample on a holey carbon Quantifoil R 2/2 grid using FEI Vitrobot Mark IV machine. Images were gained on FEI Polara F30 TEM microscope with acceleration voltage 100 KV and underfocus around 2.0 microns.

Nora(-) flies were fed 24 hours with a 1/100 dilution of the viral preparation in sucrose 50mM. In practice, 200 μL of the 1/100 dilution (in sucrose 50mM) of the viral suspension were deposited in an Eppendorf cup that was then placed in an empty fly tube. Flies (male and females) were then added in the tube. The flies were allowed to feed on the viral suspension for 24 hours and then transferred to a fly tube containing standard fly food.

200 males of Nora(+) flies were allowed to deposit their feces in a food vial for 5 days at 25°C. Nora(+) flies were then replaced by 50 males and 50 females of cured Nora(+) flies. After 5 days, the vial was emptied, and Nora virus infection status was monitored in those flies (generation G0). Once the progeny emerged, 0 to 4 days old flies were transferred to a fresh vial for 4 days and then monitored for Nora virus load or used for experiments (first generation after re-infection G1). The same procedure was repeated with the second generation after re-infection (G2).

Pure virus preparation was mixed with complete (first inoculation) then incomplete Freund’s adjuvant and was injected intraperitoneally in BALB/c mice every week over a period of three weeks prior to ultimately bleeding the mice. The specificity of the antibody was assessed by immunohistochemistry on Nora-negative and Nora-positive flies as determined using RTqPCR.

Midguts were dissected in PBS and fixed for 30 minutes with 4% paraformaldehyde. Samples were washed three times with PBS-Triton X-100 0.1% (PBT 0.1%) as described (Socha et al., 2023). For actin staining midguts were incubated for 1h30 at room-temperature or overnight at 4°C in 10 μM Fluorescein Isothiocyanate (FITC) (Sigma-Aldrich #P5282) or Texas-Red labeled phalloidin (Invitrogen TM #T7471). Samples were then washed three times with PBT 0.1%. All samples were mounted on diagnostic microscope slides (Thermo Fisher Scientific) with Vectashield plus DAPI (Vector Laboratories). Samples were observed using a LSM780 confocal microscope (Zeiss) or in Axioskop 2 microscope (Zeiss). All images were analyzed with the ImageJ/Fiji software.

Fly guts were dissected, fixed, stained by immunohistochemistry with an anti-pH3 antibody (Millipore) and mounted following standard procedures (Lee et al., 2016). Midguts were observed using a fluorescent microscope (Axioskop 2, ZEISS) and nuclei positive for pH3 staining were counted manually.

Female flies were sterilized for 30 seconds in 70% ethanol and midguts were dissected in sterile PBS (10 midguts per sample) and immediately transferred in sterile PBS. Midguts were homogenized with a sterile pestle. Serial dilutions of the suspension were performed prior to plating on specific solid media and incubated at 30°C. Acetobacteriaceae plates, Enterobacteriaceae plates and MRS plates were performed as described (Guo et al., 2014). For BHB agar plates: 37 g/L BHB (Sigma), 15 g/L agar (Sigma). Media were autoclaved 15 min, at 121°C (prior to use) and stored at 4°C.

PA14 presence in the hemolymph was assessed as described previously (Haller et al., 2014); briefly, hemolymph was collected by capillarity using a drawn-out pipette mounted on the Nanoject II microinjector in the absence of oil. Hemolymph from 10 female flies per sample was used.

Fly midguts (without crop, hindgut and Malpighian tubules) were dissected (20 per sample) and RNAs were extracted in TRI Reagent®-RT as described (Lee et al., 2016). Reverse transcription was performed using iScriptTM (BIO-RAD). Quantitative PCR was performed using iQTM SYBR® Green (BIO-RAD) and a C1000TM Thermal Cycler (BIO-RAD) device. Expression values were calculated using a standard curve (with genomic DNA) and normalized to rp49 expression level. Results presented the average ± SD of 3 independent experiments (with biological duplicates or triplicates). See primer sequences in Table 1.

Whole flies (6 per sample) were frozen at −80°C. RNA extraction with a Nucleospin® RNA kit (Macherey-Nagel), reverse transcription and quantitative PCR were performed as described (Haller et al., 2014). Quantification values were calculated using a standard curve obtained from serial dilutions of a plasmid carrying the Nora virus DNA sequence amplified by the couple of primers used for the PCR. See primer sequences in Table 1.

To check the absence of other potentially contaminating viruses in the purified Nora virus preparations, we used the set of primers described by (Wu et al., 2010).

3x 20 female flies (3-8 days old) were kept on a sucrose (50mM) only diet with addition of a 5 antibiotics cocktail: 100 μg/mL ampicillin, 50 μg/mL vancomycin, 100 μg/mL neomycin, 100 μg/mL metronidazole and 50 μg/mL tetracyclin. Tubes were changed every 3 days and flies transferred without anesthesia.

The SMURF assay was developed to observe the integrity of the intestinal barrier (Rera et al., 2011). Flies are kept on a sucrose solution colored with Blue food dye (Brilliant Blue FCF E133). After ingestion, if there is an intestinal integrity loss, the dye will diffuse in the hemolymph, coloring the fly in blue.

Used as described by the manufacturer Sigma-Aldrich for the product ApopTag Fluorescein In Situ Apoptosis Detection Kit.

Fly midguts (without crop, hindgut and Malpighian tubules) were dissected (20 per sample) and RNAs were extracted in TRI Reagent®-RT as described (Lee et al., 2016). Reverse transcription was performed using iScriptTM (BIO-RAD). Quantitative PCR was performed using iQTM SYBR® Green (BIO-RAD) and a C1000TM Thermal Cycler (BIO-RAD) device. Expression values were calculated using a standard curve (with genomic DNA) and normalized to rp49 expression level. Results presented the average ± SD of 3 independent experiments (with biological duplicates or triplicates).

All statistical analyses were performed on Graphpad Prism version 10 (Graphpad software Inc., San Diego, CA). The Mann–Whitney tests were used unless otherwise indicated. For survival experiments, the time it takes for 50% of the flies to succumb (LT50) was determined for each curve being compared. An unpaired t-test used on the LT50s from biological triplicates was used to assess the significance between two survival curves. When using parametric tests (analysis of variance (ANOVA) and t-test), a Gaussian distribution of data was checked using either D’Agostino-Pearson omnibus, Anderson-Darling, or Shapiro-Wilk normality tests. All experiments were performed at least three times. Significance values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.