Nora virus proliferates in dividing intestinal stem cells and thereby sensitizes Drosophila flies to Pseudomonas aeruginosa intestinal infection and to oxidative stress
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eLife Assessment
This important study shows that the Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the bacterium Pseudomonas. The authors provide convincing data to support their conclusions. The paper provides new insights into virus-host interactions in the Drosophila gut and serves as a warning for scientists who use the fruit fly as a model to study gut physiology.
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Abstract
The digestive tract represents the most complex interface of an organism with its biotope. Food may be contaminated by pathogens and toxicants while an abundant and complex microbiota thrives in the gut lumen. The organism must defend itself against potentially noxious biotic or abiotic stresses while preserving its microbiota, provided it plays a beneficial role. The presence of intestinal viruses adds another layer of complexity. Starting from a differential sensitivity of two lines from the same Drosophila wild-type strain to ingested Pseudomonas aeruginosa, we report here that the presence of Nora virus in the gut epithelium promotes the sensitivity to this bacterial pathogen as well as to an ingested oxidizing xenobiotic. The genotype, age, nature of the ingested food and, to a limited extent, the microbiota are relevant parameters that influence the effects of Nora virus on host fitness. Mechanistically, we detect the initial presence of the virus essentially in progenitor cells. Upon stress such as infection, exposure to xenobiotics, aging or feeding on a rich-food diet, the virus is then detected in enterocytes, which correlates with a disruption of the intestinal barrier function in aged flies. Finally, we show that the virus proliferates only when ISCs are induced to divide. We propose that enterocytes essentially get infected through lineage from progenitor cells and are not directly infected.
In conclusion, it is important to check that experimental strains are devoid of intestinal viruses when monitoring survival/life span of fly lines or when investigating the homeostasis of the intestinal epithelium as these viruses can constitute significant confounding factors.
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eLife Assessment
This important study shows that the Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the bacterium Pseudomonas. The authors provide convincing data to support their conclusions. The paper provides new insights into virus-host interactions in the Drosophila gut and serves as a warning for scientists who use the fruit fly as a model to study gut physiology.
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Reviewer #1 (Public review):
[Editors' note: The article has been improved and several points raised by the reviewers have now been addressed. The authors should ideally further improve the clarity of the figures and the description of the experimental methods. This is particularly important for an article discussing potential confounding factors.]
Summary:
This important article reveals that the Nora virus can colonize the intestinal cells of Drosophila melanogaster, where it persists with minimal immediate impact on its host. However, upon aging, infection, or exposure to toxicants, stem cell activation induces Nora virus proliferation, enabling it to colonize enterocytes. This colonization disrupts enterocyte function, leading to increased gut permeability and a significant reduction in lifespan. Results are convincing and hold …
Reviewer #1 (Public review):
[Editors' note: The article has been improved and several points raised by the reviewers have now been addressed. The authors should ideally further improve the clarity of the figures and the description of the experimental methods. This is particularly important for an article discussing potential confounding factors.]
Summary:
This important article reveals that the Nora virus can colonize the intestinal cells of Drosophila melanogaster, where it persists with minimal immediate impact on its host. However, upon aging, infection, or exposure to toxicants, stem cell activation induces Nora virus proliferation, enabling it to colonize enterocytes. This colonization disrupts enterocyte function, leading to increased gut permeability and a significant reduction in lifespan. Results are convincing and hold significant import for the Drosophila community.
Strengths:
(1) Building on previous studies by Habayeb et al. (2009) and Hanson et al. (2023), this study highlights cryptic Nora virus infection as a crucial factor in aging and gut homeostasis in Drosophila melanogaster.
(2) Consistent with the oral route of Nora virus transmission, the study demonstrates that the virus resides in intestinal stem cells, with its replication directly linked to stem cell proliferation. This process facilitates the colonization of enterocytes, ultimately disrupting intestinal function.
(3) The study establishes a clear connection between stem cell proliferation and virus replication, suggesting that various factors - such as microbiota, aging, diet, and injury - can influence Nora virus dynamics and associated pathology.
(4) The experimental design is robust, comparing infected flies with virus-cured controls to validate findings.
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Reviewer #2 (Public review):
Summary:
In this manuscript, the authors report that Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the Pseudomonas bacterium. Nora virus infection was associated with an increased proliferation of ISC and disrupted gut barrier function. Genetically, the authors show that increased ISC division in Nora virus and Pseudomonas coinfected flies is driven by signaling through the JAK-STAT pathway and apoptosis.
Accordingly, blocking apoptosis and JAK-STAT signaling reduces viral load, suggesting that in this context the JAK-STAT pathway is proviral in contrast to other previous observations in systemically infected flies. This work adds …
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors report that Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the Pseudomonas bacterium. Nora virus infection was associated with an increased proliferation of ISC and disrupted gut barrier function. Genetically, the authors show that increased ISC division in Nora virus and Pseudomonas coinfected flies is driven by signaling through the JAK-STAT pathway and apoptosis.
Accordingly, blocking apoptosis and JAK-STAT signaling reduces viral load, suggesting that in this context the JAK-STAT pathway is proviral in contrast to other previous observations in systemically infected flies. This work adds to the findings of another recent paper showing that another persistent fruit fly virus, Drosophila A virus, also increases ISC proliferation and decreases gut barrier function. Intestinal viruses should therefore be considered confounders in studies of fly intestinal physiology.
Strengths:
Overall, the data are convincing and robust, starting with two wildtype fly stocks (Ore-R strain) that differ in their Nora virus infection status, followed by experiments in which cleared stocks are reinfected with a purified Nora virus stock preparation. The conclusions of the paper will be of interest to scientists working on insect physiology, virology, and immunology, but should also serve as a warning for scientists that use the fly as a model to study gut physiology.
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Reviewer #3 (Public review):
Summary:
Franchet et al. sought to characterize the impact of Nora virus on host lifespan and sensitivity to a variety of infectious or stressful treatments. Through careful and rigorous analyses, they provide evidence that the Nora virus greatly impacts fly survival to infection, overall lifespan, and intestinal integrity. The authors have been thorough and rigorous, and the experimental evidence including proper isolation of the virus and Koch's Postulate reinoculation of the organism is excellent. The additional work is valuable and to the gold standard of the field, characterizing the pathology of the gut, including data showing gut leakage, the presence of the virus in the intestinal stem cells, and the importance of stem cell proliferation for virus replication and spread using elegant genetic tools to …
Reviewer #3 (Public review):
Summary:
Franchet et al. sought to characterize the impact of Nora virus on host lifespan and sensitivity to a variety of infectious or stressful treatments. Through careful and rigorous analyses, they provide evidence that the Nora virus greatly impacts fly survival to infection, overall lifespan, and intestinal integrity. The authors have been thorough and rigorous, and the experimental evidence including proper isolation of the virus and Koch's Postulate reinoculation of the organism is excellent. The additional work is valuable and to the gold standard of the field, characterizing the pathology of the gut, including data showing gut leakage, the presence of the virus in the intestinal stem cells, and the importance of stem cell proliferation for virus replication and spread using elegant genetic tools to block stem cell proliferation or enterocyte death.
Strengths:
The authors have been rigorous and careful. The initial finding is presented through the lens of two related strains differing in virus infection. From there, the authors characterized the virus and isolated a purified culture, which they used to reinoculate a cleared strain to demonstrate proper Koch's Postulate satisfaction. The authors have also probed various parameters in terms of dietary importance in relevant conditions for many experiments. The additional work to characterize the pathology of the gut is compelling, using genetic tools to block or allow intestinal stem cell proliferation and enterocyte death through JAK-STAT and JNK signalling alongside the tracing of virus presence using a Nora virus antibody. JAK-STAT and JNK are previously described as regulators of these processes, making these tools appropriate and convincing. It is also interesting to see good evidence that the virus itself is damaging, rather than simply permitting coinfection by gut microbes (which does happen).
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Author Response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
(1) The study does not explore or discuss how oral ingestion of Nora virus leads to the colonization of stem cells, which are located basally in the gut. This mechanism should be discussed.
We have added an additional paragraph (4th) in the Discussion dealing with this issue and are further discussing the consequences of RNAi potentially not being functional in progenitor cells in the paragraph on antiviral responses.
(2) The authors fail to detect Dicer-GFP fusion protein expression in stem cells, a finding that could explain why the virus persists in these cells. Further investigation is needed to determine whether RNAi functions are effective in stem cells compared to enterocytes. For clarification, the …
Author Response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
(1) The study does not explore or discuss how oral ingestion of Nora virus leads to the colonization of stem cells, which are located basally in the gut. This mechanism should be discussed.
We have added an additional paragraph (4th) in the Discussion dealing with this issue and are further discussing the consequences of RNAi potentially not being functional in progenitor cells in the paragraph on antiviral responses.
(2) The authors fail to detect Dicer-GFP fusion protein expression in stem cells, a finding that could explain why the virus persists in these cells. Further investigation is needed to determine whether RNAi functions are effective in stem cells compared to enterocytes. For clarification, the authors could cross esg-Gal4 UAS-GFP and Myo-Gal4 UAS-GFP with UAS GFP-RNAi and/or express a Dicer-GFP construct under a stem cell-specific driver.
Actually, it is well-known in the Drosophila literature on the intestinal epithelium that RNAi functions well in progenitor cells as the technique has been widely used to understand the control of stem cell division and differentiation in tens of articles. We provide here just a few examples: Jiang et al., Nat Commun (2025) https://doi.org/10.1038/s41467-024-55255-1; Zhai et al., PLoS Genetics (2017) https://doi.org/10.1371/journal.pgen.1006854; Wu et al., https://doi.org/10.1371/journal.pgen.1009649.
(3) The presentation of experimental parameters (e.g., pathogen type, temperature, time points) should be improved in the results section and at the top of the figures to enhance clarity. Additionally, details regarding the mode of oral infection (continuous exposure vs. single feeding on a filter) should be specified. Given that fly stock flipping frequency influences microbiota load (as noted in Broderick et al.), this should be reported, especially for lifespan studies.
P. aeruginosa oral infection was always by continuous exposure, as detailed in the Mat.& Meth. section. Nora infection was done by exposure to the viral solution for 24h, as detailed in Mat. & Meth. The flipping frequency had also been reported in that section.
(4) To confirm that enterocyte colonization requires stem cell proliferation and differentiation, the authors should analyze Nora virus localization in JAK-STAT-deficient flies infected with bacteria or toxicants. This would help determine whether the virus can infect enterocytes in the absence of enterocyte differentiation, but stimulation of stem cells.
We now provide these data (pictures and quantification) in Fig.7 G-H and discuss them in the main text.
(5) The study does not discuss the spatial distribution of Nora virus infection along the gut. Specifically, it remains unclear whether viral colonization is higher in gut regions R2 and R3, which contain proliferative stem cells. Addressing this could provide valuable insights into the virus's infection dynamics.
We have now specified that Nora virus was detected only in the posterior midgut; we are now also providing a schematic illustration in Fig. S5J.
Recommendations for the authors:
Major Suggestion
See weaknesses section for key areas requiring improvement.
Minor Suggestions
(1) Line 79: Mention Nox in the text. Key references on Nox include Jones (2013), Iatsenko (2018), and Patel (2016).
Done.
(2) Line 92: The long list of publications is unnecessary and can be shortened.
We are not sure that many investigators are aware of the scope of our studies on host-pathogen relationships and this is the adequate place for a reminder.
(3) Line 196: Cite Choi et al. (Aging Cell, 2008; 7:318-334. doi: 10.1111/j.1474- 9726.2008.00380.x) for the initial work on gut dysplasia during aging. However, note that dysbiosis in aging is demonstrated in Buchon et al. (2009, Genes and Development) and other studies.
Done.
(4) Line 265: It would be interesting to clarify whether the shortened lifespan of Norainfected flies after a clean injury is dependent on the microbiota.
The shortened life span of Nora-infected flies is not due to the injury as demonstrated in Fig. S4F. Hence, the shortened lifespan is differentially affected by the microbiota according to nutrition conditions as documented in Fig. 3D-E.
(5) Line 285: Clarify what is meant by "polyubiquitin promoter"-do the authors mean a ubiquitous Gal4 driver? Specify the Gal4 lines used in the result section.
Done. The construct is a direct fusion of the ubiquitin p63E promoter to the Dicer-fluorescent protein sequences as described in Girardi et al., Sci Rep, 2015.
(6) Line 347: Indicate the references aligning with the most recent studies on this topic.
Done.
(7) Line 373 and elsewhere: Mention studies that have shown the microbiota influence on lifespan, in relation to dietary richness.
Done.
(8) Line 588: Provide details on the method used for hemolymph collection.
Done.
(9) Line 964: Clarify the phrase "as previously shown"-where in this paper was it demonstrated?
The legends have been rewritten and the phrase has been deleted.
(10) Line 987: In "survival of non-infested with PA14," explicitly mention Nora to distinguish between different infections.
Done.
Figures & Experimental Details
(11) Figures: Improve figure legends or add information at the top of figures, specifying:
Number of flies used to monitor Nora virus titer.
Temperature conditions. o Age of flies used in experiments.
Done.
(12) Figure 2E: The lifespan of Nora-negative flies appears very short. Was this lifespan assay conducted at 29{degree sign}C? What was the fly stock flipping rate?
Correct, it was 29°C. As described in the Material and Methods section, the flies were flipped every two (29°C) to four days (25°C).
(13) Figure 4C: Improve labeling on the plate for better clarity.
Done.
(14) Figure 6C: The figure legend on the right is difficult to interpret. Clarify what "+" indicates and explicitly write out the genotype. Is NP identical to NPG4G80?
Done. NP is the NP1 driver. We usually use it in a version that also includes a Gal80ts transgene to express the gene of interest only at the adult stage.
(15) Dissection Details: Clearly state which part of the gut was dissected-midgut, entire gut, {plus minus} Malpighian tubules. This should be specified in the results section.
Done (no Malpighian tubules nor crop) for RTqPCR analyses.
(16) Clean Injury: Provide more details in the results section regarding the injury site and needle size.
Done.
(17) Use "Abx" instead of "AntiB," as the former is more commonly recognized.
Done.
Reviewer #2 (Public review):
The title does not seem to be fully supported by the data. While the authors convincingly show the increased sensitivity to Pseudomonas infection, effects on another tested bacterium, Serratia marcescens, were not significantly different between Nora-virus-infected and noninfected flies. Thus, effects of 'intestinal infection' seem to be too broad a claim.
We agree with the reviewer and have accordingly modified the title, which now explicitly refers to P. aeruginosa.
Also, whether the Nora virus increases sensitivity to oxidative stress is not so clear to me: the figure that supports this claim is the survival assay of Figure 5F. However, the difference in survival between control and paraquat-treated Nora (-) flies seems to be in the same order as between control and paraquat-treated Nora (+) flies. Rather, cause and effect seem to be the reverse: paraquat increases ISC proliferation, higher viral loads, and consequently shorter survival. I suggest rephrasing the title and conclusions accordingly.
While we usually just directly compare Nora (+) vs. Nora (-) flies with the same conditions, we note that the difference of survival between control and paraquat-treated Nora (-) flies is of about 9 days, based on LT50 values whereas it is of 8 days for Nora(+) flies. This difference is of about two days when comparing Nora (+) to Nora (-) flies exposed to paraquat. Thus, Nora does contribute to an increased sensitivity to oxidative stress likely by the process highlighted by the reviewer and also by its own detrimental action on the homeostasis of the intestinal epithelium and associated disruption of its barrier function.
Quantification of immunofluorescence microscopy is missing, rendering the images somewhat anecdotal. Quantification should be provided. It will then also be of interest to quantify the number of Nora (+) cells, and the Nora virus levels per infected cell (e.g. Figure 5H). Also, the claim that the Nora virus initially infects ISC and later (upon stress) infects enterocytes requires quantification.
Missing quantifications of pictures have been added: Figs. S5E and 7H. We are not sure we understand the reviewer comment on “Nora virus levels per infected cell”: the signal we are seeing may correspond to aggregates of the virus and would be impossible to quantify reliably, e.g., in the right-most panel of Fig. 5H. Fig. 5I clearly shows that no Nora is detected in enterocytes of young 5-day-old flies in the absence of infectious or xenobiotic challenge.
Genetic support for the role of the JAK-STAT pathway in driving ISC proliferation and supporting Nora virus replication is convincing. It would also be of interest to analyze other pathways implicated in ISC proliferation (e.g. JNK, EGFR), especially given the observations of Nigg et al, showing an involvement of STING/NF-kB and EGFR pathway in driving intestinal phenotypes of Drosophila A virus-infected flies (doi: 10.1016/j.cub.2024.05.009).
We agree with the reviewer that these would be interesting experiments to perform, especially in the light of one hypothesis that antiviral defenses may prevent the initial infection of enterocytes as discussed at length in our updated discussion on host antiviral defenses. However, we are currently unable to perform additional experiments and leave it to other interested investigators studying antiviral innate immunity to address these questions. In this work, we used the interference with the JAK-STAT pathway as a second tool to block the division of ISCs.
Figure 5E: An intriguing observation is that GFP:Dicer2 seems to be unstable in Nora virusinfected cells. Here, GFP control driven by the same driver line would be required to confidently conclude that this is due to an effect on Dicer-2 specifically.
Actually, this experiment was not performed using the Gal4-UAS system but a direct fusion. We do know that GFP is stable when expressed in enterocytes, e.g., Lee et al., Cell Host&Microbe (2016) DOI: 10.1016/j.chom.2016.10.010.
Legends are mostly conclusive, and essential information about the experimental setup is missing in the captions of multiple figures, making the interpretation of the data difficult. See my private recommendations for suggestions to improve the data presentation.
Done.
Recommendations for the authors:
Suggestions for the presentation of the data:
(1) I found the names Ore-R(SC) and Ore-R(SM) for noninfected vs infected Ore-R flies not very intuitive. I suggest renaming them into something that makes the infection status clear.
These notations refer to two distinct sub-strains that may reflect different origins with some likely genetic drift accounting for the distinct properties of the two sub-strains. As the ORE-R (SM) have different infection status: infested, cleaned, re-infected, we fear that this would not clarify the matter. Of note, ORE-R(SC) are refractory to Nora virus infection (Fig. S1I).
(2) Please define the number of flies analyzed for survival assays in the legends.
Done.
(3) The authors provide conclusions in most of the figure legends, without providing an explanation of the experiment that was done. Conclusions should be used sparingly, if at all, in legends. Also, relevant information is often missing in the legends (time points after infection, Figure 2E food source, etc.). I suggest the authors carefully double-check their legends and rephrase the conclusive legends with descriptive ones.
Done. The figure legends have been rewritten.
(4) Several of the legends indicate that 'data represent the mean of biological triplicates' however some panels do not represent triplicates (e.g. Figure 1C-E). Please correct.
Done.
(5) Legends: which multiple comparison test was used for ANOVA?
Done. Tukey’s post-hoc test was used for direct comparisons.
(6) Line 888: black arrows are not shown in the figure.
Corrected.
(7) Figure 1F: legend on the figure seems incorrect (all are labeled Nora (+)); likewise for Figure 2C (all labeled Nora (-)).
Corrected.
(8) Materials and methods: please describe how the Nora virus antibody was raised (and specify on line 271 what viral protein is recognized).
Done. As the whole virus was used for immunization, we cannot state which specific viral proteins are detected by the antibody.
(9) Please define what is presented in the box plots (mean, range, whiskers, individual data points).
Done.
(10) Figure 4 and associated text (line 221): a brief explanation of the Smurf assay would be useful.
Done.
(11) Figure 4C: I did not find the picture of the agar plate informative, as similar information is conveyed in Figure 4D. Also, the labelling cannot be clearly read.
Figure 4D provides a quantification of panel C. The readability has been improved.
(12) Figure 4C: It is suggested that Nora-positive, smurf-negative flies were analyzed, but from Figure 4B it seems that these do not exist. Please explain.
The data in Fig. 4B do not represent absolute numbers but percentages. Thus, there were at most 50% of SMURF-positive flies at the time of the assay, the rest being Smurf-negative yet Nora-positive.
(13) The abbreviations PA14 and Db11 are used in several figures. I would suggest defining the abbreviation in the legend to facilitate interpretation.
Done.
(14) Figure 5A/5G: the Nora virus RNA levels in this figure are dramatically lower than the levels in other figure panels. Please check/correct.
Done. The reviewer is indeed correct: we have forgotten to write that for these two panels, the loads are relative and not absolute as is the case in other panels. 5A: the load in whole flies was taken to be 1; 5G: untreated Nora-positive flies were taken to be 1.
(15) Figure 6A: total number of AporTag positive cells are reported. Were the same number of total cells analyzed? Please define.
We have not counted all of the cells in each midgut but provide the number of ApopTag positive cells per midgut. We thus make the assumption that the overall number of midgut cells is not varying much from one midgut to the other. Visual inspection of DAPI-stained nuclei did not reveal any obvious change in the density of enterocyte nuclei as illustrated in Fig. S6 (we guess that everyone in the field is making the same assumption when counting mitotic ISCs with PHH3 staining).
(16) Figure 6C: I find the shades of blue difficult to distinguish and suggest to us other colors.
Done.
(17) There seems to be a large mismatch between the percentage of Nora virus-positive cells in Figures 5C, 6H and the images of Figures 5G and 5H. Why?
We think there might be a mistake with the Figure numbers cited by the referee. We guess the point the referee was trying to raise is the difference of perceived Nora virus burden between Fig. 5H and Fig. 6G, a quite valid point. For Fig. 5H, we had measured the Nora-virus load by RTqPCR (Fig. 5G, relative burden) but had not quantified the images. This is now done and shown in Fig. 5I. In Fig. 5H, young flies were used and hence there was no Nora virus detected in ECs, as now quantified in Fig. 5I. For Fig. 6G, we had to use 30-day old intestines to be able to observe Nora virus in the enterocytes of the controls. We have now included this important point in the main text and in the Figure legends.
(18) The Title of the legend in Figure 7 is not supported by the data as 'spread through the intestine' has not been analyzed. Please adjust.
Done.
(19) All figures in which ANOVA is used: I assume that anything not labeled with an asterisk was found to be non-significant? If so, this should be indicated in the manuscript.
Actually, we have not highlighted obvious differences to maintain clarity (e.g., Fig. 1E between uncured Ore-R(SM) and cured Ore-R(SC). We thus have underlined the biologically relevant differences in the panels. The interested readr can refer to the primary data that are accessible on a data repository.
(20) Figure 7C: the authors may want to contrast their finding that Upd3 was not upregulated in Nora virus-infected flies (in the absence of PA14) with the findings of Kuyateh et al, who did report upregulation of Upd3 (https://doi.org/10.3390/v15091849).
We thank the reviewer for pointing out this study we were unaware of. We would like to point out that this article is difficult to follow as it is not 100% clear in which of the analyzed studies the induction of upd3 was observed and which exact experimental conditions were followed, e.g., young or old flies, whole flies or gut… We have looked in more detail at ref. 133 of this article, which refers to an unpublished study from the Hultmark laboratory that is however available online: (https://www.diva-portal.org/smash/record.jsf?aq2=%5B%5B%5D%5D&c=15&af=%5B%5D&searchType=SIMPLE&sortOrder2=title_sort_asc&query=Nora+virus&language=en&pid=diva2%3A1045375&aq=%5B%5B%5D%5D&sf=all&aqe=%5B%5D&sortOrder=author_sort_asc&onlyFullText=false&noOfRows=50&dswid=4587).
In that study, flies were “infected” with Nora virus by expressing a cDNA clone injected into embryos. The problem is that for some unknown reasons the authors used Relish mutant flies. It is thus difficult to conclude as these flies are defective for the IMD and Sting pathways whereas our flies are wild-type. We were also interested to read that genes involved in midgut stem cells differentiation were expressed in flies harboring Nora virus, which is in keeping with the data of the present study. However, it is difficult to discuss this when we know little on the background of the studies analyzed by Kuyateh et al, in as much as our Discussion is already rather long.
(21) Figure 7E: are the differences between control and Dome/Stat knockdown flies significantly different for Nora (+) flies (in the absence of Pseudomonas)? This is not clear from the data presentation.
The answer to the question is positive: the JAK-STAT pathway also contributes to the maintenance of intestinal epithelium homeostasis in the absence of bacterial infection, that is presumably basal conditions. We have modified Fig. 7E to include more comparisons.
Textual suggestions:
(22) Line 25 strives > thrives
Done.
(23) Lines 150- 152, etc are not very informative. Also, some of the viruses analyzed are not "known contaminating viruses", but viruses used experimentally (VSV, IIV6, CrPV). I suggest adjusting the phrasing.
Done.
(24) Line 862: weaker fitness > lower fitness.
Done.
(25) Virology terms:
(a) I suggest not using the term titer for qPCR readouts (which do not involve titration). Viral RNA level or viral RNA load would be more appropriate.
Done.
(b) I would propose rephrasing the Y-axis label of Figure 1C, E to Nora RNA load (same for other figures showing viral RNA).
Done.
(c) Infested: rather use the more accurate term infected.
Done.
(d) Contamination: rather use the term infection.
We have modified some but not all occurrences of this word. We believe that it is important to use the word contamination when referring to enterocytes: the enterocytes are not infected by Nora; rather, differentiated infected ISCs become contaminated enterocytes. Infection refers to an active process whereas contamination refers to a state.
(e) Proliferation: rather use the term replication.
According to our US-English dictionary, proliferation refers to the “rapid reproduction of a cell, part, or organism”, which is the meaning we intend. Replication does not have this notion of speed of reproduction.
(f) Drosophila should not be italicized in Drosophila A virus, following the ICTV convention that a "virus name should never be italicized, even when it includes the name of a host species or genus" https://ictv.global/faq/names.
Done.
(26) Line 873-975: please rephrase the legend of Figure 1F as the current one is not informative.
Done.
(27) Line 934: I suggest moving the justification of the time point chosen "= LT50 on the survival test in 935 Fig. 2E" to the main text.
Done.
(28) Line 936: with drop > with a drop.
No longer relevant.
(29) Line 940-941: the grammar of the sentence does not seem to be correct as it suggests that SDS induces Diptericin expression.
No longer relevant.
(30) Line 952-953; line 980: please correct mismatch singular/plural (antibody have, inhibition do).
Done.
(31) Line 422: "It will be interesting to determine whether the absence of a Dcr2 fluorescent proteins fusions in progenitor cells that we report in this study rules out a role for the RNAi pathway in intestinal host defense against the Nora virus". It would be of interest to discuss this finding in the context that virus-derived Nora virus siRNAs can be easily detected and that the viruses encode an RNAi antagonist (doi: 10.1371/journal.ppat.1002872).
Done. We have updated the Discussion and propose a model whereby RNAi would prevent primary infection of enterocytes and then virus replication in proliferating progenitor cells would allow the virus to effectively inhibit the RNAi machinery when the infected progenitor cells become enterocytes.
(32) Line 159: Nora virus phenotypes differ between laboratories. I would be interested to read the authors' speculations on why this would be the case.
Our work shows that the effects of Nora virus depend significantly on several parameters we have identified: nutrition quality, age, exposure to abiotic or biotic stresses, and fly genotypes with the existence of Nora-refractory strains. These parameters as well as potential differences between laboratories are actually discussed in the second paragraph of the Discussion.
(32) Line 175: capitalization of ORE-R vs Ore-R at other places in the manuscript.
Done.
(33) Line 185-194: PA14 and Pseudomonas are used interchangeably. Perhaps it is clearer to stick to a single term for consistency.
PA14 is one clinical strain used to study P. aeruginosa. There are many others such as PAO1, which is also widely used. We have decided to write P. aeruginosa PA14 the first time we are using it in each figure legend, and use only PA14 afterwards.
Reviewer #3 (Public review):
The claim that Dcr2 is not abundant in ISCs because the protein is not stable is logically consistent and reasonable. Perhaps I missed this, but the authors could additionally knock down or use somatic CRISPR to delete Dcr2 in ISCs to test whether a lack of Dcr2 underlies sensitivity. In this experiment, the expectation would be that depleting Dcr2 in ISCs genetically would make little difference to susceptibility overall compared to controls. This is not an essential experiment request.
We agree with the reviewer that these would be interesting experiments to perform. However, we are currently unable to perform additional experiments and leave it to other interested investigators studying antiviral innate immunity to address these questions dealing with the specific steps of RNA interference that may be missing in progenitor cells.
Recommendations for the authors:
(1) Line 206-207 and 214-216: the order of ideas presented here is unintuitive. In Lines 206207, it is said that ABX treatment had no effect, which is counterintuitive to the nature of infection susceptibility. But this is resolved in Lines 214-216 when the reader realizes that S3G is fed on a sucrose solution, and so likely microbiota-depleted. Perhaps more could be said to clarify this in the main text, and/or swap the order of these observations so a casual reader is not confused about the nature and extent of the microbiota contributing to the sensitivity of Nora-infected flies.
As suggested by the reviewer, we have clarified the text with respect to the food source and microbiota load; we emphasize that the microbiota plays a protective role in Nora-negative flies fed on sucrose solution even though the microbiota load is very low under these conditions. Of note, the microbiota is not depleted in sucrose-fed Nora-positive flies: we suspect that delaminating enterocytes may actually provide directly or more likely indirectly (peritrophic matrix) nutrients for the microbiota.
(2) Line 262-265: the text may be a bit exaggerated given only 3 pathogens tested, one of which was a fungal natural infection breaching the cuticle and largely bypassing the gut. This could be re-phrased.
The important point is that uninfected Nora-positive flies die with a LT50 of about 10 days even when noninfected; it has nothing to do with the number of pathogens tested. Thus, any infection that causes death with kinetics in this range may be misinterpreted in the absence of a relevant uninjured or clean injury control.
(3) Line 379-382: I don't know if citing Schissel et al. is needed here. This paper's methods and data are highly problematic, as mentioned by the authors. This is not a highly cited paper, nor does it add value to the present discussion to cite it only to discredit it. Perhaps this can be left out and the field can move on quietly - naturally, this choice is the present authors', and this is just my view.
We have actually cited this article at two other places and thus had not cited it “only to discredit it”. We have nevertheless removed the lines as suggested by the reviewer.
(4) Line 404: perhaps clarify "Interestingly, mammalian stem cells..."
Done.
(5) Line 455: my understanding of digital PCR is that it is highly useful for detecting rare variants but not particularly better than qPCR for estimating loads/titres? This is not to say dPCR is worse, just that dPCR and primer-specific RT + qPCR are comparable if load/titre is desired. For instance, Qiagen actually recommends qPCR over dPCR specifically (and pretty much exclusively) for gene expression: https://www.qiagen.com/us/applications/digitalpcr/beginners/dpcr-vs-qpcr.
(6) Perhaps Line 455 could drop the advocacy for digital PCR? I agree using dissected guts, or seemingly aged individuals per Figure 3B(?), is a valuable thing to point out. Maybe the aged individuals point could be added here? I guess the idea behind dissected guts is to have samples enriched in Nora virus.
Cleaning Nora-positive strains is really difficult and we suspect that as long as there is one viral particle left, it may be sufficient to re-ignite the contamination of the strain. Our own experience with digital PCR on the expression of AMP-like molecules in the head of flies is that we found the approach to be more sensitive than classical RTqPCR (Xu et al., EMBO Rep, 2023).
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eLife Assessment
This important study shows that the Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the Pseudomonas bacterium. The authors provide convincing data to support their conclusions. The paper provides new insights into virus-host interactions in the Drosophila gut and serves as a warning for scientists who use the fruit fly as a model to study gut physiology.
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Reviewer #1 (Public review):
Summary:
This important article reveals that the Nora virus can colonize the intestinal cells of Drosophila melanogaster, where it persists with minimal immediate impact on its host. However, upon aging, infection, or exposure to toxicants, stem cell activation induces Nora virus proliferation, enabling it to colonize enterocytes. This colonization disrupts enterocyte function, leading to increased gut permeability and a significant reduction in lifespan. Results are convincing with an important impact on the Drosophila community.
Strengths:
(1) Building on previous studies by Habayeb et al. (2009) and Hanson et al. (2023), this study highlights cryptic Nora virus infection as a crucial factor in aging and gut homeostasis in Drosophila melanogaster.
(2) Consistent with the oral route of Nora virus …
Reviewer #1 (Public review):
Summary:
This important article reveals that the Nora virus can colonize the intestinal cells of Drosophila melanogaster, where it persists with minimal immediate impact on its host. However, upon aging, infection, or exposure to toxicants, stem cell activation induces Nora virus proliferation, enabling it to colonize enterocytes. This colonization disrupts enterocyte function, leading to increased gut permeability and a significant reduction in lifespan. Results are convincing with an important impact on the Drosophila community.
Strengths:
(1) Building on previous studies by Habayeb et al. (2009) and Hanson et al. (2023), this study highlights cryptic Nora virus infection as a crucial factor in aging and gut homeostasis in Drosophila melanogaster.
(2) Consistent with the oral route of Nora virus transmission, the study demonstrates that the virus resides in intestinal stem cells, with its replication directly linked to stem cell proliferation. This process facilitates the colonization of enterocytes, ultimately disrupting intestinal function.
(3) The study establishes a clear connection between stem cell proliferation and virus replication, suggesting that various factors - such as microbiota, aging, diet, and injury - can influence Nora virus dynamics and associated pathology.
(4) The experimental design is robust, comparing infected flies with virus-cured controls to validate findings.
Weaknesses:
(1) The study does not explore or discuss how oral ingestion of Nora virus leads to the colonization of stem cells, which are located basally in the gut. This mechanism should be discussed.
(2) The authors fail to detect Dicer-GFP fusion protein expression in stem cells, a finding that could explain why the virus persists in these cells. Further investigation is needed to determine whether RNAi functions are effective in stem cells compared to enterocytes. For clarification, the authors could cross esg-Gal4 UAS-GFP and Myo-Gal4 UAS-GFP with UAS GFP-RNAi and/or express a Dicer-GFP construct under a stem cell-specific driver.
(3) The presentation of experimental parameters (e.g., pathogen type, temperature, time points) should be improved in the results section and at the top of the figures to enhance clarity. Additionally, details regarding the mode of oral infection (continuous exposure vs. single feeding on a filter) should be specified. Given that fly stock flipping frequency influences microbiota load (as noted in Broderick et al.), this should be reported, especially for lifespan studies.
(4) To confirm that enterocyte colonization requires stem cell proliferation and differentiation, the authors should analyze Nora virus localization in JAK-STAT-deficient flies infected with bacteria or toxicants. This would help determine whether the virus can infect enterocytes in the absence of enterocyte differentiation, but stimulation of stem cells.
(5) The study does not discuss the spatial distribution of Nora virus infection along the gut. Specifically, it remains unclear whether viral colonization is higher in gut regions R2 and R3, which contain proliferative stem cells. Addressing this could provide valuable insights into the virus's infection dynamics.
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Reviewer #2 (Public review):
Summary:
In this manuscript, the authors report that Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the Pseudomonas bacterium. Nora virus infection was associated with an increased proliferation of ISC and disrupted gut barrier function. Genetically, the authors show that increased ISC division in Nora virus and Pseudomonas coinfected flies is driven by signaling through the JAK-STAT pathway and apoptosis.
Accordingly, blocking apoptosis and JAK-STAT signaling reduces viral load, suggesting that in this context the JAK-STAT pathway is proviral in contrast to other previous observations in systemically infected flies. This work adds …
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors report that Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the Pseudomonas bacterium. Nora virus infection was associated with an increased proliferation of ISC and disrupted gut barrier function. Genetically, the authors show that increased ISC division in Nora virus and Pseudomonas coinfected flies is driven by signaling through the JAK-STAT pathway and apoptosis.
Accordingly, blocking apoptosis and JAK-STAT signaling reduces viral load, suggesting that in this context the JAK-STAT pathway is proviral in contrast to other previous observations in systemically infected flies. This work adds to the findings of another recent paper showing that another persistent fruit fly virus, Drosophila A virus, also increases ISC proliferation and decreases gut barrier function. Intestinal viruses should therefore be considered confounders in studies of fly intestinal physiology.
Strengths:
Overall, the data are convincing and robust, starting with two wildtype fly stocks (Ore-R strain) that differ in their Nora virus infection status, followed by experiments in which cleared stocks are reinfected with a purified Nora virus stock preparation. The conclusions of the paper will be of interest to scientists working on insect physiology, virology, and immunology, but should also serve as a warning for scientists that use the fly as a model to study gut physiology.
Weaknesses:
The title does not seem to be fully supported by the data. While the authors convincingly show the increased sensitivity to Pseudomonas infection, effects on another tested bacterium, Serratia marcescens, were not significantly different between Nora-virus-infected and non-infected flies. Thus effects of 'intestinal infection' seem to be too broad a claim. Also, whether the Nora virus increases sensitivity to oxidative stress is not so clear to me: the figure that supports this claim is the survival assay of Figure 5F. However, the difference in survival between control and paraquat-treated Nora (-) flies seems to be in the same order as between control and paraquat-treated Nora (+) flies. Rather, cause and effect seem to be the reverse: paraquat increases ISC proliferation, higher viral loads, and consequently shorter survival. I suggest rephrasing the title and conclusions accordingly.
Quantification of immunofluorescence microscopy is missing, rendering the images somewhat anecdotal. Quantification should be provided. It will then also be of interest to quantify the number of Nora(+) cells and the Nora virus levels per infected cell (e.g. Figure 5H). Also, the claim that the Nora virus initially infects ISC and later (upon stress) infects enterocytes requires quantification.
Genetic support for the role of the JAK-STAT pathway in driving ISC proliferation and supporting Nora virus replication is convincing. It would also be of interest to analyze other pathways implicated in ISC proliferation (e.g. JNK, EGFR), especially given the observations of Nigg et al, showing an involvement of STING/NF-kB and EGFR pathway in driving intestinal phenotypes of Drosophila A virus-infected flies (doi: 10.1016/j.cub.2024.05.009).
Figure 5E: An intriguing observation is that GFP:Dicer2 seems to be unstable in Nora virus-infected cells. Here, GFP control driven by the same driver line would be required to confidently conclude that this is due to an effect on Dicer-2 specifically.
Legends are mostly conclusive, and essential information about the experimental setup is missing in the captions of multiple figures, making the interpretation of the data difficult. See my private recommendations for suggestions to improve the data presentation.
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Reviewer #3 (Public review):
Summary:
Franchet et al. sought to characterize the impact of Nora virus on host lifespan and sensitivity to a variety of infectious or stressful treatments. Through careful and rigorous analyses, they provide evidence that the Nora virus greatly impacts fly survival to infection, overall lifespan, and intestinal integrity. The authors have been thorough and rigorous, and the experimental evidence including proper isolation of the virus and Koch's Postulate reinoculation of the organism is excellent. The additional work is valuable and to the gold standard of the field, characterizing the pathology of the gut, including data showing gut leakage, the presence of the virus in the intestinal stem cells, and the importance of stem cell proliferation for virus replication and spread using elegant genetic tools to …
Reviewer #3 (Public review):
Summary:
Franchet et al. sought to characterize the impact of Nora virus on host lifespan and sensitivity to a variety of infectious or stressful treatments. Through careful and rigorous analyses, they provide evidence that the Nora virus greatly impacts fly survival to infection, overall lifespan, and intestinal integrity. The authors have been thorough and rigorous, and the experimental evidence including proper isolation of the virus and Koch's Postulate reinoculation of the organism is excellent. The additional work is valuable and to the gold standard of the field, characterizing the pathology of the gut, including data showing gut leakage, the presence of the virus in the intestinal stem cells, and the importance of stem cell proliferation for virus replication and spread using elegant genetic tools to block stem cell proliferation or enterocyte death.
Strengths:
The authors have been rigorous and careful. The initial finding is presented through the lens of two related strains differing in virus infection. From there, the authors characterized the virus and isolated a purified culture, which they used to reinoculate a cleared strain to demonstrate proper Koch's Postulate satisfaction. The authors have also probed various parameters in terms of dietary importance in relevant conditions for many experiments. The additional work to characterize the pathology of the gut is compelling, using genetic tools to block or allow intestinal stem cell proliferation and enterocyte death through JAK-STAT and JNK signalling alongside the tracing of virus presence using a Nora virus antibody. JAK-STAT and JNK are previously described as regulators of these processes, making these tools appropriate and convincing. It is also interesting to see good evidence that the virus itself is damaging, rather than simply permitting coinfection by gut microbes (which does happen).
Weaknesses:
The claim that Dcr2 is not abundant in ISCs because the protein is not stable is logically consistent and reasonable. Perhaps I missed this, but the authors could additionally knock down or use somatic CRISPR to delete Dcr2 in ISCs to test whether a lack of Dcr2 underlies sensitivity. In this experiment, the expectation would be that depleting Dcr2 in ISCs genetically would make little difference to susceptibility overall compared to controls. This is not an essential experiment request.
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