Natural variation in infection specificity of Caenorhabditis briggsae isolates by two RNA viruses

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   May 2, 2024

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   Reply to the reviewers

   We are very grateful to the reviewers for their positive appraisal of the manuscript and for their useful comments and suggestions. Below are our answers and corresponding modifications of the manuscript.

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   Reviewer #1

   1 - Figures 1&4 focus on JU1264 as the primary double-sensitive strain. However, the authors built their RILs with HK104 by crossing with JU1498 in Figures 7&8. In the results section and/or methods, the authors should provide some justification for this strain switch. Alternatively, the equivalent analysis of Figure 1 focusing on JU1498 would be valuable to demonstrate that the effects of both viruses on fitness are similar to JU1264. I …

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   Reply to the reviewers

   We are very grateful to the reviewers for their positive appraisal of the manuscript and for their useful comments and suggestions. Below are our answers and corresponding modifications of the manuscript.

   ---

   Reviewer #1

   1 - Figures 1&4 focus on JU1264 as the primary double-sensitive strain. However, the authors built their RILs with HK104 by crossing with JU1498 in Figures 7&8. In the results section and/or methods, the authors should provide some justification for this strain switch. Alternatively, the equivalent analysis of Figure 1 focusing on JU1498 would be valuable to demonstrate that the effects of both viruses on fitness are similar to JU1264. I am not recommending that the JU1264xHK104 crosses be performed or that Figures 7&8 be repeated with JU1264xHK104 lines, but that more explanation for strain selection for RIL generation should be provided.

   JU1264 and JU1498 are the strains where SANTV and LEBV were found, respectively. The experiments were performed over the years by different authors and were designed to answer different questions. JU1264 was the strain where the first virus was found and was used as a doubly sensitive strain in Figure 1 and the small RNA experiment. The main reason we chose JU1498 for genetic crosses to discover the genetic basis of LEBV sensitivity is that LEBV was detected and isolated from JU1498. Note that the JU1264 and JU1498 strains come from France and are in the same isotype group at CaeNDR (see also Figure 3) so the two strains may be interchangeable (although we cannot be sure).

   We added in the text concerning the RIL construction: "We chose to use JU1498 as the LEBV-sensitive strain as it was the original strain in which LEBV was discovered."

   2-The authors reasonably claim that the resistance of tropical strains like AF16 could be due to blocking viral entry or early inhibition of replication before the small RNA response is activated. Could the authors test this by directly microinjecting virus (in combination with a dye as a control for successful injection) into the intestine? I understand this could not be done on a scale that would allow for small RNA sequencing, but one could perform small-scale FISH to determine if LEBV or SANTV are replication-competent if the entry barrier is artificially overcome. Such an experiment may require considerable technical development. It may be beyond the scope/timing of this specific study, but it is worth considering to gain some insight into the possible resistance mechanisms observed.

   Although the suggested experiment is in principle a great approach, it is difficult to perform without losing animals during the FISH staining. In addition, in this manuscript we are not particularly searching for the resistance mechanisms of AF16 but trying to present a wider perspective concerning viral infections of C. briggsae and their specificity. We performed small RNA analysis for AF16 together with the sensitive strains and therefore we commented on the lack of small RNA response in AF16 comparing to the sensitive strains. We thus consider that setting up intestinal injections at this point is arduous and beyond the scope of this manuscript.

   Minor Comments: Line 78 - provide the full genus name for Caenorhabditis elegans at first appearance, as done for Caenorhabditis briggsae

   This was modified. Line 117 - The description of cul-6 could also reference Bakowski et al. 2014. This study is referenced more generally as a player in proteostasis a few lines below but could be more explicitly tied to cul-6-mediated resistance to ORV (Bakowski et al. 2014 - see Fig. 7A) This section focus on the use of natural polymorphisms but we added this reference, which is indeed key for the effect of *cul-6 *knockdown on viral infection in C. elegans. Line 197-198 - The authors could consider adding sequences for FISH probes as part of Table S2. This information could add value to the present study even if previously listed in Frézal et al. We actually removed them from an earlier version since these sequences are already published: here and in further work, it seems preferable to refer to the primary study where these probes were designed, Line 263 - Were embryos obtained by bleaching of gravid adults, or was an egg lay performed, and the embryos were collected from plates? This is potentially an important distinction and should be clarified briefly in the methods. In the section “Preparation of small RNA libraries”, we obtained embryos by bleaching gravid adults.

   We changed the first sentence to “Gravid hermaphrodites from uninfected cultures (AF16, HK104 and JU1264) were harvested using M9 solution, then bleached and washed twice using nuclease-free water. Embryo concentrations were estimated by counting embryos under the dissecting microscope and diluted to 2 embryos per mL of nuclease-free water. 200 embryos of each strain (AF16, HK104 and JU1264) were then plated onto 55 mm NGM plates seeded with *E. coli *OP50.” We also added “The embryos were obtained by bleaching gravid hermaphrodites.” to the Figure S5 legend. Line 330 - Provide justification for using JU1498 to make these RILs (see comment above). We added this sentence in the Results section. "We chose to use JU1498 as the LEBV-sensitive strain as it was the original strain in which LEBV was discovered." Line 446-Refer to the methods section for full clarity on the role of FISH in this set of experiments or reword for improved clarity. At first read-through, this phrasing made me expect some FISH experiments associated with Fig. 1, which does not appear to be the case.

   We did perform FISH experiments as control that the cultures were infected, as explained in the Methods. We removed this mention from the Results section. Line 478 - The supplementary figure callouts are misaligned with the provided documents. S2A in the text appears to refer to S3A RT-qPCR results. Changed. Line 483 - Similar to above, the text suggests serial dilutions should refer to S4, not S3. Changed. Line 498 - Modify the text to 'Figure 2C and Figure 3' for clarity. Changed. Line 531,535 - viRNAs are defined in line 535 but this should be moved to 531 above at first appearance in the text. Changed. Line 593 - Typo in 'Logarithm of Odds?' Corrected. Line 621-624 - I recommend the authors include the data for the LEBV control experiments with NIL strains, either as a supplementary table, an additional panel for Fig. 6, or represented as done in Figure 8. We removed this sentence. Line 625-632 - How many total genes are represented in the QTL on IV? The reasoning behind testing rde-11 and rsd-2 is sound, but readers might want to know other potential candidates within this region (perhaps something the authors could also speculate on in the discussion). A similar comment applies for # genes in the QTLs on II and III.

   We added in Table S7 the list of detected SNPs and short indels in the chromosome IV region and now indicate in the text "among them over 2700 SNPs and short indels (Table S7)." We added Table S11 with the polymorphisms in the chromosome II QTL region. We note that these tables do not include possible structural variants. The chromosome III QTL being weak, we abstained for this one but the data can now be found using CaeNDR.

   Line 991-992 - Figure 1B - LEBV, SANTV, and co-infection effects on body size are mentioned but not quantified. Has this phenotype been quantified elsewhere? If so, the authors should reference it in the results section or Fig. 1 legend. Alternatively, body size could be quantified as part of this study and added to Fig. 1.

   Because we do not have a large amount of data on body size, we removed "Body size quantification” from Figure 1B legend. Line 1001 - There is a typo in the first sentence; the period after LEBV should be removed. Small suggestion: Figure 2A - While described in the methods, I recommend that the authors briefly reiterate in the figure legend that the white/yellow boxes are intended to indicate serial chunking for clarity.

   We removed the typo and explained the agar chunk representation in the figure legend: "The transfer by chunking a piece of agar is indicated by beige rectangles cut out from one plate and transferred to the next plate." Line 1034 - Small formatting note for Figure 4B - percentages of reads mapping to RNA1 and RNA2 appear underneath gridlines for the graph which obscures visibility and is inconsistent with the other graphs presented.

   This was modified and is indeed clearer. Line 1094 - Figure S1 - this analysis could be strengthened by RT-qPCR represented as fold change in viral load instead of, or in addition to, the agarose gel image (like Fig. S3). Doing so would also allow for the normalization of eft-2 control across individual samples (e.g.: particularly low eft-2 amplification in ED3073). However, these results are sufficiently convincing that LEBV does not replicate in C. elegans, but a more quantitative approach is recommended if feasible for the authors. Alternatively, an additional figure panel and/or repeat of this analysis with C. elegans infected with ORV would also be beneficial as an additional control.

   We do not understand how we can estimate a viral load by a ratio when we do not seem to see any significant amplification. Of course, a RT-qPCR would provide a finite Ct value and a ratio but they are likely to be meaningless. The ED3073 sample did not amplify for *eft-2 *either and calculating a ratio of high Ct values in a RT-qPCR would be misleading. We could remove the two ED3073 lanes but prefer to leave them.

   Line 1112 - "Experiments using RNA2 primers gave similar results" - if this data isn't included in the study, this text should be removed.

   Removed. Line 1141 - Figure S6 - For full transparency, the authors could consider including HK104 infected with LEBV to show minimal (zero) reads align to the RNA1/RNA2 segments using scales consistent with JU1264 infected with LEBV (S6C) The proportion of reads mapping (0%) are provided in Figure 4A and supplementary tables. We do not show the distribution of antisense 22G and sense 23nt along the LEBV genome for the HK104 (co)infections for the following reasons. 0% of these reads map to LEBV in HK104 monoinfection, and only 0.02% antisense 22G in coinfection. Moreover, the 23nt reads mapping to LEBV-RNA2 in the HK104 coinfection (16.54%;1931 reads) correspond to a 41 bp region with 85% nucleotide similarity between SANTV-RNA2 and LEBV-RNA2. Overall, the few 23nt (+) reads mapping to LEBV in HK104 coinfection are most likely a spillover of the HK104 antiviral response to JUv1264 entry into the intestinal cells.

   Reviewer #2

   Main points:

   1. In figure 1C and D, is more than 1 biological replicate performed? Ideally multiple independent infections would be performed which would increase confidence in these experiments, but minimally the authors should make clear that this data was from an experiment only performed once. The conclusion from the life span assays is unlikely to change, but given the variance of the brood size assays within replicates, the conclusions that LEBV infection reduces the brood size is weakly supported.

   We added “Panels C-D correspond to a single experiment (see Methods).” to the legend of Figure 1. We changed the wording to "LEBV and especially the co-infection appeared to lower brood size." We do not have data for independent experiments.

   If the authors want to claim that there is a defect in viral entry in the resistant strains, they should perform infections experiments at an earlier time point that could capture viral invasion. In C. elegans with Orsay virus these experiments have been done as early as 18 hours by FISH. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011120 The way the assays are currently set up, if the infection was cleared it wouldn't be observed.

   The strongest point that indicates that the virus does not replicate is the small RNA experiment, in which the animals were collected on the initial plate inoculated with the virus. We think that our wording was careful:

   We further amended it:

   • in Results " The animals were collected for sRNA sequencing on the plates onto which the viral inoculate was added and where they were constantly exposed to the virus".

   • in Discussion " Indeed, as we did not assay viral entry by sensitive FISH or RT-PCR at early timepoints, it is possible that the viruses are cleared without production of small RNAs."

   The evidence that the region on chromosome III contributes to susceptibility is weak. The analysis in figure 5B does not identify this region and it is not clear to me how to read the scale in figure 5C to determine that a region on chromosome III is significant.

   We added in the Figure legend: "with a LOD score of 10.5, above the threshold calculated by simulations (see Methods)." and detailed the method in the Methods section (see reply to Reviewer 3 below).

   In figure 6 using a more appropriate statistical test such as one way ANOVA with multiple hypothesis testing is necessary to determine if there is a difference between JU2832 and JU2916. It would be helpful if the authors could add more discussion of the evidence that they feel that supports this region being involved in susceptibility.

   We do not think that an ANOVA is appropriate to analyze these proportions which cannot have normal distributions of residuals, therefore we used a generalized linear model, taking genotype and block (day of experiment) into account. This was only explained in the legend and is now explained in the Methods section as well. Maybe the reviewer suggests us to us a global analysis with strain as a factor. We could do this but we do not think that it applies well to this situation: here we test for a specific hypothesis for each one-QTL strain. We have corrected for multiple testing as explained next. The legend now reads: " The significance p values were obtained in a generalized linear model (glm) taking independent experimental blocks and infection replicates into account, testing NILs against their relevant background parent. The p values using the two strains testing for the QTL on chromosome IV and those using the two-QTL strain JU2832 are corrected for multiple testing." In addition, we now provide p values rather than three stars, which reinforce the point (they are very low).

   Minor points

   1. In figure 1B it would be helpful to provide more information on the animals chosen to display. Are these representative examples or extreme examples?

   These are representative examples. This detail was added in the legend.

   In figure 2B, adding a legend for the colored dots would be helpful.

   We had indicated: "Dots are replicates within a block, with 100 animals scored per replicate (see Table S4 for the detailed results and Figure S2 and Methods for the experimental design). Experimental blocks are represented by colors and the bar indicates the grand mean of the blocks."

   1. In figure 2C, the definitions for a strain to be labeled as belonging to each category should be provided.

   The categorization method is now explained in the Methods section. In addition, Figure 2C legend now refers to Table S4 for the category of each strain.

   1. Could the data in figure 2 be used for genome-wide association mapping and compared to the RIL QTL experiments? Adding comment on this would be helpful to understanding the usefulness of this data.

   There are too few strains here to test genome-wide for association. If we had the causative SNP, it would be interesting to assess its frequency but this is beyond the focus and scope of this work, which focused on the outlier phenotype of the HK104 strain.

   1. In figure 4b, in HK104 LRBV the numbers in top right corner are not defined.

   We added to the legend of Figure 4B: “For the HK104 infection with LEBV, the number of read counts is provided in the top right corner to signal their rarity compared to ca. 107 in the other conditions. See Table S5 for all read counts. ”

   1. Line 1001 remove period from "LEBV.of" and add period after isolates. Removed.

   Reviewer #3 Major comments • The authors provide most data in both a processed and raw format, which is helpful. In two cases (data from 3 DPI, line 492 and LEBV infections in the AF16xHK104 NILs, line 621), the authors state their results, but the data seems not to be provided in the document (at least no direct reference is provided). These are supporting results and do not affect the main conclusions, nevertheless providing the data in form of a table or supplementary figure would be required. Generally, it may help to include a data availability statement to have a combined overview of where data can be found.

   As noted by the reviewer, we tried to provide the data in raw format, but did not judge it necessary when the experiment had two datapoints that are provided in the text. We added the number of animals in the instance where it was missing.

   Minor comments • Line 97-126: Here the manuscript fully focuses on the work in C. elegans. It would be interesting to make clear links to the work in C. briggsae (e.g. mention if homologs are present). The paragraph in line 127 clarifies advantages of studying viral infection in C. briggsae compared to C. elegans. It may be logical to place this information early in the text.

   We added a sentence to link the *C. elegans *work and C. briggsae. • Line 166 and results from this experiment: Is the LEBV-SANTV mixture consisting of 50uL of both viruses or a total of 50uL (so 25uL of both)? This is also important for the interpretation of results.

   To clarify, we changed to: “50 l ... of an equivolume mix of SANTV and LEBV”. • Line 167: The text says the culture is maintain for 4 days, but then also mentions day 5. Figure 2 clarifies the experimental setup later, but the text could be clearer here.

   Thank you for noticing this. We changed the 4 to 7. • Line 172: What are the nine starter cultures?

   The nine starting cultures were those obtained as described in the paragraph preceding this line in the manuscript. From a plate of infected animals (five L4 larvae), we propagated the infected population by chunking over 3 plates (day 3) and 3*3 plates (day 5). To make this point clear, we have added above: "to generate for the following experiments nine starter cultures for each of the four conditions " • Line 185: 'Infection of the set of C. briggsae natural isolates'. From the text it is not clear what set the authors refer to.

   We changed to "a set" and refer to Figure 2B and Table S4 in the sentence below for the list of natural isolates. • Line 223: 'The proportion of infected animals were overall higher in Batch3 but the qualitative results are similar'. It is unclear why this statement is here instead of in the result section and it is also not clear what the authors mean by the second part of the sentence.

   We moved the sentence to Results and changed it to: " The proportion of infected animals were overall higher in Batch 3 but the relative results of the different strains were similar for the three batches." • Line 326: Is 'the same method as above' using FISH or RT-qPCR?

   Changed to "using FISH as above". • Line 382: What do the authors mean by 'two cross directions'?

   We removed this mention as the method is better explained in the next sentence.

   • Line 454-458: The data presented here does not appear well integrated in the storyline. It does not fit under the subheading. Perhaps it would be a better fit under the subheading of line 462? We moved it below the subheading. • Line 478: Reference to Fig S2 should be reference to Fig S3

   Changed. • Line 483: Reference to Fig S3 should be reference to Fig S4

   Changed. • Line 540-544: The sentence reads as a contradiction (C. elegans defends itself using RNAi, C. briggsae blocks viral infection during entry). As a result, the sentence reads as if RNAi is not of much antiviral importance in C. briggsae, but that cannot be concluded from this data. I am not sure if this is what the authors aim to suggest, but another word choice (e.g. changing 'whereas' and 'this does not seem the case for C. briggsae') may be considered.

   We changed the wording to " whereas the* C. elegans* N2 reference strain allows for viral entry and defends itself against ORV via its small RNA response (Félix et al. 2011; Ashe et al. 2013; Shirayama et al. 2014; Coffman et al. 2017), in the tested resistant *C. briggsae *strains, the viruses appeared to be blocked at entry or at early steps of the viral cycle." • Line 585 and 592: There are two QTL approaches being applied and referred to as 'the one- and two-QTL analyses'. The description in this part is rather technical and the terminology is not clear. As a result, for readers not familiar with QTL mapping, the biological interpretation may become obscured.

   We now explain in Methods: " ...scanning each pair of positions for several models, including single-QTL, full, additive and epistatic. The significance threshold LOD score of each model was estimated via 1,000 permutation tests with a coefficient of risk a=0.05. The threshold was 4.91 for the additive model and 6.09 for the full model. The LOD score of each pair of position is represented by a color scale in Figure 5C). The combination of the chromosomes III and IV QTLs had a LOD score of 10.5 in the full and additive models. No epistatic interaction was detected. The LOD score of the single-QTL model comparison was below the threshold."

   • Line 659: The authors end the section about natural genetic variation in the response to SANTV with candidate genes and a CRISPR experiment. As the authors identify a small genetic region associated with LEBV susceptibility, it would be interesting to hear about any candidate genes in this region. There are still many genes and more importantly, many polymorphisms in this region (ca. 700 single-nucleotide polymorphisms and short indels). Because structural variants are difficult to call (long-read sequencing has not been performed on the parents), we had preferred to abstain to provide a list of polymorphisms that would be incomplete and preferentially point towards SNPs. However, because of the reviewer's query, we now provide it in Table S11.

   • Line 674: The authors make use of HK104 strain in this study as it is the exception in their dataset that provides resistance against LEBV, but not SANTV. Possibly, the genetic variation linked to viral susceptibility uncovered using HK104 may therefore be relatively uncommon in C. briggsae. The implications of this choice and option for other studies using different genotypes could be interesting to discuss in this short paragraph. The aim in here is to discover why HK104 is specifically resistant to one virus and not the other. There is a possibility of uncovering a specific mechanism that is present in only two or three strains of our 40-strain dataset but we find this specificity particularly

   interesting, regardless of its prevalence. We explore in the Discussion which of the two crosses may reveal the specificity.

   • Line 774: The IPR is already described on abbreviated in line 742. As a reader, we prefer having the abbreviation explained twice than not understanding it. • Overall, to reach a broader audience, the manuscript can expand explanations in the discussion. E.g. statements in line 695 and 773, refer to previous observations, but do not explain them in enough detail to understand parallels between this and previous studies without prior knowledge.

   We added some explanations, specifically for lines 695 and 773 (of previous version). • Figure 2: Only HK104 is labelled in the figure, it would be useful to also see HK105 as this strain is also explicitly mentioned in the text.

   We now included HK105 and strains that are used in further experiments.

   • Figure 2: It is not clear from the results or methods how strains as designated into a certain class. The figure legend says variability in the data is taken into account and that is why some strains are close to each other, yet distinct in class, but how this works is not described. We now explain our criteria. See above in the response to Reviewer 2. • Figure S3: The strain JU1264 and JU1498 are mentioned thrice (as '2', 'rep' and 'ref'). These annotations should be clarified.

   These explanations were indeed missing. We now explain them in the figure legend. • Figure S4: The figure would benefit from a division in panels per strain to facilitate comparisons across strains.

   Indeed. We now added a division in panels per strain. • Figure S4: Have the authors correlated viral loads with the number of infected animals? This could result in addition information if not all individuals are infected equally.

   We have not done so in this precise experiment but preferred to use the number of infected animals in most other experiments, in particular because it is less subject to outlier effects. • Figure S4: Could the authors clarify the meaning of JU1264 Rep?

   It is explained in the legend: "The undiluted viral preparations on JU1264 are used to normalize and are indicated as "JU1264 1/1". A separate replicate was performed and indicated as "JU1264 Rep"."

   • Figure 8: The meaning of the stars in this figure is a bit confusing and the description of these stars in the legend is not clear. Indeed. We changed the legend to: " ***: p<0.001 comparing JU4034 with its parent strain HK104 using a generalized linear model."

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   Referee #3

   Evidence, reproducibility and clarity

   Summary

   The manuscript provides new and detailed insight into two viruses infecting Caenorhabditis briggsae, a close relative of the widely studied model organism Caenorhabditis elegans. The authors study infection from a host perspective using two out of three viruses known to infect C. briggsae. The study mostly focuses on unravelling genetic variation within the host that links to viral susceptibility. They identify and confirm three QTL locations. They subsequently create CRISPR mutations to study candidate genes. Moreover, the study provides novel molecular insight into the C. briggsae antiviral RNAi pathway. Overall, the …

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   Referee #3

   Evidence, reproducibility and clarity

   Summary

   The manuscript provides new and detailed insight into two viruses infecting Caenorhabditis briggsae, a close relative of the widely studied model organism Caenorhabditis elegans. The authors study infection from a host perspective using two out of three viruses known to infect C. briggsae. The study mostly focuses on unravelling genetic variation within the host that links to viral susceptibility. They identify and confirm three QTL locations. They subsequently create CRISPR mutations to study candidate genes. Moreover, the study provides novel molecular insight into the C. briggsae antiviral RNAi pathway. Overall, the study provides a good basis to continue using C. briggsae to study viral infection.

   Major comments

   • The authors provide most data in both a processed and raw format, which is helpful. In two cases (data from 3 DPI, line 492 and LEBV infections in the AF16xHK104 NILs, line 621), the authors state their results, but the data seems not to be provided in the document (at least no direct reference is provided). These are supporting results and do not affect the main conclusions, nevertheless providing the data in form of a table or supplementary figure would be required. Generally, it may help to include a data availability statement to have a combined overview of where data can be found.

   Minor comments

   • Line 97-126: Here the manuscript fully focuses on the work in C. elegans. It would be interesting to make clear links to the work in C. briggsae (e.g. mention if homologs are present). The paragraph in line 127 clarifies advantages of studying viral infection in C. briggsae compared to C. elegans. It may be logical to place this information early in the text.
   • Line 166 and results from this experiment: Is the LEBV-SANTV mixture consisting of 50uL of both viruses or a total of 50uL (so 25uL of both)? This is also important for the interpretation of results.
   • Line 167: The text says the culture is maintain for 4 days, but then also mentions day 5. Figure 2 clarifies the experimental setup later, but the text could be clearer here.
   • Line 172: What are the nine starter cultures?
   • Line 185: 'Infection of the set of C. briggsae natural isolates'. From the text it is not clear what set the authors refer to.
   • Line 223: 'The proportion of infected animals were overall higher in Batch 3 but the qualitative results are similar'. It is unclear why this statement is here instead of in the result section and it is also not clear what the authors mean by the second part of the sentence.
   • Line 326: Is 'the same method as above' using FISH or RT-qPCR?
   • Line 382: What do the authors mean by 'two cross directions'?
   • Line 454-458: The data presented here does not appear well integrated in the storyline. It does not fit under the subheading. Perhaps it would be a better fit under the subheading of line 462?
   • Line 478: Reference to Fig S2 should be reference to Fig S3
   • Line 483: Reference to Fig S3 should be reference to Fig S4
   • Line 540-544: The sentence reads as a contradiction (C. elegans defends itself using RNAi, C. briggsae blocks viral infection during entry). As a result, the sentence reads as if RNAi is not of much antiviral importance in C. briggsae, but that cannot be concluded from this data. I am not sure if this is what the authors aim to suggest, but another word choice (e.g. changing 'whereas' and 'this does not seem the case for C. briggsae') may be considered.
   • Line 585 and 592: There are two QTL approaches being applied and referred to as 'the one- and two-QTL analyses'. The description in this part is rather technical and the terminology is not clear. As a result, for readers not familiar with QTL mapping, the biological interpretation may become obscured.
   • Line 659: The authors end the section about natural genetic variation in the response to SANTV with candidate genes and a CRISPR experiment. As the authors identify a small genetic region associated with LEBV susceptibility, it would be interesting to hear about any candidate genes in this region.
   • Line 674: The authors make use of HK104 strain in this study as it is the exception in their dataset that provides resistance against LEBV, but not SANTV. Possibly, the genetic variation linked to viral susceptibility uncovered using HK104 may therefore be relatively uncommon in C. briggsae. The implications of this choice and option for other studies using different genotypes could be interesting to discuss in this short paragraph.
   • Line 774: The IPR is already described on abbreviated in line 742.
   • Overall, to reach a broader audience, the manuscript can expand explanations in the discussion. E.g. statements in line 695 and 773, refer to previous observations, but do not explain them in enough detail to understand parallels between this and previous studies without prior knowledge.
   • Figure 2: Only HK104 is labelled in the figure, it would be useful to also see HK105 as this strain is also explicitly mentioned in the text.
   • Figure 2: It is not clear from the results or methods how strains as designated into a certain class. The figure legend says variability in the data is taken into account and that is why some strains are close to each other, yet distinct in class, but how this works is not described.
   • Figure S3: The strain JU1264 and JU1498 are mentioned thrice (as '2', 'rep' and 'ref'). These annotations should be clarified.
   • Figure S4: The figure would benefit from a division in panels per strain to facilitate comparisons across strains.
   • Figure S4: Have the authors correlated viral loads with the number of infected animals? This could result in addition information if not all individuals are infected equally.
   • Figure S4: Could the authors clarify the meaning of JU1264 Rep?
   • Figure 8: The meaning of the stars in this figure is a bit confusing and the description of these stars in the legend is not clear.

   Significance

   The study contains a large amount of experimental data that provides a solid basis for using C. briggsae as a model to study viral (co-)infections. Interesting comparisons to C. elegans that is more thoroughly studied are drawn and used to advance understanding of viral infection for both organisms. Diverse experimental approaches have been taken to support conclusions and the data is thoughtfully considered throughout the manuscript. Sometimes, the text or presentation of the figures could be improved for clarity. The current manuscript will be of most interest for an audience with some knowledge about viral infections in nematodes and/or an interest in natural genetic variation or RNAi in C. elegans. Moreover, further development of model organisms like the Caenorhabditis nematodes for study of viral infection is of broad interest to virologists.

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   Referee #2

   Evidence, reproducibility and clarity

   Viruses are common parasites of most animals and hosts have evolved a variety of mechanisms to defend against viruses. C. elegans and its natural Orsay virus have been used to discover novel mechanisms of viral immunity. Understanding the genetic basis why some hosts get infected and others do not can lead to a better mechanistic understanding of viral infection. In this manuscript, the authors describe their characterization of strain-specific differences in immunity to Santeuil and Le Blanc viruses in their natural nematode host C. briggsae. They found that particular strains of C. briggsae were sensitive or resistant to either …

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   Referee #2

   Evidence, reproducibility and clarity

   Viruses are common parasites of most animals and hosts have evolved a variety of mechanisms to defend against viruses. C. elegans and its natural Orsay virus have been used to discover novel mechanisms of viral immunity. Understanding the genetic basis why some hosts get infected and others do not can lead to a better mechanistic understanding of viral infection. In this manuscript, the authors describe their characterization of strain-specific differences in immunity to Santeuil and Le Blanc viruses in their natural nematode host C. briggsae. They found that particular strains of C. briggsae were sensitive or resistant to either or both viruses corresponding to the geographic origins of the strains. Resistant strains were determined to lack a small RNA response to infection suggesting an alternate, pre-invasion method of resistance. QTLs corresponding to resistance in both viruses were identified through utilization of Advanced Intercrossed Recombinant Inbred Lines (RILs).

   Main points:

   1. In figure 1C and D, is more than 1 biological replicate performed? Ideally multiple independent infections would be performed which would increase confidence in these experiments, but minimally the authors should make clear that this data was from an experiment only performed once. The conclusion from the life span assays is unlikely to change, but given the variance of the brood size assays within replicates, the conclusions that LEBV infection reduces the brood size is weakly supported.
   2. If the authors want to claim that there is a defect in viral entry in the resistant strains, they should perform infections experiments at an earlier time point that could capture viral invasion. In C. elegans with Orsay virus these experiments have been done as early as 18 hours by FISH. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011120 The way the assays are currently set up, if the infection was cleared it wouldn't be observed.
   3. The evidence that the region on chromosome III contributes to susceptibility is weak. The analysis in figure 5B does not identify this region and it is not clear to me how to read the scale in figure 5C to determine that a region on chromosome III is significant. In figure 6 using a more appropriate statistical test such as one way ANOVA with multiple hypothesis testing is necessary to determine if there is a difference between JU2832 and JU2916. It would be helpful if the authors could add more discussion of the evidence that they feel that supports this region being involved in susceptibility.

   Minor points

   1. In figure 1B it would be helpful to provide more information on the animals chosen to display. Are these representative examples or extreme examples?
   2. In figure 2B, adding a legend for the colored dots would be helpful.
   3. In figure 2C, the definitions for a strain to be labeled as belonging to each category should be provided.
   4. Could the data in figure 2 be used for genome-wide association mapping and compared to the RIL QTL experiments? Adding comment on this would be helpful to understanding the usefulness of this data.
   5. In figure 4b, in HK104 LRBV the numbers in top right corner are not defined.
   6. Line 1001 remove period from "LEBV.of" and add period after isolates.

   Significance

   Overall, this is an interesting and well-carried out study that describes a new system for understanding the genetic basis to viral infection. Using C. briggsae as a comparative system to C. elegans is likely to gain further insight into the specificity of viral infections and if mechanisms of resistance are unique or shared between these two nematodes. This study is likely to be interesting to virologists, evolutionary biologists, and those studying host-pathogen interactions.

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   Review Commons

   May 2, 2024

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Alkan et al. investigate natural variation in the susceptibility of C. briggsae nematodes to two naturally occurring Noda-like RNA viruses, the Le Blanc (LEBV) and Santeuil (SANTV). Compared to the related nematode species, C. elegans, considerably less attention has been paid to immunity to viral infections, or causative genes, in other nematode species. Taking advantage of a large, globally distributed set of C. briggsae natural isolates, the authors infected these strains with LEBV, SANTV, or both viruses to comprehensively analyze natural variation in viral susceptibility. They generally find that strains isolated from …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Alkan et al. investigate natural variation in the susceptibility of C. briggsae nematodes to two naturally occurring Noda-like RNA viruses, the Le Blanc (LEBV) and Santeuil (SANTV). Compared to the related nematode species, C. elegans, considerably less attention has been paid to immunity to viral infections, or causative genes, in other nematode species. Taking advantage of a large, globally distributed set of C. briggsae natural isolates, the authors infected these strains with LEBV, SANTV, or both viruses to comprehensively analyze natural variation in viral susceptibility. They generally find that strains isolated from temperate regions are sensitive to both viruses, while tropical strains are resistant. However, excitingly, they identify several strains (focusing specifically on HK104 from Japan) with virus-specific susceptibility. Using this observation, the authors rigorously investigate a suite of existing RILs and generate their own RILs/NILs to identify QTLs of chromosomes II, III, and IV with likely roles in LEBV and SANTV resistance. The authors could not narrow these QTLs to causative alleles in specific genes, but this work sets up future studies to further elucidate molecular mechanisms of resistance.

   Additionally, the authors identify an interesting distinction between C. briggsae strains that are resistant to viruses compared to the more commonly studied C. elegans and its natural pathogen, the Orsay virus. Alkan et al. employ small RNA sequencing to demonstrate that LEBV and SANTV-resistant strains do not elicit a small RNA response. This suggests immunity occurs by blocking viral entry or early replication steps that precede RNAi induction. This contrasts with some C. elegans strains resistant to Orsay virus, like the N2 strain, in which a small RNA response is detected. Such a result highlights the value of investigating immune responses across distinct nematode species, as there are clearly different resistance mechanisms at play. Future work building on this study will further demonstrate the value of C. briggsae and other nematodes as valuable comparative models with C. elegans.

   Major Comments:

   Overall, I found this study's data convincing, statistically rigorous and well-executed. The author's conclusions are largely fair and supported by the presented data. I appreciated that the authors performed infection screens across multiple independently generated virus preps (a notoriously variable process) to increase confidence in the results. I have two suggestions that the authors should consider addressing before publication:

   1. Figures 1&4 focus on JU1264 as the primary double-sensitive strain. However, the authors built their RILs with HK104 by crossing with JU1498 in Figures 7&8. In the results section and/or methods, the authors should provide some justification for this strain switch. Alternatively, the equivalent analysis of Figure 1 focusing on JU1498 would be valuable to demonstrate that the effects of both viruses on fitness are similar to JU1264. I am not recommending that the JU1264xHK104 crosses be performed or that Figures 7&8 be repeated with JU1264xHK104 lines, but that more explanation for strain selection for RIL generation should be provided.
   2. The authors reasonably claim that the resistance of tropical strains like AF16 could be due to blocking viral entry or early inhibition of replication before the small RNA response is activated. Could the authors test this by directly microinjecting virus (in combination with a dye as a control for successful injection) into the intestine? I understand this could not be done on a scale that would allow for small RNA sequencing, but one could perform small-scale FISH to determine if LEBV or SANTV are replication-competent if the entry barrier is artificially overcome. Such an experiment may require considerable technical development. It may be beyond the scope/timing of this specific study, but it is worth considering to gain some insight into the possible resistance mechanisms observed.

   Minor Comments:

   Line 78 - provide the full genus name for Caenorhabditis elegans at first appearance, as done for Caenorhabditis briggsae

   Line 117 - The description of cul-6 could also reference Bakowski et al. 2014. This study is referenced more generally as a player in proteostasis a few lines below but could be more explicitly tied to cul-6-mediated resistance to ORV (Bakowski et al. 2014 - see Fig. 7A)

   Line 197-198 - The authors could consider adding sequences for FISH probes as part of Table S2. This information could add value to the present study even if previously listed in Frézal et al.

   Line 263 - Were embryos obtained by bleaching of gravid adults, or was an egg lay performed, and the embryos were collected from plates? This is potentially an important distinction and should be clarified briefly in the methods.

   Line 330 - Provide justification for using JU1498 to make these RILs (see comment above).

   Line 446-Refer to the methods section for full clarity on the role of FISH in this set of experiments or reword for improved clarity. At first read-through, this phrasing made me expect some FISH experiments associated with Fig. 1, which does not appear to be the case.

   Line 478 - The supplementary figure callouts are misaligned with the provided documents. S2A in the text appears to refer to S3A RT-qPCR results.

   Line 483 - Similar to above, the text suggests serial dilutions should refer to S4, not S3.

   Line 498 - Modify the text to 'Figure 2C and Figure 3' for clarity.

   Line 531,535 - viRNAs are defined in line 535 but this should be moved to 531 above at first appearance in the text.

   Line 593 - Typo in 'Logarithm of Odds?'

   Line 621-624 - I recommend the authors include the data for the LEBV control experiments with NIL strains, either as a supplementary table, an additional panel for Fig. 6, or represented as done in Figure 8.

   Line 625-632 - How many total genes are represented in the QTL on IV? The reasoning behind testing rde-11 and rsd-2 is sound, but readers might want to know other potential candidates within this region (perhaps something the authors could also speculate on in the discussion). A similar comment applies for # genes in the QTLs on II and III.

   Line 991-992 - Figure 1B - LEBV, SANTV, and co-infection effects on body size are mentioned but not quantified. Has this phenotype been quantified elsewhere? If so, the authors should reference it in the results section or Fig. 1 legend. Alternatively, body size could be quantified as part of this study and added to Fig. 1.

   Line 1001 - There is a typo in the first sentence; the period after LEBV should be removed. Small suggestion: Figure 2A - While described in the methods, I recommend that the authors briefly reiterate in the figure legend that the white/yellow boxes are intended to indicate serial chunking for clarity.

   Line 1034 - Small formatting note for Figure 4B - percentages of reads mapping to RNA1 and RNA2 appear underneath gridlines for the graph which obscures visibility and is inconsistent with the other graphs presented.

   Line 1094 - Figure S1 - this analysis could be strengthened by RT-qPCR represented as fold change in viral load instead of, or in addition to, the agarose gel image (like Fig. S3). Doing so would also allow for the normalization of eft-2 control across individual samples (e.g.: particularly low eft-2 amplification in ED3073). However, these results are sufficiently convincing that LEBV does not replicate in C. elegans, but a more quantitative approach is recommended if feasible for the authors. Alternatively, an additional figure panel and/or repeat of this analysis with C. elegans infected with ORV would also be beneficial as an additional control.

   Line 1112 - "Experiments using RNA2 primers gave similar results" - if this data isn't included in the study, this text should be removed.

   Line 1141 - Figure S6 - For full transparency, the authors could consider including HK104 infected with LEBV to show minimal (zero) reads align to the RNA1/RNA2 segments using scales consistent with JU1264 infected with LEBV (S6C)

   Significance

   C. elegans has received considerable attention as a model for host-natural pathogen interactions, including the Orsay virus, microsporidia species, oomycetes, and others. However, the field would benefit from increased diversification into related nematodes, as there is likely much more exciting biology to uncover beyond C. elegans. This study exemplifies the genetic advantages of nematodes for this purpose, given the diverse nematode strains available from the CaeNDR (Crombie et al. 2023, PMID: 37855690), rapid growth/genetics of nematodes, and ease of infection by naturally relevant pathogens. Anyone interested in innate immunity mechanisms to viruses or other intracellular pathogens will find this study valuable, as well as those generally interested in traits under selective pressures. My field of expertise is microsporidia as parasites of nematodes, which also act as intracellular pathogens of the intestine but are eukaryotic. Surprisingly, viruses and microsporidia overlap considerably in host immune response (Bakowski et al. 2014, Chen et al. 2017, Reddy et al. 2019 referenced in Alkan et al.). To date, this has been largely explored using C. elegans as a model, but microsporidia that infect C. elegans also infect C. briggsae (Wan et al. 2022 PMID:36534656, Wadi et al. 2023: 3741459). Thus, I view the work of Alkan et al. as opening the door to exciting new directions that could similarly be executed with microsporidia pathogens for comparative analysis in C. elegans, C. briggsae, and related nematodes.

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5. 

   Version published to 10.1101/2024.02.10.579610 on bioRxiv

   Feb 12, 2024