White spot syndrome virus (WSSV) modulates lipid metabolism in white shrimp

In addition to the Warburg effect, which increases the availability of energy and biosynthetic building blocks in WSSV-infected shrimp, WSSV also induces both lipolysis at the viral genome replication stage (12 hpi) to provide material and energy for the virus replication, and lipogenesis at the viral late stage (24 hpi) to complete virus morphogenesis by supplying particular species of long-chain fatty acids (LCFAs). Here, we further show that WSSV causes a reduction in lipid droplets (LDs) in hemocytes at the viral genome replication stage, and an increase in LDs in the nuclei of WSSV-infected hemocytes at the viral late stage. In the hepatopancreas, lipolysis is triggered by WSSV infection, and this leads to fatty acids being released into the hemolymph. β-oxidation inhibition experiment reveals that the fatty acids generated by WSSV-induced lipolysis can be diverted into β-oxidation for energy production. At the viral late stage, WSSV infection leads to lipogenesis in both the stomach and hepatopancreas, suggesting that fatty acids are in high demand at this stage for virion morphogenesis. Our results demonstrate that WSSV modulates lipid metabolism specifically at different stages to facilitate its replication.

In this manuscript, the authors reported that WSSV caused a reduction in LDs in hemocytes at the viral genome replication stage, and an increase in LDs in the nuclei of WSSV-infected hemocytes at the viral late stage. In the hepatopancreas, lipolysis was triggered by WSSV infection, and this led to fatty acids being released into the hemolymph. Aseries of -oxidation inhibition experiments revealed that the fatty acids generated by WSSV-induced lipolysis can be diverted either into -oxidation for energy production or else used as material for virion morphogenesis. At the viral late stage, WSSV infection led to lipogenesis in both the stomach and hepatopancreas, suggesting that fatty acids are in high demand at this stage for virion morphogenesis. On the whole, the authors investigated the mechanisms behind these lipid metabolism shifts in WSSV-infected shrimp. The amount of work is substantial and seems to be of good quality. However, there are still some questions and suggestions regarding the experimental design and article presentation.
Some comments: 1. In Figure 1B (i), Cells with LDs decreased at 12h, while in (iii) and (iv), neither the cytoplasm nor the nucleus decreased significantly. Is it reasonable? 2. Figure 1A (vii-x), What is the significance of LD diameter and area measurements? Why was there a significant decrease in both LD diameter and area at 24h? 3. Figure 2, the authors examined the lipase activity in three different tissues of WSSV-infected shrimp. At the viral genome replication stage, lipase activity was elevated in the WSSV-infected hepatopancreas and WSSV-infected hemocytes at the late stage, However, there was no corresponding upregulation of the corresponding free fatty acids in Figure2C, What explains this phenomenon?
4. Line281-284, "FFAs were significantly decreased in WSSV-infected hepatopancreas at the viral late stage (Fig. 2C), which might be a consequence of lipid depletion during the viral genome replication stage". Why is FFA not reduced by viral utilization in other cell types? 5. Line287-289, "Under normal circumstances, lipid droplets and triglycerides are the substrates of lipase, and they can be degraded into free fatty acids which are then transported into the mitochondria and used to produce energy by -oxidation". , However, in Figure 2A, the ATP/ADP level was increased when the β-oxidation was inhibited, is the result reasonable? 6. Line309-311, "To account for this, we hypothesized that the LCFAs produced by the lipase activity might be shifted to β-oxidation, while noting that this would also be consistent with the increased virion morphogenesis seen in Figure 3B".As shown in Figure3, -oxidation appears to inhibit WSSV replication. What is the logic of this sentence, please reformulate it? 8.The detection method in this article is relatively single, especially the detection part of lipid droplets, and some effects are not obvious enough to explain the conclusion.
Reviewer #2 (Remarks to the Author): What are the main claims of the article? 1-The authors have found evidence of the regulation of fluid metabolism in the host during WSSV infection. 2-Lipolysis and lipogenesis are controlled in the early phase of and in the late phase, respectively, during WSSV infection. Are they novel and will they be of interest to others in the community and the wider field? 1-They postulate that this new knowledge can help to understand the evolution of the infection. 2-They have shed light on how the virus diverts lipid metabolism to obtain energy during the early stages of infection and finally on the need to generate specific lipids that will allow it to achieve the lipid envelope of the mature virion. Is the work convincing and, if not, what additional evidence would be required to strengthen the conclusions? The analytical techniques used by the authors are adequate and reliable for obtaining the results they describe, of which they have proposed their discussion compared with other families of viruses such as the flaviviridea that hijack the metabolism of lipids in arthropods such as the mosquito, Similarly. On a more subjective note, do you feel the paper will influence thinking in the field? The authors suggest that such results can help to better understand the distribution of fatty acids in such conditions, but that more studies are needed, such as the use of isotopes to trace their course and define whether essential lipids such as cholesterol are involved in the process.
Reviewer #3 (Remarks to the Author): The manuscript provide a broad picture on lipid metabolism during WSSV infection in shrimp using a sound experimental approach with several linked methodologies. The manuscript is well written, supported and discussed leading to an integrated conclusion, although some details could be improved as suggested below.

Material and Methods
There is an excessive use of "The" at the beginning of each sentence Line 102 and 103 I think this sentence is useless, you can just mention the GenBank reference on line 106 after Taiwan isolate Line 113 Time elapsed from infection and hemolymph collection should be mentioned here (12 and 24 hpi) ATP/ADP methodology is confusing, and no reference is provided. I think that more details should be provided to clearly understand the meaning of data A, B, C and D

Results
Several portions of the results section seem to be more suitable in other sections. For example, sentences on lines 266-267; 281-282; 283-284; 301-303; 335-337, 342-345 provide an interpretation of results and therefore should be in the Discussion section. Alternatively, Results and Discussion should be fused if the journal allows a combined Results and Discussion section. Besides, other sentences explain why a particular experimental approach was used. This is the case for the beta oxidation inhibition and the subsequent determination of ATP/ADP ratio (lines 287-292), viral replication (lines 295-296) and fatty acid content (lines 307 and 311), as well as for fatty acid synthesis (lines 325-330). I think that these sentences are better suited in the Material and Methods section as in a first instance, I did not understand why beta oxidation was inhibited when reading this section.

Discussion
Results of ATP/ADP ratio are nor discussed. If we look only at data with solvent only, ATP/ADP ratio However, in this case, although the nucleus LDs was not increased in size at 24 hpi, we can only speculate that the size decrease of cytoplasm LDs might be due to the redistribution of lipid to the nucleus, which facilitates the virion assembly at the late stage. Response: The hepatopancreas is thought to be a lipid-storage organ in shrimp. When lipolysis occurred in the WSSV-infected hepatopancreas at 12 hpi, free fatty acids (FFAs) were released into hemolymph and taken up by hemocytes (Fig 2c). This might explain why there was no upregulation of FFAs in the infected hepatopancreas. We discuss this phenomenon in line 233 to line 237. (This passage has been slightly revised from the originally submitted version.) " Figure 2a shows that lipase activity was triggered in shrimp hepatopancreas, which is a lipidstoring tissue. Unlike the total fatty acids, which Hsieh et al. (2015) found to be decreased in the stomach at 12 hpi and increased at 24 hpi, what we found here suggests that at 12 hpi, free fatty acids (FFAs) might have been released from the hepatopancreas into the hemolymph and then taken up by hemocytes (Fig. 2c)." Comment 4: Line281-284, "FFAs were significantly decreased in WSSV-infected hepatopancreas at the viral late stage (Fig. 2C), which might be a consequence of lipid depletion during the viral genome replication stage". Why is FFA not reduced by viral utilization in other cell types?
Response: This is a deeply interesting question! Although WSSV invades every tissue of shrimp with different degree of infectivity, we hypothesize that the virus might specifically target the hepatopancreas through unknown mechanisms, and that this in turn activates a higher activity of lipase relative to the lipase activity in other infected tissues. This can ensure the sufficient supply of fatty acid for the virus replication in other tissues. We note that selective infection is also found in HIV where it selectively infects effector memory T cells, which have higher glycolysis and oxidative phosphorylation than other T cells subsets (Valle-Casuso et al., 2018). However, more work will need to be done to understand whether there is any hepatopancreas-specific mechanism that can employed by WSSV to elevate the lipase activity. Response: It is interesting to observe the ATP/ADP level was increased after β-oxidation inhibition in infected and non-infected group at 24 hpi (Figure 3a, Right panel). We speculate that other energy pathways might be activated after the long duration of β-oxidation inhibition in order to maintain the viability of the cell. This has now been added to the discussion about ATP/ADP levels at 24 hpi in line 227 to line 232" "As discussed earlier, β-oxidation might be providing ATP for virus replication at the genome replication stage. Surprisingly however, at 24 hpi, β-oxidation inhibition resulted in elevated ATP levels regardless of infection status (Figure 3a, Solvent group vs Etomoxir group at 24 hpi). We speculate that the long duration of β-oxidation inhibition might have led to the activation of other energy pathways in order to maintain cell viability." Comment 6: Line309-311, "To account for this, we hypothesized that the LCFAs produced by the lipase activity might be shifted to β-oxidation, while noting that this would also be consistent with the increased virion morphogenesis seen in Figure 3B".As shown in Figure3, β-oxidation appears to inhibit WSSV replication. What is the logic of this sentence, please reformulate it? Response: We agree that there is a problem with our argument here. This passage has been rewritten as follows (line 143 to line 146). "Lipase was activated in the hepatopancreas at the viral genome replication stage (Fig. 2a), and we hypothesized that the LCFAs produced by the lipase activity might be shifted to β-oxidation. This would also be consistent with Hsieh et al. (2015), who reported that various LCFAs were decreased in infected stomach at the same stage."

Comment 8: The detection method in this article is relatively single, especially the detection part of lipid droplets, and some effects are not obvious enough to explain the conclusion.
Response: We appreciate that there were limitations to our means of detection. However, as described in methods (lines 292-300), the lipid droplets (LDs) in the hemocytes were stained using BODIPY 493/503 (Invitrogen). And with the staining of cytoplasm and nucleus, we were able to distinguish whether the LDs were from the cytoplasm or nucleus.
1-The authors have found evidence of the regulation of fluid metabolism in the host during WSSV infection.

2-Lipolysis and lipogenesis are controlled in the early phase of and in the late phase, respectively, during WSSV infection. Are they novel and will they be of interest to others in the community and the wider field?
Response: Our study demonstrated that WSSV induces lipolysis and lipogenesis in viral genome replication stage (12 hpi) and viral late stage (24 hpi), respectively. This dual regulation of lipid metabolism is also reported in flaviviruses, a single-stranded RNA virus. To our knowledge, our study is the first to report that double-stranded DNA virus like WSSV also could trigger a similar lipid regulation as flaviviruses. Additionally, we demonstrated the timepoint for lipolysis and lipogenesis occurrence during the first virus replication cycle, which has not previously been reported in other virus research. We believe our study supports the idea that viruses, regardless of DNA virus or RNA virus, modulate the lipid metabolism specifically at different stages of infection in order to obtain necessities at that timepoint for their replication. B. What is the role of the accumulated nucleus LDs in WSSV replication? We observed that LDs were accumulated in the nucleus of the cells from the viral late stage, but we still don't know how this is involved in virion assembly. Two hypotheses have been made to explain this phenomenon. (1) the accumulated LDs might regulate gene expression that facilitates viral morphogenesis. (2) the accumulated LDs serves as a viral protein-storage depot for virus assembly. To address these questions, Transcriptomics analysis combined with LD formation inhibition might give us an insight into which gene expressions are affected when the LDs can't be accumulated. Identification of LD proteins that interact with viral proteins might also help us to understand the role of LDs during virus replication. Response: Thank you for these comments. After discussed with other co-authors, we decided not to change our format. We think that using these brief introductory sentences in the results section can help the reader to understand the progression of the experiments in this study, which first showed that WSSV modulated LDs during infection, and then followed this up with further investigations into this phenomenon. We agree however that it would be helpful to introduce the methods of the β-oxidation experiments in the same way. In order to explain the reasons behind the β-oxidation experiments, we have therefore added "To examine the role of β-oxidation and the LCFAs in WSSV replication, we used the CPT1 inhibitor Etomoxir and FAS inhibitor C75, respectively." at line 392. The results with Eto also should be discussed as the contribution of Beta-oxidation to the increase in ATP production (ATP/ADP ratio) during the early phase of infection which is in accordance with the general conclusion of the present study. However other results remain to be explained; for example, why such increase in ATP production is sustained at later stages of infection and apparently is independent of beta oxidation.