Powassan Virus Infects Mouse Testes and Reveals a Protective Role for Phagocytic Cells

R1 C1

148) Why was 0.9% sodium chloride used as the mock-infection control instead of the virus growth medium? Since the virus stocks were propagated in cell culture, using the corresponding culture medium as the control would better account for any effects associated with the inoculum itself.

addressed

0.9% sodium chloride was chosen as the mock-infection control since the viral stock was diluted 1:500 in 0.9% sodium chloride prior to challenge. This detail was included in the manuscript to make more clear in lines 147-148.

R1 C2

262) The infectious dose ranged from 1.9–2.7 log₁₀ PFU/mouse. Could the authors explain the rationale for using this dose range? Were additional infection doses tested before selecting this inoculum?

addressed

We thank the reviewer for this question. The inoculum was selected to approximate the magnitude of viral exposure that may occur during natural tick transmission of POWV, which is estimated to be between 100- 10,000 PFU. The variation from 1.9–2.7 log₁₀ PFU/mouse reflects the experimentally determined titers of the inocula used across independent experiments. We have clarified the rationale for the selected infectious dose in lines 266-267.

R1C3

289–291) Please include representative immunofluorescence images from the uninfected control mice. These images are important for evaluating the specificity of the staining and for comparison with the infected groups.

addressed

We thank the reviewer for this suggestion. Representative immunofluorescence images from uninfected control mice have now been included in the revised figure to demonstrate staining specificity and facilitate comparison with the infected groups. The corresponding figure legend and manuscript text have also been updated accordingly. See lines 308-314.

R1C4

The presentation of Figure 3 could be improved to facilitate interpretation:

• Please indicate the magnification and/or scale (zoom) used for panels 3D and 3E.
• Although the immunofluorescence markers are described in the Methods section, it would greatly improve readability if the identities of each fluorescent channel (e.g., marker/color) were indicated directly in the figure or figure legend.

addressed

The magnification and scale bars for panels in figure 3 have now been specified in the revised figure legend, thank you for the suggestion. In addition, the identities and corresponding colors of each fluorescent marker have been indicated directly in the figure and described in the figure legend to improve readability and facilitate interpretation.

R2C1

The introduction summarizes correctly the background information and the aims of the study, although the last section (from line 76) actually anticipates the results, and is thus not truly appropriate for introduction. This section should be rewritten dealing with aims only, and the results should be only mentioned in later chapters (e.g., discussion, conclusions).

addressed

We thank the reviewer for this helpful comment. We agree that the final paragraph of the Introduction in the original manuscript anticipated the study results rather than focusing exclusively on the study rationale and objectives. We have therefore revised this section to remove statements describing the study findings. The revised paragraph now focuses solely on the aims of the study, including characterization of POWV infection within the male reproductive tract, investigation of the potential contribution of macrophages to testicular infection, and characterization of the transcriptional response. The corresponding results are now presented only in the Results and discussed in the appropriate subsequent sections of the manuscript. See lines 505-515 for a summary of main conclusions.

R2C2

furthermore, since in the study two different lineages are used, introducion should introduce the reader to the philogeny of the virus and provide some explanations about the two specific lineages of interest. 

addressed

We appreciate this suggestion and agree this would strengthen the manuscript to highlight. We have revised the Introduction to provide additional background on POWV phylogeny and the two viral lineages used in this study. This can be found inlines 43-46.

R2C3

The methods are described with sufficient details. Please take care about formal aspects (e.g., consistency in writing names and localization of companies. Always in complete form the first time, company name only in later uses).

addressed

We thank the reviewer for pointing out this inconsistency. Attention has been given to make these consistent and are now referenced as suggested. Lines 94-103, 124-125, 144-145, 147, 157, 169, 176, 182, 183, 194, 196, 200-202, 204-205, 207, 212, 213, 215, 217, 218.

R2C4

Actually, the presentation seems to highlight some limitations in the study design, as mouse model infections seem to have been arranged in a way that every condition (administration route, lineages, timepoint...) have a too low number of replicates.

Addressed

We thank the reviewer for highlighting this limitation. We are in agreement that the combinations of inoculation route, viral lineage, and time point tested limit the extent to which direct comparisons can be made among these conditions. We have revised the manuscript throughout to clarify the exploratory nature of these comparisons and have tempered our conclusions accordingly. Specifically, we now state that although infection of the male reproductive tract was observed across the evaluated inoculation routes and viral lineages, the limited sample sizes preclude definitive conclusions regarding route- or lineage-dependent differences. These limitations have also been explicitly acknowledged in the Discussion.

Specifically regarding the data for the IP injection of POWV, this study was repeated thrice with similar results.

Specific changes can be found on lines 282-284, 493-496

R2C5

It is not possible to properly understand the choice of one lineage for the following experiments. Looking at experiments, it really seems that particular strain and administration route were pre-selected, and only for them the timepoint observation is complete. By the way, this selection, though acceptable, seems questionable since the 2016 human case was infected with DTV, not LB strain. So, the viral strain choosen for following experiments must be motivated in a better way, and the fact of not considering more intersting the DTV should also be explained in the discussion.

addressed

We thank the reviewer for noting this. We have clarified in the methods section in lines 112-115 the rationale for selecting the LB strain for the majority of experiments. LB was selected as the primary experimental strain because it is a well-characterized prototype POWV strain that has been extensively utilized in established murine models of POWV infection, allowing our findings to be interpreted in the context of previous experimental studies. DTV was included in the initial experiments to determine whether male reproductive tract infection was also observed with a lineage II virus, since most of the previous murine data from infection by tick feeding used DTV. We wanted to show that the results described by the Hermance and Thangamani groups with DTV were more representative of the virus, not artifact of the lineage.

R2C6

Another questionable point is about the clodronate experiment. This set of experiments makes sense if there is a documented depletion of macrophages in the textes. But, as shown in cytometry, the preliminar experiment doesn't confirm this, possibly becasue of the too low content of macrophages in the non-infected testis. So, this preliminar experiment provides no information. Cytometry, or any other technique, should be used to confirm macrophages absence from testis post-infection, in contrast with untreated infected animals.

addressed

We thank the reviewer for raising this important point. We agree that the preliminary flow cytometry experiment did not demonstrate depletion of the resident macrophage population within the testes; however, the clodronate experiment was designed to test whether systemic phagocytic cells contribute to POWV dissemination to the testes, rather than to specifically determine the role of resident testicular macrophages during established infection. In this context, the observed depletion of splenic macrophages was used to confirm systemic phagocytic-cell depletion at the time of viral challenge.

We have revised the manuscript to clarify this experimental rationale and to avoid interpreting the results as evidence of testicular macrophage depletion. We have also explicitly acknowledged that macrophage depletion was not demonstrated within the testes and that these experiments therefore cannot determine the specific contribution of resident testicular macrophages. This was done on lines 166-167, 498-500.

R2C7

About this set of experiments; I personal consider unproper to resume together the results of gene expression analysis of infected vs non-infected animals with the comparison between clodronate + vs - animals. The two comparisons are not biological related, since one is based on the effects of infetion itself, the other on the role of macrophage signaling in gene expression. The second condition, in particular, is highly articifious, and since this study shows that, as expected, clodronate enhances infection, part of the effects shown in the clodronate vs liposome comparison may actually be due barely to the higher level of virus in the tissue. In any case, putting the results from the two experimental comparisons in the same graphs may result unproper, in my opinion.

addressed

We thank the reviewer for this thoughtful comment and agree that the increased viral burden observed following phagocytic-cell depletion is an important consideration when interpreting the transcriptional differences between clodronate- and control liposome-treated infected animals. We agree that this was downplayed in the manuscript and that these differences cannot be attributed solely to the absence of phagocytic cells, as the substantially greater viral burden in clodronate-treated animals may itself contribute to the enhanced inflammatory and antiviral transcriptional response.

We respectfully believe, however, that the two comparisons remain informative when presented together, as they address related but distinct aspects of the testicular response to POWV infection. The infected versus uninfected comparison characterizes the transcriptional response associated with POWV infection itself, whereas the clodronate-treated versus control liposome-treated comparison characterizes the transcriptional state associated with infection under conditions of systemic phagocytic-cell depletion and increased viral burden. Presenting these analyses together therefore allows the transcriptional changes associated with the clodronate-treated condition to be interpreted in the context of the response observed during POWV infection.

We have revised the manuscript to more clearly distinguish the biological questions addressed by these comparisons and have tempered our interpretation of the clodronate-associated transcriptional changes. In particular, we now acknowledge that the enhanced transcriptional response observed following phagocytic-cell depletion may reflect the increased viral burden in these animals, in addition to effects associated with depletion itself, and therefore cannot be attributed specifically to the absence of phagocytic-cell signaling. This has been updated on lines

Relevant changes to text that reflect this can be found in lines 356-359, 461-467, 500-502.

R2C8

Discussion is in general terms consistent with data shown in results, although the part dealing with gene expression is definitely too long, also because relatively few genes are actually modulated in a strong and significant way, especially when comparing clodronate vs non-clodronate infected mice. In the end, no conclusions are provided, neither as an indipendent chapter, nor as a final period of the discussion, nor are study limitations. These elements must be added to complete the discussion.

addressed

We thank the reviewer for these thoughtful comments. We acknowledge the reviewer’s concern regarding the length of the discussion of the gene expression findings. We have retained the detailed discussion of these results because we believe that consideration of the antiviral, inflammatory, and immune-regulatory pathways identified provides important biological context for understanding the host response associated with POWV infection of the testes. However, in response to this and the reviewer’s preceding comment, we have revised the interpretation of these findings to more clearly distinguish transcriptional changes associated with POWV infection from those observed under conditions of phagocytic-cell depletion. We now also explicitly acknowledge that the increased viral burden in clodronate-treated animadls may contribute substantially to the enhanced transcriptional response and that these differences therefore cannot be attributed solely to phagocytic-cell depletion.

We acknowledge the absence of an explicit discussion of study limitations and an overall concluding statement. We have therefore added a dedicated limitations paragraph. We have also added a concluding paragraph to the Discussion that summarizes the principal findings of the study and places them in the context of the remaining questions regarding POWV infection of the male reproductive tract. These can be found in lines 505-513.

R2C9

- line 43: 63 cases worldwide, or in a specific country? please specify

addressed

This was clarified as worldwide, thank you. Line 43.

R2C10

- line 102: please define BHKsa (it is absent also from the abbreviation final list), and write the source company or institution.

addressed

We thank the reviewer for noting this omission. The Materials and Methods section has been revised to clarify that BHK-SA cells were obtained from an existing laboratory stock maintained at the University of Texas Medical Branch (UTMB, Galveston, TX). The particular significance of “SA” is undetermined and considered to be an internal identifyer of this cell stock. Line 103-104.

R2C11

- line 110: please add cells LLC-MK-2 to the paragraph (definition, source, media, etc)

addressed

We appreciate the chance to clarify that LLC-MK-2 cells were not used experimentally in the present study. Their mention in the Materials and Methods refers exclusively to the documented passage history of the POWV LB virus stock prior to its use in this study. We have revised the text to make this distinction explicit. As LLC-MK-2 cells were not maintained or used in the experiments described here, culture conditions and media for this cell line have not been added.109-112.

R2C12

- line 120: the cited paper makes use of this technique, but doesn't describe its protocol in the methods. A reference is appropriate in the methods section only when citing the original source of a protocol. This is not the case.

addressed

We thank the reviewer for this comment and apologize for the lack of clarity regarding the purpose of this citation. The reference was not intended to serve as the source of the plaque assay protocol used in this study, but rather to document the previous use of Vero cells for DTV propagation and titration. The cited study describes DTV growth in Vero cells and subsequent quantification of viral titers by plaque assay on Vero cells. The plaque assay procedure used in the present study is described directly in the Materials and Methods. Line 122.

R2C13

- line 122: inoculum is removed (I suppose)

addressed

Removal of inoculum was included in the methods section, thank you. Lines 136-137.

R2C14

- line 126: the expression of viral titre in  n log10 PFU/ml, albeit biologically acceptable, is not formally correct. A mathematically correct expression (i.e. 10^n PFU/ml), would be highly preferable, here and in the entire paper.

addressed

We thank the reviewer for this comment. Viral titers are presented as log10-transformed PFU/mL (or PFU/g for tissue samples). Reporting plaque-assay titers in log10 PFU/mL or log10 PFU/g is a commonly used convention in related virology literature, although we do recognize there is some inconsistency in our field related to the best practices to present these numbers. As this notation is used consistently throughout the text and figures and does not alter the interpretation of the data, we have elected to retain the log₁₀ notation throughout the manuscript.

R2C15

- line 129-132: this period appears off-topic in this paragraph, please move it to a more related one.

addressed

We agree with the reviewers suggestion and have moved this paragraph to lines 152-155.

R2C16

- line 134: identify cell lines and viral lineage(s) used.

addressed

This line was elaborated to include cell lines and viruses, thank you. The correction can be found on line 132.

R2C17

- line 147: please check for viral dose. 100 log10 PFU is an astronomic amount.

addressed

We appreciate this catch and have corrected the error. The correction can be found on line 146.

R2C18

- line 155: the entire period is not very clear, both in biological meaning and in phrasing.

addressed

We thank the reviewer for noting the lack of clarity in this passage. We have rewritten this section to more clearly identify which experimental cohorts underwent PBS perfusion and to clarify the biological rationale for perfusion, namely to minimize the contribution of circulating virus to measured tissue viral titers in animals in which viremia was expected or could be altered by experimental treatment. The correction can be found on line 159-163.

R2C19

- line 243: fig 1A actually shows 10^9 pfu/mL already at 1dpi, which sincerely seems an incredibly fast replication rate.please check. Also, it is not clear if titre at 0 dpi is referred to virus in the supernatant after inoculum removal (as it should), or to viral inoculum itself. 10^3/4 is a very high titre for a 0 dpi. 

addressed

We appreciate the feedback, and we have reviewed the data and confirmed that 10^9 titer after 24 hours is indeed the reported value. Materials and methods have been updated to explicitly state that inoculum was removed and washed prior to collection of the time point. The correction can be found on line 137.

R2C20

- line 249: ZIKV infection is completely forgotten in the methods section. please include relevant information. alternatively, if this experiment comes from previous work (i.e. ref 23), omit it from the results and only mention it in the discussion. In both cases, there is no point in having a reference in the results section, so remove or displace it.

addressed

We updated the materials and methods section to properly include ZIKV, and we thank the reviewer for catching this oversight. The correction can be found on line 107.

R2C21

- line 250: fig 1 completely lacks error bars.

addressed

We thank the reviewer for this consideration, and would like to clarify that error bars are present in Figure 1.

R2C22

- line 278: LB strain. Anyways, the conclusion that infection route and lineage is not affecting the outcome is not supported by the data that are shown.

addressed

We appreciate the feedback and have altered the language in lines 282-284 to include the consideration that a small group number has on making definitive conclusions.

R2C23

- line 289: fig 3 A-C is too poorly resolved and lacks any highlighting sign, so it is hard to find there what is described in the text.

addressed

We thank the reviewer for this comment. Panels 3A–C are representative H&E-stained sections included to illustrate the overall testicular architecture in uninfected and POWV-infected mice. Because the blinded pathological assessment identified no focal inflammation, tubular hyalinization, or other discrete histopathological lesions, there is no specific abnormality to indicate with arrows or other highlighting symbols. The reported measurements of seminiferous tubule diameter, spermatogenic potential, and Leydig cell abundance were determined by a blinded board-certified pathologist across the examined sections and are not intended to be derived from the individual representative fields shown.

R2C24

- line 292: fig2 is too complex and not very well organized. probably, some different choices could be made to simplify the presentation. Also, some points must be clarified: in fig 2B, why are time points organized in inconsistent way across different infection conditions? and why is there no DTV ID?0.

addressed

We appreciate the feedback and agree with the assessment that Figure 2 is overly complex. We have adjusted this figure so that all POWV IP experimental results are included in one line, and we have adjusted the labeling and titles to make clear the distinctions. We have included language to address the inconsistent timing for viremia, which reflects the different scheduling of sampling and euthanasia between different cohorts of mice. DTV ID was not included because DTV was incorporated specifically to determine whether male reproductive tract infection was also observed with a second POWV lineage, while the effect of inoculation route was evaluated independently using POWV LB. This has been updated on lines 287-289

R2C25

- line 315: figure S2 contains errors, since the identification of different experimental conditions is provided in three unconsistent ways (figure titles, figure caption, file names within the figure).please check and correct.

addressed

We thank the reviewer for noticing this inconsistency and have updated the language to be fully consistent and accurate. This has been updated in the accompanying Supplemental figure 2 headings.

R2C26

- line 458: table 1 contains results, so it should be part of the results chapter, and mentioned there. 

addressed

We agree with this suggestion and added language referencing Table 1 in the Results section at line 354-356.

R2C27

- line 460: which of the data shown is this sentence referring to?

addressed

We thank the reviewer for identifying this ambiguity. The sentence referred specifically to the observed changes in Socs3, Il1b, and Elane expression. We have revised the sentence (line 455-458) to explicitly identify these findings and to clarify that, while these transcriptional changes involve pathways associated with testicular immune homeostasis and epithelial barrier regulation, they do not directly demonstrate disruption of the BTB.

R2C28

- line 465: the reference numer is wrong, since it comes after others with higher numbers.

addressed

Reference number has been updated, thank you.

R2C29

- line 490: in international articles written in english, even if the reference article is written in a different language, authors names, journal references, and possibily title translation must be provided in english. in this case, pubmed entry can be useful as a source (https://pubmed.ncbi.nlm.nih.gov/203101)

addressed

We thank the reviewer for providing guidance on addressing this, and we have made the suggested change of including the international article in English. This has been updated on lines 661-662.

R2C30

- line 496: co-author OAS is absent from the authors contribution

addressed

We appreciate this catch and have included OAS in the authors contribution. This has been updated on line 520.

Comment 1

I would like to invite them checking for some residual mistypes, also in newly introduced text (e.g., caption of fig 3

Response

We thank the reviewer for noticing this error, we have reread the manuscript and noticed the error in Figure 3 pertaining to the images referenced and updated accordingly in line 313.

Comment 2

table 1, which should be moved to the results also as a graphic object

Response

The table has been moved accordingly between line 344 and 345, thank you.

Comment 3

line 461 (former 460). In this case maybe I didn't exlain well the problem: this sentence describes some kind of an experiment showing the amount of phagocytes during infection experiments, but no similar experiment is documented in the results, including the supplementary files. So, this sentence should be removed, or additional results be shown.

Response

We appreciate this clarification by the reviewer and agree that the language as it stood was unclear and misleading. We have updated the language on line 465-466 to clarify that the experiment we were referring to was separate from the infection experiment, and related data can be found in Supplementary figure 2 detailed on lines 322-328.