Review Reports - Fetal Lymphoid Organ Immune Responses to Transient and Persistent Infection with Bovine Viral Diarrhea Virus

Round 1

Reviewer 1 Report

The possibility of differentiating between persistent infections (PIs) and transient infections (TIs) by focusing on the fetal immune response is certainly an interesting concept. However, the authors did not include enough detail (i.e., experimental plans and methods) that would be required to support their hypothesis. In addition, the quality of the figures was extremely low, and as such the results were difficult to comprehend. I managed to interpret Figs. 1 and 6; however, the other figures did not provide enough information and were unclear. All the figures require major revisions.

Similarly, most of the results could not be understood. I have commented on the parts that were decipherable (up to line 170, description of Figs. 1 and 6):

The authors need to provide direct evidence of PI and TI. These data would include quantitative evaluation of viral RNA as well as antibody titers in fetal blood. Without these findings, it is not possible to interpret any of the results. It should be possible to perform blood withdrawal from a fetus at 190 days of gestation.

Line 133; “2.5. Morphogenesis of thymus during bovine fetal development and effect of in utero BVDV infection” → There are no data associated with this section; these need to be provided.

Lines 141~; “2.6. Statistical analysis” → What is 2-ΔΔCq? I checked the designated reference ([34]) but did not understand the term Cq. Lines 152–155 are also difficult to understand. The standard term ΔΔCt is more familiar and easier to understand.

The quality of the graphs is extremely low (Figs. 1–5). Furthermore, it is not clear which graphs are labeled A, B, or C. It is also unclear why the graphs are divided into yellow, red, and blue; the significance of the color schemes needs to be explained in the Figure Legend. Similarly, the vertical axes (y-axes) should include a numerical scale, while the name of each sample should be included where appropriate on the horizontal axes (x-axes). Additionally, please indicate the degree of significant difference (using one or more asterisks) in the graphs. None of the graphs presented reach even the minimum level of quality required for paper submission.

Does the graph on the right in Fig. 1 present CD46 expression levels? Although this was not specified, I interpreted blue as control, yellow as TI, and red as PI. When the ΔΔCt method is used (see point #3), the expression levels of the control (or standard) are defined as 1.0. Why has the expression level reported for the control samples increased? Is this due to the use of the ΔΔCq method? Further explanation and clarification of all issues related to this graph are required.

It is not appropriate to draw conclusions such as those shown in Fig. 6 after the examination of mRNA expression levels alone. The expression levels of encoded proteins as well as mRNA should be considered in any conclusion or final analysis. Furthermore, CD4 and CD8 T lymphocytes are more appropriately evaluated using flow cytometry. Although the authors state that significant changes occur in the mRNA expression, it is necessary to determine whether these changes affect the protein expression. In any case, Fig. 6 is an overstatement. Please revise or delete it.

Author Response

Introductory remarks: We thank the reviewers for their detailed review of our manuscript. We apologize for the appearance of the figures. The manuscript was generated on a Mac and we did not realize that corruptions occurred in the figures when the word file was opened on some, but not all Windows operating systems. The revised manuscript is submitted as a pdf and doc file and the figures are submitted separately as tiff files in a compressed Zip folder. We request that the PDF file be submitted for review, because it seems to be stable in context of retaining high resolution figures from operating system to operating system.

Response to Reviewer 1

Comment: Authors did not include enough detail (i.e., experimental plans and methods

Comment: The possibility of differentiating between persistent infections (PIs) and transient infections (TIs) by focusing on the fetal immune response is certainly an interesting concept. However, the authors did not include enough detail (i.e., experimental plans and methods) that would be required to support their hypothesis.

Response: We have provided more detail on experimental plan and methods as suggested and referenced the previous article describing the experiment.

Concerns with Figures:

Comment: The quality of the figures was extremely low.

Comment: In addition, the quality of the figures was extremely low, and as such the results were difficult to comprehend. I managed to interpret Figs. 1 and 6; however, the other figures did not provide enough information and were unclear. All the figures require major revisions.

Comment: The quality of the graphs is extremely low (Figs. 1–5). Furthermore, it is not clear which graphs are labeled A, B, or C. It is also unclear why the graphs are divided into yellow, red, and blue; the significance of the color schemes needs to be explained in the Figure Legend. Similarly, the vertical axes (y-axes) should include a numerical scale, while the name of each sample should be included where appropriate on the horizontal axes (x-axes). Additionally, please indicate the degree of significant difference (using one or more asterisks) in the graphs. None of the graphs presented reach even the minimum level of quality required for paper submission.

Comment: Does the graph on the right in Fig. 1 present CD46 expression levels? Although this was not specified, I interpreted blue as control, yellow as TI, and red as PI. When the ΔΔCt method is used (see point #3), the expression levels of the control (or standard) are defined as 1.0. Why has the expression level reported for the control samples increased? Is this due to the use of the ΔΔCq method? Further explanation and clarification of all issues related to this graph are required.

Response: Again, we apologize for the quality of figures in the manuscript. The figures for this manuscript were corrupted after submission from a Mac-based platform to the journal (please see introductory statements to this review). This affects the Word Document that was uploaded. The figures in the PDF file that were uploaded to the journal for review of this manuscript appear to be fine. To resolve this problem, the revised manuscript is submitted as a Word Document from a Windows platform computer and a PDF is supplied for review by the reviewers. High quality Tiff files for the figures are attached in a zip folder. Presentation of the uncorrupted figures should address many of the reviewer’s questions including the gene/graph identities, axis labels, and significant differences (***< p values).

Comment: Similarly, most of the results could not be understood. I have commented on the parts that were decipherable (up to line 170, description of Figs. 1 and 6):

Response: We thank the reviewer for continuing with the review in light of the terrible electronic corruption of figures in the original manuscript. This has now been resolved. In the revision, we present non-corrupted figures that should help with interpretation of the results. Also, text has been added to these results for clarification.

Comment: The authors need to provide direct evidence of PI and TI. These data would include quantitative evaluation of viral RNA as well as antibody titers in fetal blood. Without these findings, it is not possible to interpret any of the results. It should be possible to perform blood withdrawal from a fetus at 190 days of gestation.

Response: The quantitation of BVDV RNA (expressed as 2^-ΔΔCq) in fetal thymuses and spleens for PI, TI and controls is shown in Fig. 1A-B. The uncorrupted figures in this revision should address this concern. Thank you for your kind patience when reviewing the original submission. As shown in Figure 1B, control fetuses had a 2^-ΔΔCq value = 1 compared to 130 in TI fetal spleens and 3,037 in PI fetal spleens. These values establish the presence of greater amounts of BVDV RNA in PI fetuses at day 190, 115 days post-maternal inoculation and the presence of lower amounts of BVDV RNA in TI fetuses on day 190, 15 days post-maternal inoculation on day 175,confirming fetal infection. Antibody titers were not analyzed because of known lack of antibodies in PI fetuses. TI fetuses are born with BVDV-specific antibodies. However, in this model, the dams of TI fetuses were inoculated on day 175 of gestation and their fetuses were collected on day 190 of pregnancy, which was 15 days post-maternal inoculation. Based on prior studies, the fetuses were likely infected 10 to 14 days post-maternal inoculation. Consequently, these TI fetuses were unlikely to have had sufficient time to elaborate antibodies 1 to 5 days later when they were collected by C-section.

Comment: Line 133; “2.5. Morphogenesis of thymus during bovine fetal development and effect of in utero BVDV infection” → There are no data associated with this section; these need to be provided.

Response: Section 3.2.3 summarizes the findings associated with section 2.5. No morphological differences were seen between control and PI thymuses.

Comment: Lines 141~; “2.6. Statistical analysis” → What is 2-ΔΔCq? I checked the designated reference ([34]) but did not understand the term Cq. Lines 152–155 are also difficult to understand. The standard term ΔΔCt is more familiar and easier to understand.

Response: Our group is following the “Minimum Information for Publication of Quantitative Real-Time PCR Experiments” (MIQE) guidelines, reference 33. Due to drastic differences in RT-qPCR data presentation in publications, the MIQE guidelines have been adopted by labs and journals to try and standardize how these data are presented. One issue is that different RT-qPCR instrument manufacturers use different terms for cycle quantification. These different terms include threshold cycle (C_t), crossing point (C_p), and takeoff point (TOF). MIQE recommends a more concise term, quantification cycle (C_q), that does not pertain to specific instruments or companies. These terms are all synonymous for the same quantified number, which is the first cycle that amplification occurs above background level. Additionally, reference 34 explains the use of 2^-^DD^Cq for RT-qPCR analysis instead of DDC_q. We have added statements to the methods for clarification to those who may not be familiar with MIQE guidelines.

Comment: It is not appropriate to draw conclusions such as those shown in Fig. 6 after the examination of mRNA expression levels alone. The expression levels of encoded proteins as well as mRNA should be considered in any conclusion or final analysis. Furthermore, CD4 and CD8 T lymphocytes are more appropriately evaluated using flow cytometry. Although the authors state that significant changes occur in the mRNA expression, it is necessary to determine whether these changes affect the protein expression. In any case, Fig. 6 is an overstatement. Please revise or delete it.

Response: We have couched this presentation in Fig 6 as hypothetical based on RT-qPCR analysis. We have also stated that while valid, RT-qPCR data are somewhat limiting in scope and require further study using other approaches such as western blot, immunohistochemistry and flow cytometry, which are beyond the scope of these studies. Unfortunately, the sampling of the fetal tissues was not conducive for immunohistochemistry or flow cytometry and bovine western blot because validated antibodies for these bovine targets were unavailable.

Sampling of fetal tissues was limited as was availability of validated and specific antibodies against bovine proteins encoded by the genes described in Figure 6. Our group investigated several western blot antibodies for cross reactivity but were not successful because of non-specific immunoreactive proteins and were not comfortable including those results. Flow cytometry studies would have been excellent, but also were beyond the scope of our fetal collections following C-Section at the time when we completed these experiments. Also, the multiplexing of targets with more recent flow cytometry approaches was not available at the time when we completed these studies. With new approaches in flow cytometry, we agree with the reviewer and in future animal studies we will include plans for flow cytometry. Figure 6 was designed as a simple outline of results, which will lead to future studies to investigate “BVDV clearance” or “immunotolerance to BVDV” in postnatal animals. The authors included text in the figure legend to clarify that these concepts will be further examined in future studies.

Reviewer 2 Report

It is widely accepted that persistently infected (PI) animals play a central role in the epidemiology of BVDV, and that the interaction of pestiviruses with the host’s immune response is of upmost importance. Accordingly, inhibition of innate and adaptive immunity is a prerequisite to establish persistent infections, but in vivo data are sparsely available. In this study, transcripts of a set of genes involved in innate and adaptive immunity were quantified by RT-PCR in PI and transiently infected (TI) fetuses upon in utero infection at different time points of pregnancy. This is an interesting study investigating these transcripts in thymus and spleen, showing somewhat different results between these two organs. Accordingly, care should be taken in generalizing the results, and as the issue of inhibition of the immune responses (including e.g. the extent if IFN inhibition) is indeed a matter of controversy, the data should be discussed more critically and not biased by the own hypotheses.

Specific comments:

1) Title / abstract: It is implied that innate and adaptive immune responses are downregulated in spleen and thymus, but according to the results, transcripts related to innate immunity were not reduced in PI spleens.

2) Introduction, lines 67-71. The authors report that in a previous analysis, it was shown that the viral RNA decreased in a PI animal between day 97 and 245. However, in reference 27 provided, no viral RNA was analyzed. On the one hand, it probably should have been reference 29 (Virus Res). On the other hand, the claim that this is proof for an active immune response in the PI animal is not justified, as during development of the fetus, e.g. a change in number of susceptible cells might also be responsible (see also section 4.2).

3) Introduction, lines 76-78. To conclude from the chapter described above that this shows that inhibition of innate immune response by ncp BVDV is unlikely and does not explain persistence is, at least to the opinion of this reviewer, not justified. It is well debatable whether e.g. the inhibition of the IFN response is absolute or partial, but the facts that (i) PI animals represent the reservoir that maintain the virus in the host population and thus display the evolutionary strategy of “infect & persist”, (ii) the IFN antagonists E^rns and N^pro are absolutely conserved in BVDV, and (iii) mutant viruses lacking these IFN antagonists are unable to induced persistence (ref 26) are among the most important indications that inhibition of the IFN response does play a role. Accordingly, experimental infection of fetuses with ncp, but not cp, BVDV failed to induce an IFN response (Charleston et al., 2001, JGV 82:8, 1893-1897) and results in this study confirm that inhibition of the expression of innate immune genes indeed occurs.

4) Introduction, last paragraph. It is not clear why the authors postulate that adaptive immunotolerance is a “second explanation for viral persistence”. The fact that PI animals do not mount neither a humoral nor a cellular immune response to the infecting strain (which is rather unique upon fetal viral infections) is a clear indication that central immunotolerance is part of persistence. In addition, it should be specified whether in the model of the authors, BVDV interferes with the general development of T-cells (line 83) or only with the development of BVDV-reactive T-cells (e.g. line 386). Thus, do the authors postulate another mechanism of prevention of adaptive immune responses compared to classical central immunotolerance (development of self tolerance)? It should be kept in mind that PI are not necessarily immunosuppressed, as older PI cows that gave birth to several PI calves were reported that must thus be in a generally could health status. Thus, inhibition of innate immunity and central immunotolerance are not mutually exclusive in causing persistence, on the contrary.

5) Table 1. The PCR efficiencies are sometimes rather high, up to 160%. This might be observed if values are measured in the nonlinear phase of the amplification or if inhibitors are present in the reaction. With such high differences, care should be taken as delta-delta-Ct calculations should only be performed with similar PCR efficiencies to avoid erroneous results.

6) Fig. 2. The figure is difficult to read (see minor comments), but the statement that only NF-kB and IFNbeta were reduced in PI fetal thymus compared to control might be due to the fact that the other genes were already very low in controls !?

7) Section 4.2. What is the evidence that persistence might be caused by exhaustion of the initial innate immune response? An initial slight increase followed by a decrease does not necessarily present a casual relationship. In addition, why should this exhaustion (reduction) in innate immune response lead to a decrease in viral titer. Finally, can it be excluded that an increase in ISG expression as shown in previous studies originated from maternal of placental expression of IFNs?

Minor comments:

- Line 74. The term RLR3 is rarely used and rather unknown. Replace or complement by DHX58 and LGP2.

- Figures: At least in the pdf file available for review, the label of the axis and the indication of significance was lacking, making proper evaluation rather difficult.

- Line 161: BVDV-RNA in PI fetal spleens is reported to be 3037-fold higher than in controls. However, how can this be as the control (mock) should be negative?

- Line 299: IRF7 and ISG15 are not really cytoplasmic sensors (such as DDX58), but rather a transcription factor and an ISG (interferon-induced ubiquitin-like modifier), respectively.

- Update ref 32 including name of Journal

- Ref 25 and 55: Rümenapf, not Rumenapf

Author Response

Response to Reviewer 2

Comment: It is widely accepted that persistently infected (PI) animals play a central role in the epidemiology of BVDV, and that the interaction of pestiviruses with the host’s immune response is of upmost importance. Accordingly, inhibition of innate and adaptive immunity is a prerequisite to establish persistent infections, but in vivo data are sparsely available. In this study, transcripts of a set of genes involved in innate and adaptive immunity were quantified by RT-PCR in PI and transiently infected (TI) fetuses upon in utero infection at different time points of pregnancy. This is an interesting study investigating these transcripts in thymus and spleen, showing somewhat different results between these two organs. Accordingly, care should be taken in generalizing the results, and as the issue of inhibition of the immune responses (including e.g. the extent if IFN inhibition) is indeed a matter of controversy, the data should be discussed more critically and not biased by the own hypotheses.

Response: Thank you for your concern about our interpretation. We have couched discussion of inhibition of the IFN immune response and have cited others (i.e., Palomares et al., 2013) who have published data that challenge this interpretation.

Specific comments

Title / abstract: It is implied that innate and adaptive immune responses are downregulated in spleen and thymus, but according to the results, transcripts related to innate immunity were not reduced in PI spleens.

Response: Thank you for pointing this out. This was an overstatement on our part and we apologize for the over-generalization. The title has been changed to “Persistent bovine viral diarrhea virus fetal infection decreases the expression of innate and adaptive immune response genes in the bovine fetal thymus and the adaptive genes in the fetal spleen” to more accurately reflect the results.

Specific comment Introduction, lines 67-71. The authors report that in a previous analysis, it was shown that the viral RNA decreased in a PI animal between day 97 and 245. However, in reference 27 provided, no viral RNA was analyzed. On the one hand, it probably should have been reference 29 (Virus Res).

Response: The reference has been corrected to reference 29. Due to the order in the manuscript, the reference number will still remain 27.

Specific comment On the other hand, the claim that this is proof for an active immune response in the PI animal is not justified, as during development of the fetus, e.g. a change in number of susceptible cells might also be responsible (see also section 4.2).

Response: The authors acknowledge that the expression of individual genes changes quite dramatically during fetal development. However, had the changes in gene expression been due to fetal development, the expression would not have been significantly different from control tissues collected at the same time point in gestation (day 190). In addition, if the expression of genes varied due to fetal development, the authors would have expected no difference between control and PI or TI fetuses or greater individual variability within each treatment group. For these reasons, we feel that our interpretation is reasonable.

Specific comment Introduction, lines 76-78. To conclude from the chapter described above that this shows that inhibition of innate immune response by ncp BVDV is unlikely and does not explain persistence is, at least to the opinion of this reviewer, not justified. It is well debatable whether e.g. the inhibition of the IFN response is absolute or partial, but the facts that (i) PI animals represent the reservoir that maintain the virus in the host population and thus display the evolutionary strategy of “infect & persist”, (ii) the IFN antagonists Erns and Npro are absolutely conserved in BVDV, and (iii) mutant viruses lacking these IFN antagonists are unable to induced persistence (ref 26) are among the most important indications that inhibition of the IFN response does play a role. Accordingly, experimental infection of fetuses with ncp, but not cp, BVDV failed to induce an IFN response (Charleston et al., 2001, JGV 82:8, 1893-1897) and results in this study confirm that inhibition of the expression of innate immune genes indeed occurs.

Response: We acknowledge the (i) persistence of BVDV as an evolutionary strategy, (ii) conservation of Erns and Npro and (ii) the experiments that show mutations in these genes result in BVDVs that are unable to establish persistence in the Introduction (see lines 59-67of the revision). However, in our previous studies, increased expression of IFN stimulated genes was demonstrated to occur in PI fetal blood and tissues collected at earlier time points (Shoemaker et al 2009; Smirnova et al 2008; Smirnova et al 2012) and from post-natal PI steers (Shoemaker et al. 2009) indicating that an IFN response to ncp BVDV occurs in bovine fetuses and post-natal PI cattle during early gestation.

In addition, the paragraph has been amended to read.

“shown to be chronically upregulated in the PI animal postnatally [28]. These results indicate that PI fetuses and placenta respond to BVDV with an innate immune response albeit somewhat reduced compared to TI fetuses [27-29]. These findings and those of indicate that the fetus responds to ncp BVDV infection with an innate immune response much like the innate response to acute BVDV infection in post-natal calves shown by Palomares et al. (2013); hence, inhibition of the innate immune response by viral proteins does not entirely explain viral persistence in vivo [30].”

Specific comment: Introduction, last paragraph. It is not clear why the authors postulate that adaptive immunotolerance is a “second explanation for viral persistence”. The fact that PI animals do not mount neither a humoral nor a cellular immune response to the infecting strain (which is rather unique upon fetal viral infections) is a clear indication that central immunotolerance is part of persistence. In addition, it should be specified whether in the model of the authors, BVDV interferes with the general development of T-cells (line 83) or only with the development of BVDV-reactive T-cells (e.g. line 386). Thus, do the authors postulate another mechanism of prevention of adaptive immune responses compared to classical central immunotolerance (development of self tolerance)? It should be kept in mind that PI are not necessarily immunosuppressed, as older PI cows that gave birth to several PI calves were reported that must thus be in a generally could health status. Thus, inhibition of innate immunity and central immunotolerance are not mutually exclusive in causing persistence, on the contrary.

Response: We do not dispute the role of central immunotolerance as a part of viral persistence. We cannot conclude whether BVDV interferes generally with the development of T-cells or specifically with the development of BVDV-reactive T-cells based on this experiment. We are limited to postulating that ncp BVDV fetal infection decreases the amount of mRNA for specific genes in the innate and adaptive immune response pathways. The first sentence in the last paragraph of the Introduction has been revised to clarify that this is just another possible component of viral persistence.

“An additional explanation for viral persistence is that the presence of BVDV during the development of the T cell repertoire permits its antigens to be accepted as “self” antigens resulting in a state referred to as immunotolerance in which elements of the adaptive immune system do not respond to viral antigens and do not clear the virus from fetal tissues [31].”

We agreed that some PI cattle remain healthy; however, many PI cattle are unhealthy and succumb to a variety of secondary infections. See references: 1) L F Taylor, E D Janzen, J A Ellis, J V van den Hurk, and P Ward. Performance, survival, necropsy, and virological findings from calves persistently infected with the bovine viral diarrhea virus originating from a single Saskatchewan beef herd. Can Vet J. 1997 Jan; 38(1): 29–37; 2) Loneragan GH, Thomson DU, Montgomery DL et al.2005. Prevalence, outcome and health consequences associated with bovine viral diarrhea virus in feedlot cattle. JAVMA 226:595-601; 3) Fulton RW, Blood KS, et al.2009. Lung pathology and infectious agents in fatal feedlot pneumonias and relationship with mortality, disease onset, and treatments. JVDI 21:464-477.

Specific comment Table 1. The PCR efficiencies are sometimes rather high, up to 160%. This might be observed if values are measured in the nonlinear phase of the amplification or if inhibitors are present in the reaction. With such high differences, care should be taken as delta-delta-Ct calculations should only be performed with similar PCR efficiencies to avoid erroneous results.

Response: The authors acknowledge that the ideal PCR efficiencies should be in the range of 90-110%. Unfortunately, there is a shortage of published bovine primer sets for the specific genes targeted and not all hand designed primers are perfect. In most cases, 2-4 primer sets were designed using ncbi primer blast then tested for the target genes. Primers were evaluated and only used if they had single amplifications, correct amplifications (products were sequenced), and single melting curves. Efficiencies were calculated and the primer sets were chosen based on all of the above as well as the efficiency closest to the appropriate range. Poor efficiencies were most likely due to complementarity of the primers. We are confident that the primers designed were the best for that target with the current design tools at our disposal. We agree with the reviewer that care should be taken in the interpretation of the results given unfavorable efficiencies, which is the reason for publishing said efficiencies.

Specific comment Fig. 2. The figure is difficult to read (see minor comments), but the statement that only NF-kB and IFNbeta were reduced in PI fetal thymus compared to control might be due to the fact that the other genes were already very low in controls?

Response: Analysis using 2^-^DD^Cqputs unchanged (control group) expression at a value of 1 (1 fold change). Expression above controls ranges from 1 to infinity. Expression below 1 is limited to between the values of 0 and 1. For example, a -2 fold change is presented as 0.5 . We acknowledge that this is limiting for interpretation however, upon consulting with a statistician and the standard for publication, we continued with 2^-^DD^Cq. C_q values of 32 or higher were considered as no expression, therefore, should controls and PIs have little to no expression, significant differences would not have been found.

Specific comment Section 4.2. What is the evidence that persistence might be caused by exhaustion of the initial innate immune response? An initial slight increase followed by a decrease does not necessarily present a casual relationship. In addition, why should this exhaustion (reduction) in innate immune response lead to a decrease in viral titer. Finally, can it be excluded that an increase in ISG expression as shown in previous studies originated from maternal of placental expression of IFNs?

Response: Our original hypothesis was that the innate immune system was exhausted. However, while doing our studies, the lack of lymphocyte response and subsequent downregulation of the innate immune response may point towards Treg cell development and T cell anergy. Text has been added for clarification.

Minor comments:

- Line 74. The term RLR3 is rarely used and rather unknown. Replace or complement by DHX58 and LGP2.

Response: RLR3 is replaced by DHX58.

- Figures: At least in the pdf file available for review, the label of the axis and the indication of significance was lacking, making proper evaluation rather difficult.

Response: We suspect that the figures were corrupted in a Mac to windows platform. We have uploaded figures as Tiffs as well as a PDF that can be opened in both platforms.

- Line 161: BVDV-RNA in PI fetal spleens is reported to be 3037-fold higher than in controls. However, how can this be as the control (mock) should be negative?

Response: The control fetal spleen was negative for BVDV and the BVDV RNA 2^-ΔΔCq value = 1.0. The 2^-ΔΔCq value for the PI fetal spleen was 3037. This sentence has been edited for clarity.

“ PI fetal spleens had significantly increased amount of BVDV RNA (p < 0.001, 2^-ΔΔCq = 3037 fold greater) compared to controls (2^-ΔΔCq =1.0) and very low amounts of BVDV RNA in TI fetal spleens (Fig.1B).”

- Line 299: IRF7 and ISG15 are not really cytoplasmic sensors (such as DDX58), but rather a transcription factor and an ISG (interferon-induced ubiquitin-like modifier), respectively.

Response: Thank you for catching this. This oversight has been edited for clarification.

- Update ref 32 including name of Journal

Response: Journal name has been included.

- Ref 25 and 55: Rümenapf, not Rumenapf

Response: Thank you for catching this oversight. This has been corrected.

Round 2

Reviewer 1 Report

To the author:

I have checked reference no. 32 of the revised manuscript (Hanah M Georges at al., Attenuated lymphocyte activation leads to the development of immunotolerance in bovine fetuses persistently infected with bovine viral diarrhea virus., Biology of Reproduction., 2020. 1-12.) and found that this reference has many similarities with your manuscript, such as the PI production method and the RT-qPCR analysis. In addition, the immune-related genes assayed using RT-PCR are almost the same. There is no problem with the materials and methods being the same, but the analysis data of PI fetal spleen overlap with that of reference no. 32 (Figs. 2 and 3; the data of 190 gestational days).

Reference no. 32 has already reported decreased expression of genes associated with innate and adaptive immune responses in the spleen of PI fetuses (collected on gestational day 190) by BVDV inoculation on gestational day 75. Therefore, even if the experiment in this manuscript was conducted independently of that in reference no. 32, it is problematic to interpret the findings regarding the PI fetal spleen (Figs. 4 and 5 of the revised version) as being the novelty of this study.

The manuscript should be checked to confirm that it adheres to the guidelines of this journal. Additionally, the main text (including figures) and title require significant modification to establish the novelty of this study.

Author Response

General Comment: In regards to the concerns raised by reviewer 1; although you have indicated that this study is a separate one from your newly published manuscript in reproduction biology, and you now have the inclusion of a transiently infected subject group, your abstract mainly defers to the outcomes of the persistently infected group as the novelty of this manuscript. However, these findings overlap significantly with the above mentioned manuscript. Can you please outline the significant difference in this data, and make sure this is reflected in the abstract. Additionally, could you please include some caveats in your discussion surrounding the lack of information in regards to protein levels of the transcripts you have measured, and how this lack of information may impact the results and conclusions presented in this manuscript.

Response: We apologize for this oversight. The experiments in the current manuscript and the Georges et. al. paper are two separate studies with different animals and different locations (University of Wyoming vs. Colorado State University). The experiment in the current manuscript, performed in Wyoming, was originally designed to compare TI vs. PI fetal responses to BVDV (only one fetal collection day: day 190 of gestation) whereas the Georges et. al. experiment, performed in Colorado, was designed to examine PI fetal immune responses to BVDV compared to uninfected controls over different timepoints of fetal development (days 82, 97, 190, and 245; No TIs were generated). The abstract was edited accordingly, to reflect the original goal of this study (Lines: 23-26 and 40-41). This was also reflected in the discussion at several points. Direct comparisons between the results of the Georges et al. paper and the PI spleen results of the current study were also outlined in the discussion (Lines 426-430, 454-458, 489-491). Finally, we disclosed that lack of protein data does limit interpretation in the final paragraph of the discussion (Lines: 510-518). We understand and agree that this is a limitation of this study that was unfortunate and out of our control.

Reviewer 1

Reviewer Comment. I have checked reference no. 32 of the revised manuscript (Hanah M Georges at al., Attenuated lymphocyte activation leads to the development of immunotolerance in bovine fetuses persistently infected with bovine viral diarrhea virus., Biology of Reproduction., 2020. 1-12.) and found that this reference has many similarities with your manuscript, such as the PI production method and the RT-qPCR analysis. In addition, the immune-related genes assayed using RT-PCR are almost the same. There is no problem with the materials and methods being the same, but the analysis data of PI fetal spleen overlap with that of reference no. 32 (Figs. 2 and 3; the data of 190 gestational days).

Response: Thank you for your comment on this matter. The authors should have been clearer on the differences between the two studies. The study performed in reference 32 (now changed to reference 35) was done at Colorado State University and only had 2 treatment groups (controls and PIs) of which pregnant cattle were inoculated on day 75 of gestation but had several days of fetal collections on days 82, 97, 190, and 245. The current manuscript/study was performed at the University of Wyoming (lines 117-119), had 3 treatment groups (controls, PIs (inoculated on day 75) and TIs (inoculated on day 175) with fetuses collected on only one day (day 190). The objective of the study in reference 32, now 35, was to understand how BVDV affects fetal immune development and responses to persistent infection over time (in utero), while the current study was to compare the different infection types on day 75 (PI) and 175 (TI) with fetal collection on day 190. The fact that there are similarities in the spleen data between the 2 papers, i.e., corroboration of findings, only strengthen the results for both studies. Although significant results differ slightly between the two papers, we have added text to clarify some differences between the two (Lines 426-430, 454-458, 489-491). Title has been changed as suggested.

Reviewer 2 Report

The authors submitted a revised manuscript, which clearly removed some ambiguities and clarified difficult to understand issues. In addition, the figure captions clearly improved. Finally, the authors agreed to broaden possible interpretations of various findings, which are indeed (and still are) a matter of debate, and they even adjusted their working hypothesis as indicated in the discussion (lines 364-366). However, there appears to be a discrepancy in the argumentation, as on the one hand, the authors agree that inhibition of IFN response is a part of pestiviral persistence, but on the other hand, they refute that IFN inhibition plays a role in vivo in later sections of the manuscript. Thus, this appears incoherent to any reader.

Specific comments:

1) line 71/72 ("…suggesting 71 partial viral clearance by an active immune response in the PI fetus):
The authors discuss in the response to this issue that "had the changes in gene expression been due to fetal development, the expression would not have been significantly different from control tissues collected at the same time point in gestation (day 190). In addition, if the expression of genes varied due to fetal development, the authors would have expected no difference between control and PI or TI fetuses or greater individual variability within each treatment group".

However, this reviewer cannot clearly follow this line of arguments, and is still of the opinion that the mere decrease in viral titer over time does not necessarily suggest an active immune response being responsible. It might not only be a matter of "gene expression" (which genes ?) but the number of susceptible cells and/or even the type of cells present at different stages of development might be the cause of such a difference. Thus, at the different time points of infection (TI vs. PI), various numbers and types of susceptible cells might be present that will possibly change until d190, and the change might even be influenced by the presence of the virus. Accordingly, the number of infected cells in a given PI animal remains rather constant over time, but with large variabilities between different animals (e.g. JGV 68, 1987, 1971-82), further demonstrating the importance of the number of susceptible cells present at a given time. A similar situation might already be present in PI fetuses late in gestation.

This reviewer will not insist on this, as the authors suggestion is indeed possible. But especially in an introduction section, previously published results should be presented as such, avoiding one-sided opinions and excluding additional interpretations.

2) lines 76-82. Innate immune response in fetuses.
The authors obviously agree that inhibition of the IFN response by ncp BVDV is indeed required for successful establishment of PI, and write in the revised version that this cannot "entirely explain persistence in vivo", a statement that can be fully agreed on. By contrast, the addition of the work or Palomares et al. (2013) does not add to this argument, as in that work, only postnatal infections were analyzed. The extent of an innate immune response cannot be extrapolated to the one observed in PI fetuses, as observed be the authors in previous studies to be present but clearly reduced to TI.

3) Fig. 2: Apology for the misunderstanding. There was no doubt that in PI, DDX58, IRF-7 and ISG15 are not significantly different from the controls. Rather, the comment should have indicated that because the controls were already very low in expression, a further down-regulation cannot be demonstrated. Thus, the statement in line 188 that " Neither DDX58, IFNB nor ISG15 were significantly different than controls" indirectly implies that the expression of these genes is not actively altered in PI animals (in contrast to NF-kB and IFNbeta), which would somehow unnecessarily weaken the conclusion of the author (similarly for Fig. 4).

4) Lines 337-348. The authors argue that in the study by Charleston et al. in 2001, the samples were taken to early (d3, d5 and d7) for successful fetal infection (Ref 47). However, this is not correct, as in that study, in utero infections were performed, thus bypassing the 7-14 days required upon infection of the mother. In addition, the "several in vivo studies" cited are all from the author's group, and in their own studies, they showed several times that the IFN response is clearly reduced compared to a TI infection. Thus, the "conclusion that the fetal IFN response is inhibited by BVDV infection was refuted" is simply not valid. As discussed above and agreed on by the authors, persistence is probably not relying on a single factor, and thus, the inhibition of IFN response does occur – to an extent that can be discussed – but it's by no means refuted. Even the authors themselves state in the conclusion that "Specifically, the down-regulation of genes including type I IFN transcriptional regulators, antigen presentation, and T cell markers in PI fetal tissues was found.", which would completely contradict their own conclusion of "refusal to inhibit an IFN response".

Minor comments:

- Ref. 7: All other Journal names are abbreviated (Vet. Clin. North Am. Food Anim. Pract., compare Ref 17). Similar for Ref 14, 24, 32, 33, 39-44, 47, 51, 54, 55, 57, 63.

- Ref. 8: Please edit names (Brownlie, J.; Clarke, M. C.; Howard, C. J.; Pocock, D.H.,), year is in double.

Author Response

Reviewer 2:

Reviewer Comment. The authors submitted a revised manuscript, which clearly removed some ambiguities and clarified difficult to understand issues. In addition, the figure captions clearly improved. Finally, the authors agreed to broaden possible interpretations of various findings, which are indeed (and still are) a matter of debate, and they even adjusted their working hypothesis as indicated in the discussion (lines 364-366). However, there appears to be a discrepancy in the argumentation, as on the one hand, the authors agree that inhibition of IFN response is a part of pestiviral persistence, but on the other hand, they refute that IFN inhibition plays a role in vivo in later sections of the manuscript. Thus, this appears incoherent to any reader.

Response: Please see response to comment 4 below.

Reviewer Specific Comment 1. line 71/72 ("…suggesting 71 partial viral clearance by an active immune response in the PI fetus):
The authors discuss in the response to this issue that "had the changes in gene expression been due to fetal development, the expression would not have been significantly different from control tissues collected at the same time point in gestation (day 190). In addition, if the expression of genes varied due to fetal development, the authors would have expected no difference between control and PI or TI fetuses or greater individual variability within each treatment group".

Response: Thank you for this insight. Text has been added to line 85 to include different interpretations of the data. The Bielefeldt-Ohmann paper referenced has been added and the authors agree that there may be different cell populations present/infected in the TI and PI fetuses due to the timing of the initial inoculation as well as variability between individual animals.

Reviewer Comment 2. lines 76-82. Innate immune response in fetuses.
The authors obviously agree that inhibition of the IFN response by ncp BVDV is indeed required for successful establishment of PI, and write in the revised version that this cannot "entirely explain persistence in vivo", a statement that can be fully agreed on. By contrast, the addition of the work or Palomares et al. (2013) does not add to this argument, as in that work, only postnatal infections were analyzed. The extent of an innate immune response cannot be extrapolated to the one observed in PI fetuses, as observed be the authors in previous studies to be present but clearly reduced to TI.

Response: Lines 68-102 were written to introduce the two main theories of BVDV mechanisms of infection. We have presented what we can, as unbiased as we can. However, our own previous work indicates an IFN response in PI fetuses at earlier gestational development and time following infection with BVDV in that section of the manuscript. In those previous studies, our group proposed that PI fetuses have a chronically upregulated IFN response. Since then, we have done additional work and altered our hypotheses to the current hypotheses/theory introduced in lines 103-112. We don’t believe this brings in bias but instead, highlights the natural evolution of thought that is consistent with scientific exploration. We have added text to the section (lines 68-102) to hopefully point out to the reader that results are also open to their interpretation with the information given in the introduction.

Reviewer comment 3. Fig. 2: Apology for the misunderstanding. There was no doubt that in PI, DDX58, IRF-7 and ISG15 are not significantly different from the controls. Rather, the comment should have indicated that because the controls were already very low in expression, a further down-regulation cannot be demonstrated. Thus, the statement in line 188 that " Neither DDX58, IFNB nor ISG15 were significantly different than controls" indirectly implies that the expression of these genes is not actively altered in PI animals (in contrast to NF-kB and IFNbeta), which would somehow unnecessarily weaken the conclusion of the author (similarly for Fig. 4).

Response: Thank you for this insight, we agree that control levels are inherently low. However, NFKB and IFNB, 2 strong IFN response players, were significantly downregulated in PIs, supporting our conclusions of a partially inhibited innate immune response. Text has been edited to reflect that PI concentrations of non-significant genes were at control/basal levels, line 212.

Reviewer Comment 4. Lines 337-348. The authors argue that in the study by Charleston et al. in 2001, the samples were taken to early (d3, d5 and d7) for successful fetal infection (Ref 47). However, this is not correct, as in that study, in utero infections were performed, thus bypassing the 7-14 days required upon infection of the mother. In addition, the "several in vivo studies" cited are all from the author's group, and in their own studies, they showed several times that the IFN response is clearly reduced compared to a TI infection. Thus, the "conclusion that the fetal IFN response is inhibited by BVDV infection was refuted" is simply not valid. As discussed above and agreed on by the authors, persistence is probably not relying on a single factor, and thus, the inhibition of IFN response does occur – to an extent that can be discussed – but it's by no means refuted. Even the authors themselves state in the conclusion that "Specifically, the down-regulation of genes including type I IFN transcriptional regulators, antigen presentation, and T cell markers in PI fetal tissues was found.", which would completely contradict their own conclusion of "refusal to inhibit an IFN response".

Response: Thank you for that clarification on the Charleston paper, the experimental model with amniotic inoculation, as opposed to a maternal inoculation, was an oversight on our part. We believe that the differences in results between our data and others may be differences in BVDV strain or experimental models (ex: maternal inoculation vs. inoculation via amniotic fluid) but the different studies do refute each other. Our model of infection and BVDV strain have been used several times and has shown a fetal IFN response soon after PI fetal infection (22 days post maternal inoculation). We have shown this in previous papers through RNA and ISG15 protein (please see reference 35, Georges et. al. 2020). This current study examined thymic and splenic tissues 115 days post maternal inoculation. We conclude that early in the fetal response (22 days post maternal inoculation), there is an IFN response (ref 35 and others) however, there is an IFN inhibition in PIs ~90 days after that initial response (current manuscript). In the current manuscript, the initial IFN response is supported by the TI fetus having an upregulated expression of ISG15 in response to BVDV. The authors believe that the IFN inhibition shown in PIs in the current manuscript is not a direct inhibition by BVDV but rather, an inhibition by the fetus’ own immune system becoming tolerant to the virus. Text has been edited in lines 371-375 for clarification.

Reviewer Comment. Ref. 7: All other Journal names are abbreviated (Vet. Clin. North Am. Food Anim. Pract., compare Ref 17). Similar for Ref 14, 24, 32, 33, 39-44, 47, 51, 54, 55, 57, 63.

Response: Thank you, these corrections in how references are cited has been addressed.

Reviewer Comment. Ref. 8: Please edit names (Brownlie, J.; Clarke, M. C.; Howard, C. J.; Pocock, D.H.,), year is in double.

Response: Thank you, we made these corrections in this reference.