Onset and Progression of Infection Based on Viral Loads in Rhesus Macaques Exposed to Zika Virus

Round 1

Reviewer 1 Report

In the presented manuscript, the authors analyzed the serum viral load observed in Rhesus macaques exposed to Zika virus (ZIKV). The aim of the study was to characterize the progression of infection based on viral load in serum in ZIKV-infected RMs using unvaccinated or sham-vaccinated control animals from previous studies. Please see some comments below.

General comments

All abbreviations should be explained when appear for the first time in the manuscript and used consistently thereafter.

Specific comments

Introduction

Line 68: please explain abbreviation NIAID.

Line 73: please explain abbreviation FDA.

Table 1: please add Age (years)

Results

Line 170: please delete LOD=685 copies/ml, it is explained in the RT-qPCR paragraph (line 124).

Line 185: please delete LOD=61 PFU/ml, it is explained in the Plaque Assay paragraph (line 134).

Line 187: please delete (LLOQ=195 PFU/mL), it is explained in the Plaque Assay paragraph (line 134).

Discussion

Line 263: please correct RT-qPCR.

Line 292: please delete "blood" and correct "... and human plasma is transient ..."

References

Reference 13: please check and correct.

Reference 29: please check and correct.

Author Response

Response to Review 1 Comments:

In the presented manuscript, the authors analyzed the serum viral load observed in Rhesus macaques exposed to Zika virus (ZIKV). The aim of the study was to characterize the progression of infection based on viral load in serum in ZIKV-infected RMs using unvaccinated or sham-vaccinated control animals from previous studies. Please see some comments below.

General comments

All abbreviations should be explained when appear for the first time in the manuscript and used consistently thereafter. – Checked and addressed.

Specific comments

Introduction

Line 68: please explain abbreviation NIAID. – Abbreviation explained.

Line 73: please explain abbreviation FDA. – Abbreviation explained.

Table 1: please add Age (years) – Specification that age is in years is added to the table.

Results

Line 170: please delete LOD=685 copies/ml, it is explained in the RT-qPCR paragraph (line 124). – “LOD=685 copies/mL” was deleted from Line 166.

Line 185: please delete LOD=61 PFU/ml, it is explained in the Plaque Assay paragraph (line 134). – “LOD=61 PFU/mL” deleted from Line 180.

Line 187: please delete (LLOQ=195 PFU/mL), it is explained in the Plaque Assay paragraph (line 134). – “(LLOQ=195 PFU/mL)” deleted from Line 182.

Discussion

Line 263: please correct RT-qPCR. – “PCR” updated to “RT-qPCR” in Line 261.

Line 292: please delete "blood" and correct "... and human plasma is transient ..." – Deleted “blood” from Line 270.

References

Reference 13: please check and correct. – Reference checked, and no error identified.

Reference 29: please check and correct. – Reference checked and corrected.

Reviewer 2 Report

In this article, the authors statistically evaluate the Rhesus macaque model to evaluate ZIKV progression, as to determine if it is a good model in the search for novel vaccines. They compare data from three studies where either viral load in serum as determined with RT-qPCR and viremia in serum as determined with plaque assay are evaluated, and further determine if sex, age, or weight influence outcome. Specifically, they model progression of infection and time to virus detection. The statistical methods are sound and the figures clear. As it is encouraged that research is also focused on infectious diseases in periods of limited number of cases, this is a nice study. However, the way it is written now it is unclear if this manuscript adds much novelty to the field, as the RM ZIKV model is regularly used, and it also seems out of scope of this Journal’s aims.

Introduction: first two paragraphs are too stretched. The last paragraph on the other hand could be expanded and better structured, with more emphasis on currently used ZIKV (NHP) models or potential vaccines. Also, the connection of the study with where it fits in one of the three strategies is not very clear from the text.

M&M: why are the three studies not specifically mentioned?

Results:

Line 171: 21?

Fig 3 vs Fig4: why is viral rna evaluated over only 2 days and viremia over 27 days?

Discussion: this should be better structured and also made more clear what this analysis contributes. Human and NHP findings are mixed up which disturbs the reading flow and makes it unclear if it concerns humans or NHP (eg line 252).

Line 256: could you discuss if the absence of viremia (as detected by plaque assay) in some animals exerts a problem to be used as model?

Line 260: Is it impossible to include clinical signs in this study, as these are probably also used as end-points in the three separate studies?

Line 270: this seems contradictory to what is mentioned earlier in the discussion.

Line 276: where is this finding shown in the results?

Line 299: contradictory to line 274

Author Response

Response to Review 2 Comments:

In this article, the authors statistically evaluate the Rhesus macaque model to evaluate ZIKV progression, as to determine if it is a good model in the search for novel vaccines. They compare data from three studies where either viral load in serum as determined with RT-qPCR and viremia in serum as determined with plaque assay are evaluated, and further determine if sex, age, or weight influence outcome. Specifically, they model progression of infection and time to virus detection. The statistical methods are sound and the figures clear. As it is encouraged that research is also focused on infectious diseases in periods of limited number of cases, this is a nice study. However, the way it is written now it is unclear if this manuscript adds much novelty to the field, as the RM ZIKV model is regularly used, and it also seems out of scope of this Journal’s aims.

Introduction: first two paragraphs are too stretched. The last paragraph on the other hand could be expanded and better structured, with more emphasis on currently used ZIKV (NHP) models or potential vaccines. Also, the connection of the study with where it fits in one of the three strategies is not very clear from the text. – The first two paragraphs have been condensed. The relevance of this study to the third strategy (application of the animal rule) is made more specific.

M&M: why are the three studies not specifically mentioned? – The three studies are referred to as Study 1, Study 2, and Study 3 as the specific internal study numbers would have no meaning outside the research institution where the studies were conducted. None of the individual research studies is published.

Results:

Line 171: 21? – We believe the reviewer may be questioning whether N should be 21 instead of 26 for Figure 1. The N = 26 is correct, while N = 21 is correct for Figure 2. At noted in the text (Line 114), RT-qPCR was measured in all 3 studies (N = 26) while plaque assay was measured in only 2 studies (N = 21).

Fig 3 vs Fig4: why is viral rna evaluated over only 2 days and viremia over 27 days? – In Figure 3, all animals exhibited positive viral RNA within 2 days, and the Kaplan Meier plots reach 1.00 detection probability within this timeframe. In contrast, not all animals exhibited viremia within the 27 days on study, so Figure 4 extends over this longer timeframe to display the censoring at 27 days.

Discussion: this should be better structured and also made more clear what this analysis contributes. Human and NHP findings are mixed up which disturbs the reading flow and makes it unclear if it concerns humans or NHP (eg line 252). – Line 253 has been clarified to refer to humans. The goal of the discussion is to compare human presentation to RM presentation side-by-side. The authors believe that the remaining text of the discussion clearly indicates human or RM for each finding.

Line 256: could you discuss if the absence of viremia (as detected by plaque assay) in some animals exerts a problem to be used as model? – Text is added to the manuscript to describe the impact of the absence of viremia on use of the model. It is noted that the viremia model is less sensitive than viral RNA and that study design adjustments would be needed with this endpoint.

Line 260: Is it impossible to include clinical signs in this study, as these are probably also used as end-points in the three separate studies? – Clinical signs were recorded but no clinical signs of disease were observed in the infected RM on any of the three studies included in the analysis, so cannot be added to this manuscript.

Line 270: this seems contradictory to what is mentioned earlier in the discussion. – Earlier in the discussion at Line 255 the text refers to persistence of viremia, where text at Line 270 refers to viral RNA.

Line 276: where is this finding shown in the results? – This finding is not shown in the results, and the statement has been removed.

Line 299: contradictory to line 274 – While there are some cases reported of longer duration of viral RNA in plasma, as noted in Line 277, viral RNA generally clears within 10 days as noted in Line 275. The text has been updated in both places to clarify the typical duration of 10 days with some exceptions of longer duration.