Experimental Yellow Fever in the Squirrel Monkey (Saimiri spp.): Hematological, Biochemical, and Immunological Findings

Round 1

Reviewer 1 Report

This manuscript describes a potential NHP model of YFV infection. It has some novelty since a new NHP squirrel monkey was used, and infection was with a different strain of YFV isolated from South America.

The studies are in general reasonably designed and conducted. Data are clearly presented.

Major issue is the way how the samples were collected. During the first seven days and at 10th, 20th and 30th dpi, one infected animal was randomly selected to obtain ten mL of blood. Therefore, for viremia evaluation (and many other parameters), the data do not represent real kinetics. It is unclear how big is the animal-to-animal variation. That being said, it is unreasonable to ask for redo the experiment, but the authors should not over-interpretate their data. Limitation of their study should be clearly discussed.

Figure 1. line graph is somewhat misleading because each sample was from different animals. Figure 1 legend is inconsistent with figure panels and text. The corresponding M&M part for hemagglutination inhibition needs more details.

It would be interesting to specifically mark which samples were from the two sick animals.

Figure 2 and 3. Unit of Y-axis is unclear. If they are relative numbers, please describe how the numbers are normalized. Please indicate what is the grey area (normal range?).

Figures 4-6. legend should indicate panel A-D. For the marker expression, please explain how the error bars were generated (assuming there was only one sample for each time point). If they were assay multiplex, please indicate how many replicates.

The author stated the detection of cross-reactivity with other flaviviruses (DENV 1-4, ILHV, SLEV and ROCV) between 5-30 dpi. Data should be shown.

Author Response

Thanks for the suggestions. The answers to the questions are below.

 

This manuscript describes a potential NHP model of YFV infection. It has some novelty since a new NHP squirrel monkey was used, and infection was with a different strain of YFV isolated from South America.

The studies are in general reasonably designed and conducted. Data are clearly presented.

Major issue is the way how the samples were collected. During the first seven days and at 10th, 20th and 30th dpi, one infected animal was randomly selected to obtain ten mL of blood. Therefore, for viremia evaluation (and many other parameters), the data do not represent real kinetics. It is unclear how big is the animal-to-animal variation. That being said, it is unreasonable to ask for redo the experiment, but the authors should not over-interpretate their data. Limitation of their study should be clearly discussed.

R.: The limitation of the study was included in the discussion as requested.

Figure 1. line graph is somewhat misleading because each sample was from different animals. Figure 1 legend is inconsistent with figure panels and text. The corresponding M&M part for hemagglutination inhibition needs more details.

R.: Text and figure have been corrected for better comprehension

It would be interesting to specifically mark which samples were from the two sick animals.

R.: The text has been changed to become clear.

Figure 2 and 3. Unit of Y-axis is unclear. If they are relative numbers, please describe how the numbers are normalized. Please indicate what is the grey area (normal range?).

R.: The correction was included in the manuscript

Figures 4-6. legend should indicate panel A-D. For the marker expression, please explain how the error bars were generated (assuming there was only one sample for each time point). If they were assay multiplex, please indicate how many replicates.

Performing each sample in technical and biological triplicate increased the reliability of the generated data.

The author stated the detection of cross-reactivity with other flaviviruses (DENV 1-4, ILHV, SLEV and ROCV) between 5-30 dpi. Data should be shown.

R.: Data were included as supplementary material

Author Response File: Author Response.docx

Reviewer 2 Report

The submitted manuscript by Ferreira et al describes clinical and immunologic observation of squirrel monkeys experimentally infected with yellow fever virus (YFV).  These studies are a fist in kind for this model and demonstrate that squirrel monkeys could be a reasonable model for yellow fever (YF) in humans.  Overall, the data provided here are very helpful in understanding this model and may provide some insight into YF in humans.

 

There are some aspects of this submission that need to be improved, including a careful editing and proof reading to improve grammar and language use. Additionally, I find the plotting of clinical data as bars graphs and as ratios to baseline problematic, especially since absolute values are referenced in the discussion.  If I read the methods correctly, each of the bars represents a ‘randomly selected’ animal at each day.  How is comparing back to baseline appropriate since the same animals are not being compared?  These are outbred animals and their response to the infection will be different.  Also, scientists at Fiocruz published ‘normal’ clin path values for animals in their squirrel monkey colony (Mem Inst Oswaldo Cruz 2004 Oct;99(6):581-9. Epub 2004 Nov 18. PMID: 15558168). The ‘normal’ range should be included as reference.

 

Specific comments:

 

Title:  Should be ‘hematological’ rather than ‘sematological’.

Line 27:  “in” humans

Line 43-44:  Should this be ‘detection of YFV genome in Ae. albopictus’?

Line 54:  IS the BeH655417 a contemporary isolate or has it been around a while?  Do you know the passage history before the authors received the virus?

Line 62:  What was the acclimatization period for the NHPs?

Line 65:  Should be Saint Louis encephalitis virus

Lines 70-71:  I interpret this to mean staff were on site 24/7 for 30 days to perform the animal observations.  It this correct?

Line 77: ‘randomly selected’ could mean that the same animal was bled repeatedly by happenstance.  Please explain how this was avoided.

Figure 1:  Please provide the limit of detection for the assays plotted here.  This can be added to the plots.

Lines 252-253: ‘for at least’

Line 261:  Was there gross or histologic evidence of hemorrhage?  If so, please mention it here.

Line 262:  I don’t understand what is meant by ‘the white series presented…’

Lines 271-273:  I don’t think DENV infection of NHPs is relevant here.  It is a different virus with a very different disease.

Lines 260-287:  Absolute clinical values are provided here, but the presented data is in ratios.  They are not comparable.  The presented data in the figures should be in absolute values.

Line 291:  ‘normal’ should be ‘baseline’

Line 307: ‘Antigen presentation to the bile duct cell’?  I don’t understand what this means.

Lines 319-321:  As with the comment above, DENV and YFV are different viruses and the models should not be compared.  The diseases in humans are quite different.

Author Response

Thanks for the suggestions. The answers to the questions follow below.

 

The submitted manuscript by Ferreira et al describes clinical and immunologic observation of squirrel monkeys experimentally infected with yellow fever virus (YFV).  These studies are a fist in kind for this model and demonstrate that squirrel monkeys could be a reasonable model for yellow fever (YF) in humans.  Overall, the data provided here are very helpful in understanding this model and may provide some insight into YF in humans.

            There are some aspects of this submission that need to be improved, including a careful editing and proof reading to improve grammar and language use. Additionally, I find the plotting of clinical data as bars graphs and as ratios to baseline problematic, especially since absolute values are referenced in the discussion.  If I read the methods correctly, each of the bars represents a ‘randomly selected’ animal at each day.  How is comparing back to baseline appropriate since the same animals are not being compared?  These are outbred animals and their response to the infection will be different.  Also, scientists at Fiocruz published ‘normal’ clin path values for animals in their squirrel monkey colony (Mem Inst Oswaldo Cruz 2004 Oct;99(6):581-9. Epub 2004 Nov 18. PMID: 15558168). The ‘normal’ range should be included as reference.

 

R.:  The absolute values cited in the discussion referred to humans. To improve understanding we have excluded it from the text. Blood samples were collected before and after infection. For comparison, data obtained before and after infection of the same animal were considered.

We adopted bar graphs because we believe that this is the best way to present our data. The data are easy to understand.

 

Specific comments:

 

Title:  Should be ‘hematological’ rather than ‘sematological’.

 

R.: Thank you. The text was corrected.

 

Line 27: “in” humans

 

R.: Thank you. The text was corrected.

 

Line 43-44:  Should this be ‘detection of YFV genome in Ae. albopictus’?

 

R.: Thank you. The text was corrected.

 

Line 54:  IS the BeH655417 a contemporary isolate or has it been around a while?  Do you know the passage history before the authors received the virus?

 

R.: The BeH655417 sample was isolated (one passage) from a human case of severe YF from the State of Roraima - Brazil. It was used with a single passage, and an additional passage to amplify was done.

 

Line 62:  What was the acclimatization period for the NHPs?

 

R.: The acclimatization of all animals was of 2 months before the experiment.

 

Line 65:  Should be Saint Louis encephalitis virus

 

R.: Thank you. The text was corrected as needed.

 

Lines 70-71:  I interpret this to mean staff were on site 24/7 for 30 days to perform the animal observations.  It this correct?

 

R.: Yes, survived infected and control animals were observed since 24/7 for 30 days.

 

Line 77: ‘randomly selected’ could mean that the same animal was bled repeatedly by happenstance.  Please explain how this was avoided.

R.: No. The animals were randomly selected in the first seven days and 10, 20 and 30 dpi. Then, the selected animal was anesthetized, bled and later sacrificed for tissue collection. Please, see the published study::

 

Ferreira MS, Júnior PSB, Cerqueira VD, Rivero GRC, Júnior CAO, Castro PHG, Silva GAD, Silva WBD, Imbeloni AA, Sousa JR, Araújo APS, Silva FAE, Tesh RB, Quaresma JAS, Vasconcelos PFDC. Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp.) I: gross anatomical and histopathological findings in organs at necropsy. Mem Inst Oswaldo Cruz. 2020 Nov 9;115:e190501. doi: 10.1590/0074-02760190501. PMID: 33174908; PMCID: PMC7651848.

 

Figure 1:  Please provide the limit of detection for the assays plotted here.  This can be added to the plots.

 

R.: Thank you very much. The figure has been corrected as necessary.

 

Lines 252-253: ‘for at least’

 

R.: Thank you. The text was corrected.

 

Line 261:  Was there gross or histologic evidence of hemorrhage?  If so, please mention it here.

 

R.: The text was excluded from the paper since they were previously published (see Ferreira et al., 2020. Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp.) I: gross anatomical and histopathological findings in organs at necropsy. Mem Inst Oswaldo Cruz. 2020 Nov 9;115:e190501. doi: 10.1590/0074-02760190501. PMID: 33174908; PMCID: PMC7651848.)

 

Line 262:  I don’t understand what is meant by ‘the white series presented…’

 

R.: Thank you. The text was corrected as needed.

 

Lines 271-273:  I don’t think DENV infection of NHPs is relevant here.  It is a different virus with a very different disease.

 

R.: Thank you very much. The text was excluded from the paper.

 

Lines 260-287:  Absolute clinical values are provided here, but the presented data is in ratios.  They are not comparable.  The presented data in the figures should be in absolute values.

 

R.: Thank you very much. The text was excluded from the paper.

 

Line 291:  ‘normal’ should be ‘baseline’

 

R.: Thank you. The text was corrected as needed.

 

Line 307: ‘Antigen presentation to the bile duct cell’?  I don’t understand what this means.

 

R.: Thank you very much. The text was excluded from the paper.

 

Lines 319-321:  As with the comment above, DENV and YFV are different viruses and the models should not be compared.  The diseases in humans are quite different.

 

R.: Thank you very much. The text was excluded from the paper.

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Adjustments to the manuscript were completed as suggested.