A STAT1-Knockout Mouse Model for Chapare Virus Infection and Pathogenesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Monticelli et al. describes a novel small animal model of Chapare virus. The study finds that wild-type Chapare virus is partially lethal in STAT-1 knockout mice, and that passaging the virus through mice results in a fully lethal strain. The data show that there is a biphasic disease course with accompanying histopathology, dysregulated cytokine expression, and neurological infection. The development of a small animal model for Chapare virus is an important advance in the field, as the only other small animal model is in Strain 13 guinea pigs, which are difficult to obtain.  Only minor comments are listed below.

 

Minor comments:

1)  Figure 2--please define what is meant by pfu/mL in the tissue viral loads. Is there a way to express this in pfu/g?

2) Figure 3--how many mice per group?

3)  The authors should discuss if different routes of infection or infectious doses could alter pathology

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

“A STAT1 Knockout Mouse Model for Chapare Virus Infection 2 and Pathogenesis” by Monticelli et al. is a comprehensive well written and designed study to develop a mouse model for Chapare hemorrhagic fever (CHHF). This report presents a substantial and well-executed body of work evaluating infection of STAT1 knockout mice with a human patient–derived CHAPV isolate, along with the generation of a mouse-adapted CHAPV (ma-CHAPV) that produces more uniform lethal disease following three sequential passages using brain homogenates in STAT1 knockout mice. The study is timely and represents a meaningful contribution to the literature. The authors provide a level of methodological detail that is not always included in model development studies, and this transparency is particularly valuable. The thorough reporting of experimental design and procedures enhances the utility of the work and will greatly benefit the field.

Based on the data presented, a logical next step would be to evaluate ma-CHAPV in immunocompetent mouse strains. If lethality is maintained, this could broaden the model’s applicability and provide additional options for investigators. However, given the scope and depth of the current study, this is better framed not as a limitation, but as a natural and important direction for future work building on a strong foundation.

Overall, the study has many notable strengths. The specific comments below are offered to further strengthening the manuscript. Careful consideration of these points would enhance the clarity, impact, and long-term value of the work as efforts continue to advance model development for CHAPV and other pathogenic arenaviruses.

Specific comments:

-Line 92 – A space is needed between “strain 13” and “guinea pig.”

-Line 111 – Please clarify what the delta (Δ) symbol preceding “FBS” denotes.

-Lines 115–123 – Add GenBank accession numbers for strain 810419 or refer readers to Table 2.

-Line 126 – A wide age range of mice was used. Please clarify whether different ages were used for specific study components (e.g., serial euthanasia vs. terminal/outcome studies), or whether ages were mixed and proportioned equally within each experiment. When reporting individual studies, specify the ages used.

-Line 144 – Was 1000 PFU the target dose or the confirmed dose based on back-titration? Were inocula back-titered for this work? Please clarify.

-Lines 158–164 – The description of how mice were weighed is not entirely clear. While the general concept is understandable, the use of two pans, group sizes, and number of mice per pan is difficult to follow. Please revise for clarity.

-Line 170 – Define “severe” weight loss as used here (e.g., “> X% loss”).

-Line 177 – Was sequencing performed on viruses from each passage, particularly given that lethality increased with passage number and then diminished?

-Lines 247–248 – Specify whether the assay was designed to detect mRNA/cRNA or vRNA.

-Line 294 (Table 2) – Presumably wt-CHAPV refers to strain 810419; please make this explicit in the table or legend. Consider expanding the legend to provide additional clarification overall, including a brief description of ma-CHAPV (e.g., ma-CHAPV, 3rd passage of brain homogenates from STAT-1 KO mice with clinical signs, inoculated into naïve STAT-1 KO mice).

-Figure 1 – The weight and clinical score graphs display large error bars. As presented, it is unclear whether this reflects substantial variation between the two independent studies. The text should state whether such variation was observed. It would be helpful to revisit data visualization to provide greater granularity and clarify what is driving the variation.

-Line 360 – Again, please define “severe” weight loss (e.g., “> X% loss”).

-Line 361 – Clinical signs should be described in greater detail. Please provide examples of the “numerous neurological signs” observed (and use “signs” rather than “symptoms” when referring to mice).

-Figure 2 – It appears that colors are intended to represent different dpi and are consistent across panels A–F, but not in G or I. Please verify color consistency. The inclusion of individual data points is very helpful.

-Anatomic Pathology section – Consider dividing this into two sections: one focused on non-CNS findings and one dedicated to CNS findings. The section contains substantial valuable information but is lengthy; dividing it would improve readability and allow readers to focus on areas of interest.

-Anatomic Pathology section – It is not always clear whether findings were observed uniformly across animals or only in a subset. Please clarify where appropriate.

-Figure 3 – Very effective presentation of the data.

-Figures 4 & 5 – The panels are quite small. Please modify the layout or size so that images are more easily interpreted.

-Line 513 – Similar to the earlier comment, was sequencing performed on viruses from different passage numbers?

-Figure 6C – clinical score data is very interesting but hard to read in current graphing approach, please consider alternatives.

-Line 540 – Again, define “severe” weight loss (e.g., “> X% loss”).

-Line 541 – As noted earlier, please provide more detailed descriptions of clinical signs. Were these comparable in nature and severity to those observed with wt-CHAPV? Were there any qualitative differences?

-Figure 8 – individual data with mean/median line would be more helpful to visualize here. Please clarify if values are mean and consider presenting individual data as well, like figure 2.

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes the development of a partially lethal STAT1-/- mouse model for Chapare virus infection, including characterization of a mouse-adapted variant. The establishment of a small-animal model for CHAPV is an important advance for pre-clinical and translational work. Strengthening the manuscript includes more comments on the individual mouse heterogeneity in the infection response, inclusion of more detailed methods behind the sequencing analysis of maCHAPV, and explicit discussion of CNS-dominant pathology relative to human disease, and clarification of statistical and experimental design considerations.

Major comments

1. Can you comment on the choice to weigh whole groups of mice in a single pan? Is this to assess a single cage as an “experimental block”? Please clarify whether the cage was treated as the experimental unit in statistical analyses throughout the manuscript.
2. The plotted weight curves beyond day 36 indicate survivor bias. Do plotted values reflect only surviving animals at each time point? Individual weight trajectories would help determine whether distinct phenotypes (lethal versus non-lethal infection) exist.
3. It is also important to confirm whether surviving animals were productively infected. Were all mice assessed for seroconversion or neutralizing antibodies? Were antibody titers measured in animals surviving beyond day 35? The authors include there are neutralizing antibodies made exceptionally early for some animals (days 0-6 post-challenge), which seems biologically implausible for SPF mice as these same mice may not yet be viremic, but there is no presented data for after day 35.
4. Does the reported NP mutation from maCHAPV that is derived from p3 revert in p4/5? Also, is the absence of changes in other ORFs or in the L segment based on the consensus sequence? Can the authors provide additional information on how variants were called, especially on the frequency threshold and depth.
5. maCHAPV was evaluated only in STAT1-/- mice. Was infection tested in immunocompetent mice? If not, this limitation should be emphasized beyond the discussion in lines 735-740. Adaptation in an interferon-deficient background does not necessarily demonstrate broader host adaptation, and this distinction is important for translational relevance in drug/vaccine testing and the language used throughout this manuscript.
6. The model appears to cause CNS-predominant disease, whereas human infection with CHAPV is primarily described as a hemorrhagic fever syndrome with some reports of neurological involvement in the 2019 outbreak. It would be important to clarify whether there is evidence of vascular dysfunction, platelet depletion, coagulopathy, or hepatic injury during the early phase in this model. The inclusion of microscopic findings in the liver and spleen prior to day 12 are helpful, but additional systemic measures (liver enzymes, CBC data) could strengthen our understanding of the model and relevance to clinical disease course.
7. How do the cytokines and chemokines vary from an uninfected animal? Interpretation of cytokine and chemokine responses requires comparison to uninfected STAT1-/- controls, as baseline inflammatory signaling may differ substantially from immunocompetent animals. Inclusion may provide greater resolution of the biphasic disease course and the reported significant increase in LIX at day 8 post-infection.
8. Both male and female mice were reportedly used. Were equal numbers were included across all experiments and were analyses were stratified by sex? Were there any differences in survival, weight loss, viral burden, or cytokine responses? This should be addressed explicitly.

Minor comments

1. Please indicate whether pathology scoring was performed in a blinded manner and the number of folks who professionally review the pathology, and if a veterinary pathologist was consulted.
2. The term “serial sampling” may imply repeated sampling from the same animal. If samples were collected from independent mice at each time point, clearer terminology would avoid confusion.
3. Were other routes of infection attempted? Other arenaviruses, notably LCMV, have vastly different disease presentations in mice dependent on route (ip, iv, in/it, or ic) of infection.

 

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have clarified all points raised in the revised manuscript, or sufficiently explained their terminology in the response to review.