Andrographolide Suppresses Influenza A Virus-Induced Pyroptosis via PI3K/AKT-Mediated Caspase-3/GSDME Inactivation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the role of andrographolide in mitigating IAV-induced inflammation.  This study explores its mechanisms, revealing a novel host-directed approach that activates the PI3K/AKT pathway, suppressing caspase-3/GSDME-dependent pyroptosis and excessive inflammation in severe influenza, offering new therapeutic avenues. However, there are several issues that must be addressed carefully.

Comments

1. The authors should include a design strategy in the introduction section.
2. In KEGG pathway analysis, the mechanism of the PI3K/AKT signaling pathway should be illustrated.
3. Although the in-vitro and in-vivo analyses have been carried out, however, in case of KEGG pathway analysis, in-silico studies i.e., molecular docking analysis between the andrographolide and Influenza A virus would strengthen the mechanistic interpretation.
4. The conclusion section should be updated by adding key findings and future prospects.

Other Minor Remarks:

The article must be carefully proofread to avoid the similar errors:

• Line 49, 50, use of word “possesses” is not suitable for the sentence.
• Throughout the manuscript, space between number and unit needs to be checked i.e., 8µM should be written as 8 µM and 4^oC should be written as 4 ^o
• Generally, all the references are appropriate. However, in references, formatting inconsistencies have been observed which should be as per journal's format and guidelines.

Author Response

Reviewer 1:

Comment 1: This manuscript investigates the role of andrographolide in mitigating IAV-induced inflammation. This study explores its mechanisms, revealing a novel host-directed approach that activates the PI3K/AKT pathway, suppressing caspase-3/GSDME-dependent pyroptosis and excessive inflammation in severe influenza, offering new therapeutic avenues. However, there are several issues that must be addressed carefully. The authors should include a design strategy in the introduction section.

Response：We agree with the reviewer and have added a concise description of the research design in the revised manuscript，as shown below (lines 73-79).

"To systematically decipher the mechanism underlying the protective effect of andrographolide against IAV, we designed a sequential investigational strategy: a) confirming its protective efficacy in vivo and in vitro; b) employing unbiased RNA sequencing to screen for key dysregulated host pathways; c) validating the activation of the prioritized PI3K/AKT signaling pathway and its functional consequence on suppressing GSDME-mediated pyroptosis; and d) definitively establishing the pathway's necessity using combined pharmacological and genetic inhibition approaches. "

Comment 2: In KEGG pathway analysis, the mechanism of the PI3K/AKT signaling pathway should be illustrated.

Response：Thank you for this valuable suggestion. We have enhanced the manuscript to better illustrate the biological significance of the PI3K/AKT signaling pathway following its identification in our KEGG analysis. In the revised Results section, we have added a bridging paragraph (lines 340-347) that provides mechanistic context.

Comment 3: Although the in-vitro and in-vivo analyses have been carried out, however, in case of KEGG pathway analysis, in-silico studies i.e., molecular docking analysis between the andrographolide and Influenza A virus would strengthen the mechanistic interpretation.

Response：We appreciate the reviewer for his constructive suggestions. In response, we have conducted molecular docking studies to evaluate potential interactions between andrographolide and key influenza A virus proteins. The results indicated that andrographolide forms only moderate binding interactions​ with these viral targets, with no evidence of high-affinity or stable binding (Figure R1). This computational simulation result aligns closely with the key experimental findings presented in Figure 2 of the Results section. which demonstrate that andrographolide treatment does not significantly inhibit viral replication in vitro or in vivo.

 Figure R1. Molecular docking diagram of andrographolide with IAV PB2, PB1, PA, HA, NP, NA, M1, and M2 protein.

The predicted binding energies of the small molecule andrographolide with the binding pockets of eight influenza virus protein receptors are as follows: PB2: -7.518 kcal/mol; PB1: -7.776 kcal/mol; PA: -8.416 kcal/mol; HA: -6.907 kcal/mol; NP: -7.233 kcal/mol; NA: -6.839 kcal/mol; M1: -5.977 kcal/mol; M2: -8.672 kcal/mol.

Comment 4: The conclusion section should be updated by adding key findings and future prospects.

Response：Thank you for this suggestion. We have rewritten the Conclusion section (lines 500-506，lines 510-514)​ to concisely summarize the key findings of our study and to propose specific directions for future research.

Other Minor Remarks:

Comment 5: The article must be carefully proofread to avoid the similar errors: Line 49, 50, use of word “possesses” is not suitable for the sentence. Throughout the manuscript, space between number and unit needs to be checked i.e., 8µM should be written as 8 µM and 4^oC should be written as 4 ^oC.

Response：Thank you for your valuable reminder. We have carefully proofread the entire article to ensure that similar errors are avoided and the quality of the submission is maintained.

The word "possesses" has been replaced with more suitable phrasing "exhibits" in the revised manuscript (line 51)​.

The entire manuscript has been carefully checked. All instances (e.g., "8µM", "4°C ") have been corrected to include the proper space ("8 µM", "4 °C") (line 92, line 101, line 156, line 158, line 165, line 169, line 189, and line 193)​.

Comment 6: Generally, all the references are appropriate. However, in references, formatting inconsistencies have been observed which should be as per journal's format and guidelines.

Response：The reference list has been comprehensively reviewed and reformatted to ensure strict compliance with the journal's specific style guidelines (Ref 19, Ref 30, Ref 32, Ref 33, Ref 34, Ref 35, Ref 36, Ref 51, Ref 53, Ref 54, and Ref 55).

We believe these corrections have enhanced the manuscript's clarity and adherence to publication standards. Thank you for your thorough review.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The protective function of andrographolide in influenza A virus infection is examined in this publication, which also suggests a host directed mechanism that involves caspase 3/GSDME mediated pyroptosis inhibition and PI3K/AKT pathway activation. The work tackles a significant clinical issue because a significant factor influencing influenza severity is not just viral burden but also excessive inflammation. To provide a logical mechanistic framework, the authors integrate transcriptome research, in vitro cell models, in vivo mice investigations, and pharmacological inhibition. Without directly influencing viral replication, andrographolide consistently improves longevity, lowers lung pathology, and inhibits the generation of inflammatory cytokines. The discovery of GSDME dependent pyroptosis as a downstream target is novel and fits in nicely with the growing body of research on viral infections and inflammatory cell death.

Considering these advantages, a number of problems restrict the conclusions mechanistic certainty and generalizability. The causal relationship between PI3K/AKT activation and reduction of caspase 3/GSDME cleavage is weakened when pathway dependency is established with a single AKT inhibitor without additional genetic methods. Further supporting evidence for pyroptosis would come from functional or morphological confirmation of this type of cell death, which is mostly inferred from protein breakage events. The RNA seq analysis might benefit from a more thorough justification of route prioritizing and more transparent statistical reporting, and the use of a single influenza strain and male mice further limits wider applicability. The study's rigor and significance would be greatly increased if these issues were addressed, or if they were more clearly acknowledged as limitations.

 

Author Response

Reviewer 2：

Comment 1:  The protective function of andrographolide in influenza A virus infection is examined in this publication, which also suggests a host directed mechanism that involves caspase 3/GSDME mediated pyroptosis inhibition and PI3K/AKT pathway activation. The work tackles a significant clinical issue because a significant factor influencing influenza severity is not just viral burden but also excessive inflammation. To provide a logical mechanistic framework, the authors integrate transcriptome research, in vitro cell models, in mice investigations, and pharmacological inhibition. Without directly influencing viral replication, andrographolide consistently improves longevity, lowers lung pathology, and inhibits the generation of inflammatory cytokines. The discovery of GSDME dependent pyroptosis as a downstream target is novel and fits in nicely with the growing body of research on viral infections and inflammatory cell death.

Response：We sincerely thank the reviewer for the positive and insightful evaluation of our work, and for recognizing its novelty and significance in addressing host-directed therapy for severe influenza. We are particularly grateful for the reviewer's acknowledgment that our integrated approach (combining transcriptomics, in vitro and in vivo models, and pharmacological inhibition) provides a logical mechanistic framework, and that the discovery of GSDME-dependent pyroptosis as a target is a novel contribution to the field.

Comment 2: Considering these advantages, a number of problems restrict the conclusions mechanistic certainty and generalizability. The causal relationship between PI3K/AKT activation and reduction of caspase 3/GSDME cleavage is weakened when pathway dependency is established with a single AKT inhibitor without additional genetic methods.

Response：We agree with the reviewer’s concern. Following your suggestions, we performed AKT1 knockdown using siRNA.

The detailed procedures for siRNA experiments are provided in the Materials and Methods section (lines 228-234). Experimental results are presented in the Results section (lines 392-401).

"The siRNA sequence with the highest knockdown efficiency (siAKT1-1) was selected for subsequent experiments (Figure 4F). AKT1 knockdown not only reversed the andrographolide-mediated improvement in the survival of infected cells (Figure 4G) but also abolished the reduction in intracellular levels of IL-1β, IL-6, and TNF-α induced by andrographolide (Figure 4H). Furthermore, the inhibitory effect of andrographolide on virus-induced LDH release and its alleviation of cell death, as indicated by PI staining, were both abrogated upon AKT1 knockdown (Figure 4I and 4J). Western blot analysis further confirmed that the suppression of pyroptosis-related proteins, GSDME-N and cleaved caspase-3, by andrographolide was also dependent on AKT1 activity (Figure 4K). "

Collectively, these new genetic data provide direct and complementary evidence to support our previous pharmacological inhibition findings, robustly confirming that the PI3K/AKT signaling axis is essential for mediating the protective effects of andrographolide against IAV-induced pyroptosis.

Comment 3: Further supporting evidence for pyroptosis would come from functional or morphological confirmation of this type of cell death, which is mostly inferred from protein breakage events.

Response：We have performed complementary functional and morphological assays, including a key rescue experiment to test the dependency of the observed anti-pyroptotic effect on PI3K/AKT pathway activation (Figure S1A, Figure S1B, Figure 4I and 4J). These results from these new experiments strengthened the notion that andrographolide suppresses Caspase-3/GSDME-dependent pyroptosis via PI3K/AKT pathway activation. Detailed procedures are described in the Materials and Methods section (lines 219-227).

Comment 4: The RNA-seq analysis might benefit from a more thorough justification of route prioritizing and more transparent statistical reporting.

Response：We thank the reviewer for this suggestion to enhance the clarity and rigor of our bioinformatics reporting, and have revised the manuscript accordingly. In the revised Materials and Methods section, we have provided a more detailed description of the statistical analysis (lines 141-149). In the revised Results section, we have added a dedicated explanatory paragraph to justify our focus on the PI3K/AKT pathway (lines 340-347).

We hope these revisions address the request for clarity in statistical reporting and provide a well-supported rationale for our mechanistic focus, thereby strengthening the overall narrative of the manuscript.

Comment 5: The use of a single influenza strain and male mice further limits wider applicability. The study's rigor and significance would be greatly increased if these issues were addressed, or if they were more clearly acknowledged as limitations.

Response：We agree with the reviewer that these are important considerations for the generalizability of our findings (lines 489-493). The text explicitly acknowledges:

" The conclusions are based primarily on experiments from male BALB/c mice infected by a single mouse-adapted influenza strain (A/Puerto Rico/8/34, H1N1). Thus, whether the protective mechanism could be generalized to other influenza viral strains or to female hosts requires further investigation."

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

• The manuscript reports animal group sizes (e.g., survival n=13–21; BALF n=6; WB n=3) but does not state randomization or blinding for outcome assessment .
• Andrographolide dose is 150 mg/kg (oral gavage), but justification is not described humane endpoints criteria for survival studies.
• MK-2206 reversal is a strong piece of evidence; however, MK-2206 can have off-target or systemic effects. Complement pharmacologic inhibition with one of: genetic knockdown in A549 cells and use of an additional PI3K/AKT pathway inhibitor (or PI3K inhibitor) to show convergent evidence
• Include cell-type–specific markers or flow cytometry of lung immune populations or at least qPCR of interferon-stimulated genes to characterize immune modulation.
• Some statements read as definitive but the intermediate molecular steps are not directly demonstrated.
  
  

 

Author Response

Reviewer 3:

Comment 1: The manuscript reports animal group sizes (e.g., survival n=13–21; BALF n=6; WB n=3) but does not state randomization or blinding for outcome assessment.

Response：We thank the reviewer for this important methodological point. We have added a detailed description of the randomization and blinding procedures in the revised Materials and Methods section (lines 106-113).

Comment 2: Andrographolide dose is 150 mg/kg (oral gavage), but justification is not described humane endpoints criteria for survival studies.

Response：The 150 mg/kg oral dose of andrographolide was chosen based on our preliminary dose-ranging studies, where it demonstrated the most effective survival benefit in our IAV model (Figure R2 and R3). This dose is also consistent with or lower than efficacious doses reported in other murine studies (10.1016/j.micinf.2017.08.009, 10.1248/bpb.32.1385.).

Figure R2. Mice were intraperitoneally administered andrographolide at varying doses after infection with IAV.

 

Figure R3. Mice were intragastrically administered andrographolide at varying doses after infection with IAV.

We strictly followed predefined humane endpoints to minimize animal suffering. Euthanasia was performed if mice exhibited: ≥25% body weight loss, severe lethargy/inability to feed, severe respiratory distress, or irreversible moribundity. These criteria are now explicitly stated in the revised Materials and Methods section (lines 119-122).

Comment 3: MK-2206 reversal is a strong piece of evidence; however, MK-2206 can have off-target or systemic effects. Complement pharmacologic inhibition with one of: genetic knockdown in A549 cells and use of an additional PI3K/AKT pathway inhibitor (or PI3K inhibitor) to show convergent evidence.

Response：We have followed the suggestion and performed AKT1 gene knockdown experiments using specific siRNA (siAKT1) in A549 cells. We have incorporated these data into the revised Results sections (lines 392-401), which significantly strengthens the mechanistic basis of our conclusions.

Comment 4: Include cell-type–specific markers or flow cytometry of lung immune populations or at least qPCR of interferon-stimulated genes to characterize immune modulation.

Response：To characterize immune modulation, we performed multiplexed immunofluorescence on lung tissue sections using antibodies against the macrophage marker CD68​ and the neutrophil marker Ly6G (Figure 1G).

IAV infection induced substantial infiltration of both CD68 macrophages and Ly6G⁺ neutrophils into lung tissue indicating that the lung tissue is undergoing infection. Andrographolide treatment significantly reduced this infiltration. Descriptions have been added to the Materials and Methods (lines 209-218) and Results sections (lines 257-259).

Comment 5: Some statements read as definitive but the intermediate molecular steps are not directly demonstrated.

Response：We thank the reviewer for the pertinent comment. We acknowledge that some of the original claims regarding the intermediate mechanistic steps were overly definitive. In response, we have revised the manuscript to temper the language and, more importantly, have strengthened the mechanistic evidence with the following additions:

Justification for focusing on this pathway:​ In the Results section (lines 340-347), we have provided a clearer rationale for prioritizing the PI3K/AKT signaling pathway based on the RNA-seq data, detailing the reasoning behind its selection.

Direct genetic evidence:​ New data from AKT1 knockdown experiments have been included (lines 392-401). This provides key genetic evidence that the protective effects of andrographolide on cell death, inflammation, and pyroptosis markers are dependent on the PI3K/AKT signaling pathway, thereby directly linking the compound's action to this signaling pathway.

Enhanced methodological transparency:​ We have expanded the description of the RNA-seq analysis methods (lines 141-149) to provide a more detailed and reproducible account of the alignment, assembly, quantification, and differential expression analysis workflow.

In summary, these revisions—ranging from clarifying the rationale and providing genetic validation to enhancing methodological transparency—strengthen the chain of evidence. We have also revised the Discussion section (lines 483-485) to use more cautious language when inferring direct mechanistic links. We believe the revised manuscript now more accurately presents the strength of our conclusions.

 

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript is clearly written and describes a large set of experimental data, and the results obtained, in most cases, do not raise doubts. Although this work largely repeats the following article [10.1016/j.micinf.2017.08.009], it can be published in Biomedicines after taking into account the reviewer's suggestions.

1. Please, comment on the lack of activity of andrographolide in your experiments and the presence of activity against a number of IAV strains in vitro and in vivo in the following paper [10.1248/bpb.32.1385].

Also, discuss the following paper:

1. Li G, Tian T, Wang L, et al. Andrographolide Improves Influenza A virus induced Lung Epithelial Cells Injury via Regulating p38 and ERK Phosphorylation[J]. Chin J Virol, 2024, 40(3):476-483;
2. https://doi.org/10.2147/DDDT.S445788;
3. 10.1016/j.micinf.2017.08.009.
4. Lines 49-51: «Furthermore, andrographolide possesses demonstrated antibacterial, anti-inflammatory, and antitumor properties [30,31].»

Ref. 31 is about vernonioside V, not andrographolide. In Ref. 30 there is nothing about antibacterial and anti-inflammatory properties of andrographolide (only antitumor). Revise accordingly.

4. Lines 51-53: «Previous studies have demonstrated that andrographolide exerts anti-IAV effects through mechanisms that modulate Retinoic acid-inducible gene I(RIG-I)-related signaling pathways or suppress NF-κB activity[32,33].»

The second mechanism is not associated with anti-IAV effects. Revise

5. Please, clearly state in the materials and methods how many mice were in each group (the number of mice is not clearly shown in the caption to Figure 1)
6. In 2.10 Plaque reduction assay, describe in detail when the cells were treated with andrographolide. Please describe in more detail how the infection was carried out (with lung homogenates?)
7. Revised Figure E, right central box (in particular, GSDME spots). At the moment, it looks like andrographolide does not reduce GSDME-N levels.

Typos

1. Lines 158-159: … at 450 nm using a micro-158 plate reader (Epoch, BioTek).
2. Ref 15. Cite 10.1007/s13238-020-00768-w, not correction
3. Figure 4: «(A, B) Mice were pretreated with the AKT inhibitor MK-2206 (80 mg/kg, i.p.) or vehicle for 4 days before and 4 days after IAV infection. Survival rates (A) and body weight 404 changes (%) (B) were monitored for 15 days. (n = 9-10).» Weren't the mice treated with a combination?

Author Response

Reviewer 4：

Comment 1: This manuscript is clearly written and describes a large set of experimental data, and the results obtained, in most cases, do not raise doubts. Although this work largely repeats the following article [10.1016/j.micinf.2017.08.009], it can be published in Biomedicines after taking into account the reviewer's suggestions.

1.Please, comment on the lack of activity of andrographolide in your experiments and the presence of activity against a number of IAV strains in vitro and in vivo in the following paper [10.1248/bpb.32.1385].

Also, discuss the following paper:

1.Li G, Tian T, Wang L, et al. Andrographolide Improves Influenza A virus induced Lung Epithelial Cells Injury via Regulating p38 and ERK Phosphorylation[J]. Chin J Virol, 2024, 40(3):476-483;

2.https://doi.org/10.2147/DDDT.S445788;

3.10.1016/j.micinf.2017.08.009.

Response：We sincerely appreciate your insightful comments and have addressed the points as follows:

a). Regarding Innovation Relative to Ding et al. (Microbes Infect，2017). We fully understand the reviewer's concern on this point and would like to take this opportunity to further clarify. Our work aims to elucidate a previously unreported mechanism. While the seminal study by Ding et al. first demonstrated protection via inhibition of the NF-κB and JAK-STAT pathways, our study reports for the first time that andrographolide activates the PI3K/AKT pathway, specifically suppressing caspase-3/GSDME-mediated pyroptosis. This advances the mechanistic understanding from modulating cytokine expression to inhibiting a key executive process of inflammatory cell death. We believe this finding represents a significant and complementary contribution to the existing knowledge.

1. b) Comment on the Discrepancy in Reports of Antiviral Activity

Thank you for highlighting this important discrepancy. There are conflicting reports on the antiviral activity of andrographolide. Chen et al. (Biol Pharm Bull，2009) focused on direct antiviral effects, whereas Ding et al. (Microbes Infect，2017) and our study support a host-directed mechanism, as no direct antiviral activity was observed in cellular models (Figure 2). We fully endorse the consensus that andrographolide's primary benefit in influenza is host immunomodulation. Our work strengthens this view by providing novel mechanistic evidence through the PI3K/AKT/pyroptosis axis.

c)Discussion of the Suggested Literature

Li G et al. (Chin J Virol, 2024): This study shows that andrographolide alleviates IAV-induced lung epithelial injury by regulating p38 and ERK phosphorylation, highlighting the important role of MAPK signaling. This work complements our finding of the PI3K/AKT pathway, together suggesting that andrographolide may exert protection by modulating multiple host signaling pathways, further supporting its multi-target nature. We will cite this study in the Discussion to reflect this broader mechanistic context.

Yu et al. (10.2147/DDDT.S445788，2024): This study reports inhibition of the NLRP3 inflammasome by andrographolide in a Chronic Obstructive Pulmonary Disease (COPD) model, also focusing on regulating a specific inflammatory cell death pathway (pyroptosis). Although the disease model differs, its core finding highly aligns with the theme of our research, collectively enriching the understanding of andrographolide's action in suppressing pyroptosis.

Ding et al. (Microbes Infect，2017): As mentioned above, we have adequately discussed and acknowledged this foundational work in the manuscript, clearly delineating the new mechanistic dimension identified in our study.

Comment 2: Lines 49-51: «Furthermore, andrographolide possesses demonstrated antibacterial, anti-inflammatory, and antitumor properties [30,31].

Ref. 31 is about vernonioside V, not andrographolide. In Ref. 30 there is nothing about antibacterial and anti-inflammatory properties of andrographolide (only antitumor). Revise accordingly.

Response：We sincerely thank the reviewer for pointing out these citation inaccuracies. The incorrect Reference 31 has been removed, and directly relevant references supporting the antibacterial and anti-inflammatory properties of andrographolide have been added to replace the previous unsupported reference (Ref. 30, Ref. 32, Ref. 33, Ref. 34, Ref. 35, Ref. 36).

Comment 3: Lines 51-53: «Previous studies have demonstrated that andrographolide exerts anti-IAV effects through mechanisms that modulate Retinoic acid-inducible gene I(RIG-I)-related signaling pathways or suppress NF-κB activity [32,33].»The second mechanism is not associated with anti-IAV effects. Revise.

Response：We thank the reviewer for this correction. The statement has been revised. The non-supporting reference regarding NF-κB has been removed.

Comment 4: Please, clearly state in the materials and methods how many mice were in each group (the number of mice is not clearly shown in the caption to Figure 1)

Response：Thank you for highlighting the need for precise reporting of animal group sizes. In the revised manuscript, we have provided comprehensive details on sample numbers in both the Materials and Methods​ section (lines 106-109) and the corresponding Figure legends​ to ensure full transparency (lines 280-281，lines 416-420).

Comment 5: In 2.10 Plaque reduction assay, describe in detail when the cells were treated with andrographolide. Please describe in more detail how the infection was carried out (with lung homogenates?)

Response：We have added more methodological details in the Materials and Methods section (lines 203-205).

Comment 6: Revised Figure E, right central box (in particular, GSDME spots). At the moment, it looks like andrographolide does not reduce GSDME-N levels.

Response：We have repeated the key experiments​ to re-examine the effect of andrographolide on GSDME-N levels. The new results unequivocally show that andrographolide treatment leads to a marked reduction in the level of the GSDME-N fragment compared to the IAV-infected control group (Figure 3E).

Comment 7: Lines 158-159: … at 450 nm using a micro-158 plate reader (Epoch, BioTek).

Response：Thank you for this comment. We have carefully re-examined the relevant descriptions in our manuscript. However, we did not identify the specific issue that was raised (lines 184-186).

Comment 8: Ref 15. Cite 10.1007/s13238-020-00768-w, not correction.

Response：We have corrected it (Ref. 19).

Comment 9: Figure 4: «(A, B) Mice were pretreated with the AKT inhibitor MK-2206 (80 mg/kg, i.p.) or vehicle for 4 days before and 4 days after IAV infection. Survival rates (A) and body weight 404 changes (%) (B) were monitored for 15 days. (n = 9-10).» Weren't the mice treated with a combination?

Response：We sincerely thank the reviewer for raising this point. We have revised the legend for Figure 4A and 4B​ to explicitly state: “Mice were divided into four groups: Control (n = 9), IAV-infected (H1N1, n = 10), IAV-infected + Andrographolide (H1N1+And, n = 10), and IAV-infected + Andrographolide + MK-2206(H1N1+And+MK2206, n = 10). Mice in the inhibitor group were pretreated with MK-2206 (80 mg/kg, i.p.) or vehicle for 4 days before and 4 days after IAV infection. Survival rates (A) and body weight changes (B) were monitored for 15 days.” (lines 416-420)

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the manuscript and can now be accepted for publication.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors took into account the reviewer's suggestions, thank you.