Genetic Diversity and Evolutionary Dynamics of Feline Panleukopenia Virus in China: Phylogenetic Analysis and Substitution Patterns in NS1 and VP2 Proteins

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a relevant topic, namely the genetic diversity of feline panleukopenia virus (FPV/FPLV), with emphasis on NS and VP2 variation and their possible relevance to cross-species transmission. The combination of three newly isolated strains with a broader GenBank-based sequence survey is potentially valuable.

However, in its current form, the manuscript has substantial methodological, interpretative, and language-related weaknesses. The study is largely descriptive, whereas the title, abstract, and discussion imply stronger biological conclusions than the presented data can support. In particular, the claims related to cross-species transmission potential are not adequately demonstrated by the experiments or analyses performed.

Overall, the manuscript may become publishable after major revision, but it currently requires substantial improvement.

 

Major Comments

• The title and central conclusions overstate what the data actually show. The manuscript does not provide functional, structural, receptor-binding, or host range data demonstrating “cross-species transmission potential.” The study mainly identifies sequence variation and mutation frequencies. The authors should substantially tone down these claims throughout the manuscript and rephrase them more cautiously (e.g., “mutations previously reported in isolates from multiple host species” rather than “associated with cross-species transmission”).
• A major part of the manuscript is based on analysis of 157 NS sequences and 947 VP2 sequences retrieved from NCBI/GenBank, but the dataset assembly is not sufficiently described. The manuscript should clearly explain: how sequences were retrieved, inclusion/exclusion criteria, removal of duplicates, vaccine strains, laboratory constructs, partial/low-quality entries, whether host, year, and geographic origin were curated. Without this information, the large-scale sequence analysis is difficult to reproduce and interpret.
• The phylogenetic analysis is potentially useful, but currently too limited and insufficiently interpreted. The authors should clarify: the substitution model used for maximum likelihood analysis, alignment trimming / quality control, whether recombination was assessed, the basis for defining “two FPLV-China groups.” In addition, the phylogenetic results are discussed only superficially. If the manuscript aims to address host adaptation or cross-species transmission, the phylogeny should be biologically interpreted in relation to host species, geography, and lineage structure, not only clustering patterns.
• The manuscript mainly catalogs synonymous and non-synonymous substitutions and their frequencies. This can be useful as descriptive genomic surveillance, but several biological interpretations are too strong for the data presented. These interpretations should be made much more cautiously unless supported by functional assays, structural analysis or host-association statistics.
• The laboratory component (PCR screening, virus isolation, CPE observation, immunofluorescence, partial genome amplification) is acceptable as a basic virological characterization, but it is relatively limited. The experimental data do not support broad biological conclusions regarding altered host range, pathogenicity, replication fitness, or immune escape.
• The Discussion is currently too descriptive and often repeats the Results. It should be strengthened by clearer distinction between observed sequence diversity and demonstrated biological significance, a more critical discussion of study limitations, reduced speculation regarding host adaptation and transmission.

 

I have also some minor comments

1. The manuscript should use consistent terminology (FPV vs FPLV).
2. Expressions such as “as far as I know,” “were founded,” “what more,” “instability of FPLVs” are not appropriate for a scientific manuscript.
3. Some methodological descriptions need correction and standardization (e.g., centrifugation should preferably be reported in × g, not only rpm).
4. Figure legends should be more informative and self-contained.
5. Figure 4 would benefit from improved readability and clearer highlighting of the newly isolated strains.

Comments on the Quality of English Language

The English language requires major editing throughout. There are numerous grammatical, syntactic, and stylistic problems that affect clarity and scientific precision.

Author Response

Responses to Reviewers 1

Dear Reviewers:

Thank you for your letter and give some comments concerning our manuscript entitled “Genetic Divergence and Cross-Species Transmission Potential of Feline Panleukopenia Virus: Insights from NS and VP2 Protein Variations (viruses-4242030)”. Those comments are all valuable and very helpful for revising and improving my paper. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in yellow in the paper-revised. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

 

Responds to the reviewer’s major comments:

Comment 1: “The title and central conclusions overstate what the data actually show. The manuscript does not provide functional, structural, receptor-binding, or host range data demonstrating “cross-species transmission potential.” The study mainly identifies sequence variation and mutation frequencies. The authors should substantially tone down these claims throughout the manuscript and rephrase them more cautiously (e.g., “mutations previously reported in isolates from multiple host species” rather than “associated with cross-species transmission””.

Response 1:

Dear Reviewer, thank you for pointing this out. We agree with this comment and have made the following revisions:

1. We have changed the manuscript title from "Genetic Divergence and Cross-Species Transmission Potential of Feline Panleukopenia Virus: Insights from NS and VP2 Protein Variations" to "Genetic Diversity and Evolutionary Dynamics of Feline Panleukopenia Virus in China: Phylogenetic Analysis and Substitution Patterns in NS and VP2 Proteins."
2. We have toned down several claims throughout the manuscript and rephrased them more cautiously. For example, we have changed "associated with cross-species transmission" to "mutations previously reported in isolates from multiple host species" on page 1, line 13 and page 11, line 283, in the revised manuscript.

Comment 2: A major part of the manuscript is based on analysis of 157 NS sequences and 947 VP2 sequences retrieved from NCBI/GenBank, but the dataset assembly is not sufficiently described. The manuscript should clearly explain: how sequences were retrieved, inclusion/exclusion criteria, removal of duplicates, vaccine strains, laboratory constructs, partial/low-quality entries, whether host, year, and geographic origin were curated. Without this information, the large-scale sequence analysis is difficult to reproduce and interpret.

Response 2:

Dear Reviewer, thank you for this valuable comment. We have changed the text in the section 2.7 in Materials and Methods section (page 4, lines 141-153). The following details are now explicitly described:

1. Sequence Retrieval of NS1

All FPLV NS sequences (n=157) were downloaded from the NCBI/GenBank database using the search terms “Feline panleukopenia virus [title] AND (NS1[Title]” OR “Feline parvovirus [title] AND NS1[Title] " with the filter "sequence length =2007 bp" to exclude partial sequences.

The search was conducted on April 2, 2025, and metadata including accession numbers, host species, collection year, and geographic location were manually curated from GenBank records and associated publications.

Included: Complete or near-complete NS (=2007 bp) sequences with clear host and geographic annotations.

Excluded: (i) sequences short than 2007 bp; ii) sequences lacking host or collection year information for phylogeographic analyses.

2. Sequence Retrieval of VP2

All FPLV NS sequences (n=157) were downloaded from the NCBI/GenBank database

using the search terms “Feline panleukopenia virus [title] AND (VP2[Title] OR capsid[Title])” OR “Feline parvovirus [title] AND (VP2[Title] OR capsid [Title])" with the filter "sequence length =1755 bp" to exclude partial sequences.

The search was conducted on April 2, 2025, and metadata including accession numbers, host species, collection year, and geographic location were manually curated from GenBank records and associated publications.

Included: Complete or near-complete NS (=1755 bp) sequences with clear host and geographic annotations.

Excluded: (i) sequences short than 1755 bp; ii) sequences lacking host or collection year information for phylogeographic analyses.

All curated metadata and accession numbers are now provided in Supplementary Dataset 1 and 2 to ensure full transparency and reproducibility.

We believe these revisions adequately address your concerns. Please let us know if any additional details would be helpful.

Comment 3: The phylogenetic analysis is potentially useful, but currently too limited and insufficiently interpreted. The authors should clarify: the substitution model used for maximum likelihood analysis, alignment trimming / quality control, whether recombination was assessed, the basis for defining “two FPLV-China groups.” In addition, the phylogenetic results are discussed only superficially. If the manuscript aims to address host adaptation or cross-species transmission, the phylogeny should be biologically interpreted in relation to host species, geography, and lineage structure, not only clustering patterns.

Response 3:

Dear Reviewer, thank you for this constructive feedback on our phylogenetic analysis. We agree that the phylogenetic analysis requires more rigorous interpretation and biological contextualization. We have now added the following details to the revised manuscript:

1. The phylogenetic tree constructed in this study was based on three isolates from our laboratory and 104 most related FPLV complete genome or nearly full-length complete genome (>4269 bp). The tree clearly illustrates the close relationship between our three FPLV isolates and the other 104 parvovirus genomes. All the parvovirus clustered separately according to parvovirus type, host species, and geographical location, rather than reflecting cross-species transmission of FPLVs or other parvoviruses. The designation of “two FPLV-China group” was based on the phylogenetic analysis performed in this study combined with the geographical location information of FPLV isolates. FPLV isolates in the FPLV-China group within Cluster I primarily from central/southern China, whereas those in the other FPLV-China group within Cluster II were from northeastern and northwestern regions of China.

2.The host species of FPLV isolates in one FPLV-China group (cluster I) are diverse, including the common host (Felis catus) as well as other felids (lion, cheetah, jaguar, Panthera leo, giant panda, Ailurus fulgens, Ailuropoda melanoleuca, and Panthera tigris), whereas the host species in the other FPLV-China group consist primarily of the common host (Felis catus). Whether this pattern involves cross-species transmission remains unknown.

3.Lineage structure: Cluster I shares a common ancestor with the prototype strain (M38246.1) but forms a distinct sub-lineage, while cluster II represents a separate lineage with closer affinity to older Chinese FPLV isolates.

We have added a narrative in the Discussion section (line 294-299, page 11, in the revised manuscript) to discuss the difference in host species and geographical characteristics between the two clusters, rather than merely describing clustering patterns.

Comment 4: The manuscript mainly catalogs synonymous and non-synonymous substitutions and their frequencies. This can be useful as descriptive genomic surveillance, but several biological interpretations are too strong for the data presented. These interpretations should be made much more cautiously unless supported by functional assays, structural analysis or host-association statistics.

Response 4:

We sincerely thank the reviewer for this constructive feedback.

1.We agree that our manuscript primarily catalogs synonymous and non-synonymous substitutions and their frequencies, but this study is the first to systematically examine synonymous and non-synonymous substitutions in NS1 and VP2 sequences of FPLV isolates and their frequencies. Based on these finding, we also interpreted several non-synonymous substitutions (Gly299Glu, Ile100Thr, Ala91Ser, and others) that occurred in FPLV isolates, with reference to multiple published studies (line 339-343, and line 351-353, page 12). Cross-species transmission of FPLVs is evident, as FPLV variant carrying these non-synonymous substitutions were more frequently isolated from diverse hosts, including monkey, tiger, giant panda, lion, Ailurus fulgens, mink, and Ailuropoda melanoleuca), rather than the common host (Felis catus).

2. Where appropriate, we have replaced definitive conclusions with more cautious language (e.g., changing "suggests" to "may suggest," "indicates" to "is consistent with," and removing speculative claims about viral fitness or pathogenicity).
3. The limitations of our approach are obvious. Our findings are based on sequence analysis alone and interpretation them with reference to multiple published studies. There was no functional validation through experimental assays (e.g., reverse genetics, viral replication assays) is required to confirm any biological implications of the identified substitutions.

We believe these revisions appropriately address the reviewer's concern by ensuring that our biological interpretations remain proportionate to the descriptive data presented.

Comment 5: The laboratory component (PCR screening, virus isolation, CPE observation, immunofluorescence, partial genome amplification) is acceptable as a basic virological characterization, but it is relatively limited. The experimental data do not support broad biological conclusions regarding altered host range, pathogenicity, replication fitness, or immune escape.

Response 5:

We sincerely thank the reviewer for this constructive feedback. We acknowledge that the laboratory component of our study-comprising PCR screening, virus isolation, CPE observation, immunofluorescence, and partial genome amplification-represents basic virological characterization and has inherent limitations. We agree that these experimental data alone do not support broad biological conclusions regarding altered host range, pathogenicity, replication fitness, or immune escape.

1. We present our study as foundational virological surveillance and genomic cataloging that provides a basis for future hypothesis-driven functional studies, rather than as evidence of adaptive or pathogenic mechanisms.

2.We have tempered our language regarding biological implications. Statements suggesting potential effects on host range, replication efficiency, or antigenicity have been revised to reflect the observational nature of our data. For example, we now use phrases such as "is consistent with," "raises the possibility of," or "warrants further investigation" rather than definitive claims.

We believe these revisions appropriately address the reviewer's concern by ensuring that our conclusions remain proportionate to the scope of our experimental data.

 

Responds to the reviewer’s minor comments:

Comment 1: The manuscript should use consistent terminology (FPV vs FPLV).

Response 1:

We sincerely thank the reviewer for pointing out this important issue. We acknowledge that inconsistent use of "FPV" and "FPLV" throughout the manuscript could cause confusion, and we have thoroughly revised the manuscript to ensure terminological consistency.

FPLV and FPV are both the abbreviation for feline panleukopenia virus and feline parvovirus, respectively-two names commonly used by scholars in the field. In this manuscript, we uniformly use “FPLV” (referring to feline panleukopenia virus) throughout, and no instances of” FPV” appear in our text. However, some researchers have used” FPV” to refer to feline panleukopenia virus or feline parvovirus in the titles of their publications; we have retained the original “FPV” in these references, as altering cited titles would be in appropriate.

We believe these revisions fully address the reviewer's concern and improve the clarity and professionalism of the manuscript.

Comment 2: Expressions such as “as far as I know,” “were founded,” “what more,” “instability of FPLVs” are not appropriate for a scientific manuscript.

 

Response 2:

We sincerely thank the reviewer for this valuable feedback regarding the language and tone of our manuscript. 

We acknowledge that expressions such as "as far as I know," "were founded," "what more," and "instability of FPLVs" are inappropriate for scientific writing, as they introduce subjectivity, colloquialism, or imprecision. We have carefully revised the entire manuscript to eliminate such non-scientific expressions and ensure a formal, objective, and professional tone throughout. Details can be seen in the revised manuscript.

 We believe these revisions have significantly improved the professionalism and clarity of the manuscript, aligning it with the standards of scientific publishing.

Comment 3: Some methodological descriptions need correction and standardization (e.g., centrifugation should preferably be reported in × g, not only rpm).

Response 3:

    Dear reviewer, we sincerely thank you for this important methodological suggestion.

We acknowledge that reporting centrifugation parameters solely in rpm is insufficient, as the relative centrifugal force (×g) varies depending on the rotor radius and is the standard unit for ensuring reproducibility across different centrifuge models. Following conversion, the rpm values have been converted to relative centrifugal force (×g), details can be seen in line 88, page 3, in the revised manuscript.

We believe these revisions significantly enhance the clarity, completeness, and utility of our figures for the broad readership of this journal.

 

Comment 4: Figure legends should be more informative and self-contained

Response 4:

We sincerely thank the reviewer for this constructive feedback. We have revised all figure legends in the manuscript to make them more informative and self-contained. Details can be seen in the revised manuscript.

We believe these revisions significantly enhance the clarity, completeness, and utility of our figures for the broad readership of this journal.

Comment 5: Figure 4 would benefit from improved readability and clearer highlighting of the newly isolated strains.

Response 5:

Dear reviewer, we sincerely thank you for this constructive feedback. We have revised Figure 4 to improve readability and have more clearly highlighted the newly isolated strains. Details can be seen in the revised manuscript.

We believe these revisions significantly enhance the clarity our figures for the broad readership of this journal.

 

 

Special thanks to you for your good comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Major points:

This manuscript provides a comprehensive overview of the mutation spectrum of three Chinese FPLV strains by isolating them and conducting genetic variation analysis of the FPLV sequences in NCBI. It offers valuable information in this field. Although most of the descriptive data are reliable, the study has obvious limitations: the virus isolation lacks hemagglutination, titers, and electron microscopy data; the analysis only stops at the description of mutation frequencies, without conducting selection pressure, structural mapping, host association, and parallel comparisons with CPV; the discussions are mostly repetitions of the results, without in-depth exploration of key scientific questions such as why high-frequency mutations can spread widely. These limitations should be clearly stated in the revised manuscript, and it is recommended to supplement necessary experiments and analyses to enhance the depth of the research.

 

Minor points:

Page 2, Line 63: Sample information is incomplete. The 16 clinical samples are only described as collected from Shijiazhuang and Changchun, but no specific collection dates, cat breeds, ages, or vaccination histories are provided. Please supplement this information.

Page 3, Line 101 (ITR amplification method): The method of boiling for 5 minutes followed by ice‑bath incubation for 10 minutes to amplify the inverted terminal repeats is unusual and may be difficult to reproduce. Please provide a reference or explain the rationale.

Page 3, Line 115: Sample information is incomplete. No specific isolation dates, host origins, or geographic details are provided for the relevant strains. Please supplement this information.

Page 4, Line 137: The virus isolation and identification methods are incomplete. Three FPLV strains were isolated, but only CPE observation and immunofluorescence assays were performed. No data on viral titers (TCID₅₀ or PFU), hemagglutination (HA) titers, or transmission electron microscopy are provided. Please either supply these data or explain why they were not included.

Figure 2 (Direct immunofluorescence results): Only single fluorescence images are shown. No DAPI nuclear staining or merged images (green fluorescence + DAPI) are provided, making it impossible to determine the intracellular localization of viral antigens or the proportion of infected cells. Please add DAPI staining and merged images.

Page 5, Line 172: The data analysis is limited to descriptive statistics of mutation frequencies. Deeper evolutionary analyses, such as selection pressure assessment, three‑dimensional structural mapping, parallel comparison with CPV, host‑origin association analysis, temporal trend testing, and Bayesian phylodynamic analysis, have not been performed. Please explain why these were not done or provide appropriate additional analyses.

Page 11, Line 267: The discussion lacks depth. The authors state that the findings “provide useful information for vaccine development,” but they do not specify how this mutational spectrum could guide vaccine development (e.g., whether high‑frequency mutations fall within known antigenic epitopes, or how well current vaccine strains match circulating strains). Please elaborate on these points in the discussion.

Page 12, Line 331: The conclusion merely repeats the numbers already presented in the abstract and results. Please shorten this section and highlight the most core findings.

Language throughout the manuscript: The manuscript contains colloquial expressions and unclear phrasing. For example, on Page 5, Line 163, “it is a pity not to succeed amplified” is subjective, and on Page 8, Line 180, “as far as I know” expresses uncertainty. Please replace such expressions with neutral, objective, and direct statements.

Comments on the Quality of English Language

Language throughout the manuscript: The manuscript contains colloquial expressions and unclear phrasing. For example, on Page 5, Line 163, “it is a pity not to succeed amplified” is subjective, and on Page 8, Line 180, “as far as I know” expresses uncertainty. Please replace such expressions with neutral, objective, and direct statements.

Author Response

Responses to Reviewers 2

Dear Reviewers:

Thank you for your letter and give some comments concerning our manuscript entitled “Genetic Divergence and Cross-Species Transmission Potential of Feline Panleukopenia Virus: Insights from NS and VP2 Protein Variations (viruses-4242030)”. Those comments are all valuable and very helpful for revising and improving my paper. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in yellow in the paper-revised. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

 

Responds to the reviewer’s major comments:

Comment 1: “This manuscript provides a comprehensive overview of the mutation spectrum of three Chinese FPLV strains by isolating them and conducting genetic variation analysis of the FPLV sequences in NCBI. It offers valuable information in this field. Although most of the descriptive data are reliable, the study has obvious limitations: the virus isolation lacks hemagglutination, titers, and electron microscopy data; the analysis only stops at the description of mutation frequencies, without conducting selection pressure, structural mapping, host association, and parallel comparisons with CPV; the discussions are mostly repetitions of the results, without in-depth exploration of key scientific questions such as why high-frequency mutations can spread widely. These limitations should be clearly stated in the revised manuscript, and it is recommended to supplement necessary experiments and analyses to enhance the depth of the research.”.

Response 1:

Dear reviewer, we thank you very much for these insightful comments. We fully acknowledge the limitations raised in virus isolation. First, the virus morphology was not observed by electron microscopy, for the typical cytopathic effects in F81 cells is enough for indicate parvovirus infection. Hemagglutination assays were carried out on these three FPLV isolates; however, the results were not included in the original manuscript.

As to the discussions are mostly repetitions of the results, without in-depth exploration of key scientific questions, we appreciate the reviewer's critical assessment. The Discussion has been extensively revised to eliminate redundancy with the results section. We have added in-depth analysis of non-synonymous substitutions in the VP2 protein, and interpreted several non-synonymous substitutions (Gly299Glu, Ile100Thr, Ala91Ser, and others) that occurred in FPLV isolates, with reference to multiple published studies (line 339-343, and line 351-353, page 12, in the revised manuscript)

These revisions provide mechanistic insights and contextualize our findings within the current understanding of parvovirus evolution.

Responds to the reviewer’s minor comments:

Comment 1: Page 2, Line 63: Sample information is incomplete. The 16 clinical samples are only described as collected from Shijiazhuang and Changchun, but no specific collection dates, cat breeds, ages, or vaccination histories are provided. Please supplement this information.

Response 1:

Dear reviewer, we thank you very much for this important comment.

Unfortunately, detailed metadata including age, breed, and vaccination history were not recorded by the referring veterinary hospital, representing an important limitation of our sample collection protocol. We addressed this limitation in the section 2.1, line 73, page 2, in the revised manuscript.

Comment 2: Line 101 (ITR amplification method): The method of boiling for 5 minutes followed by ice‑bath incubation for 10 minutes to amplify the inverted terminal repeats is unusual and may be difficult to reproduce. Please provide a reference or explain the rationale

Response 2:

Dear reviewer, we thank you very much for this important comment.

We have added a reference to explain the rationale for the amplification protocol of the ITR regions of the FPLV genome. Details can be seen in line 112, page 3, in the revised manuscript

Comment 3: Line 115: Sample information is incomplete. No specific isolation dates, host origins, or geographic details are provided for the relevant strains. Please supplement this information.

Response 3:

Dear reviewer, thank you for this suggestion.

We have re-write the sentence in section 2.6, line 136-142, to elucidate sample information about the relevant strain of isolation year, host origins and geographic details. This information was supplied in supplementally Table 1. The revised table is provided in the updated manuscript.

 

Comment 4: Line 137: The virus isolation and identification methods are incomplete. Three FPLV strains were isolated, but only CPE observation and immunofluorescence assays were performed. No data on viral titers (TCID₅₀ or PFU), hemagglutination (HA) titers, or transmission electron microscopy are provided. Please either supply these data or explain why they were not included.

Response 4:

Dear Reviewer, thank you for this constructive comment.

While we acknowledge that additional virological assays would strengthen the phenotypic characterization of these isolates, the primary objective of this study was genomic and phylogenetic analysis of FPLV. PCR identification, CPE observation, and immunofluorescence assays were deemed sufficient to confirm successful viral isolation and identity.

Indeed, the viral titers (TCID₅₀) and hemagglutination (HA) titers of the three FPLV isolates were previously determined in a separate study. The viral titers of three Chinese FPLV isolates were 10^5.2 TCID₅₀/1.0 mL and 2⁸(ON630416.1), 10^5.5 TCID₅₀/1.0 mL and 2⁹ (ON630417.1), and 10^5.3TCID₅₀/1.0 mL and 2⁹ (ON630418.1), respectively. In addition, transmission electron microscopy was not performed, as it was deemed unnecessary for this study, because we believe that the typical cytopathic effects observed in F81 cells are sufficient to indicate parvovirus infection.

Comment 5: (Direct immunofluorescence results): Only single fluorescence images are shown. No DAPI nuclear staining or merged images (green fluorescence + DAPI) are provided, making it impossible to determine the intracellular localization of viral antigens or the proportion of infected cells. Please add DAPI staining and merged images.

Response 5:

Dear, we appreciate this constructive comment.

While we acknowledge that DAPI staining and merged images would provide additional spatial context, the single fluorescence images shown in Figure 2 were sufficient to confirm viral antigen expression in infected cells. Given that the primary objective of this study was genomic characterization rather than detailed subcellular localization, we respectfully suggest that the current data meet the requirements for demonstrating successful viral isolation and infection."

Comment 6: The data analysis is limited to descriptive statistics of mutation frequencies. Deeper evolutionary analyses, such as selection pressure assessment, three‑dimensional structural mapping, parallel comparison with CPV, host‑origin association analysis, temporal trend testing, and Bayesian phylodynamic analysis, have not been performed. Please explain why these were not done or provide appropriate additional analyses.

Response 6:

Dear reviewer, thank you for this insightful comment.

We agree that deeper evolutionary analyses would strengthen this study. The primary objective of this study was to characterize the genomic features and phylogenetic relationships of three Chinese FPLV isolates. Compared to the protype FPLV strain (M38246.1), six non-synonymous substitutions were found in the NS1 and the VP2 sequences of these FPLV variants, some of them were species-specific. Accordingly, we conducted a comprehensive analysis of synonymous and non-synonymous mutations in the NS1 and VP2 genes of all FPLV isolates deposited in the NCBI database. We also simply interpreted several species-specific non-synonymous substitutions by reference to multiple published studies, employing structural mapping approaches (three‑dimensional structural mapping), no functional or genetic evolutionary mechanism studies were conducted in this study.

Indeed, selection pressure assessment, parallel comparison with CPV, host origin association analysis, temporal trend testing, and Bayesian phylodynamic analysis are all valuable methods for studying FPLV genetic evolution. Functional characterization of these species-specific non-synonymous mutations will require a reverse genetics system, and all these approaches will be employed in future studies."

"Finally, we wish to extend our sincere appreciation to the reviewers for their comprehensive methodological suggestions. Their valuable insights have substantially strengthened the quality and rigor of this research in future studies.

Comment 7: Line 267: The discussion lacks depth. The authors state that the findings “provide useful information for vaccine development,” but they do not specify how this mutational spectrum could guide vaccine development (e.g., whether high‑frequency mutations fall within known antigenic epitopes, or how well current vaccine strains match circulating strains). Please elaborate on these points in the discussion.

Response 7:

Dear reviewer, thank you for this insightful comment.

We have substantially revised and expanded the Discussion to address correction between our findings and vaccine development. First, we found the genetic distance between foreign FPLV isolates and our three isolates, highlight the necessity of screening local vaccine strains. Details can be seen in line 299-302, page 11, in the revised manuscript.

Second, mutations occurred 585 in the NS1 sequences, augment the viral replication ability, enhance the expression of interferon-stimulated genes (ISGs), this inference can be applied to the genetic engineering of vaccine strains, furthermore, these insights could be leveraged to optimized vaccine manufacturing processes. Details can be seen in line 308-310, page 11, in the revised manuscript.

Finally, certain non-synonymous substitutions may modulate the antigenicity and receptor-binding ability of FPLV variants, although these inferences remain speculative as it derives from structural analysis of species -specific FPLV variant. If these inferences are proven correct, they will provide essential theoretical foundations for vaccine research. Details can be seen in line 341-344, and line 352-355, page 12, in the revised manuscript.

 

Comment 8: Language throughout the manuscript: The manuscript contains colloquial expressions and unclear phrasing. For example, on Page 5, Line 163, “it is a pity not to succeed amplified” is subjective, and on Page 8, Line 180, “as far as I know” expresses uncertainty. Please replace such expressions with neutral, objective, and direct statements.

Response 8:

Dear reviewer, thank you very much for pointing out the language mistakes throughout the manuscript.

We have improved language quality of this manuscript, replaced many colloquial and subjective expressions, such as “as far as I know”,” it is a pity not to success amplified” and others, with neutral, objective, and direct statements, for example, from the manuscript, Details can be seen in the revised manuscript.

Special thanks to you for your good comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript investigates genetic variation in feline panleukopenia virus (FPLV), with particular emphasis on mutations in the NS and VP2 proteins. By analyzing both newly isolated strains and publicly available sequences, the study identifies patterns of synonymous and non-synonymous substitutions. The authors report that most mutations occur at low frequency; however, a subset of amino acid substitutions is more widely distributed and may be associated with host range or cross-species transmission. Overall, the findings provide an overview of FPLV genetic diversity and highlight mutations of potential biological significance.

 

Specific comments:

 

1. The Abstract (page 1, lines 11-27) is currently difficult to interpret due to grammatical errors and unclear phrasing. It should more clearly differentiate between findings obtained from the three isolates and results derived from database analysis. A substantial rewriting is recommended to improve clarity, ensure logical flow and explicitly highlight the main contributions of the study. This will help readers quickly understand the key findings and their significance.

 

2. The Introduction (page 1-2, lines 31-61) provides general background information but does not clearly define the specific research gap addressed by the study. Although cross-species transmission is mentioned, the rationale for focusing on the NS and VP2 proteins remains unclear. The authors should explicitly identify the knowledge gap and explain how their study addresses it, moving beyond a simple descriptive catalogue of mutations to provide a clearer scientific justification for their approach.

 

3. The Materials and Methods section lacks sufficient detail to ensure reproducibility. In the sample collection section (page 2, lines 63-69), “faces’ swabs” should be corrected to “fecal swabs,” and the storage conditions and handling procedures need further clarification. In the PCR identification section (page 2, lines 70-77), details such as cycling conditions, reaction volumes and controls are incomplete. Similarly, in the phylogenetic analysis section (page 4, lines 120-127), the criteria for sequence inclusion and exclusion are not described. The authors should specify how the 100 sequences were selected and indicate whether any redundancy filtering was applied.

 

4. The Results section contains useful observations, but often lacks depth in interpretation. For instance, in the phylogenetic analysis (page 6-7, lines 180-193), the clustering of isolates is described, but the biological significance of these groupings is not explored in detail. The authors should consider discussing how these clusters relate to geographic distribution, host range or evolutionary history. In Section 3.5 (page 8, lines 199-200) and in the Discussion (page 11, lines 281 and 284), the phrases “as far as I know” or “to my knowledge” should be removed. All claims should be supported by references rather than personal statements. More generally, the discussion of mutations would benefit from additional context, particularly regarding their potential structural or functional implications. The large-scale mutation analysis presented in Sections 3.6 and 3.7 (pages 8-11) is comprehensive, but largely descriptive. The manuscript would be strengthened by more advanced analyses, such as selection pressure (dN/dS), structural mapping of mutations or statistical testing of mutation frequency. Without such analyses, the conclusions remain largely observational.

 

5. In the Discussion section (page 11-12, lines 267-330), the authors frequently speculate on the functional significance of mutations without direct evidence. Statements regarding the impact of mutations on host range or pathogenicity should be clearly framed as hypotheses unless experimentally validated. Additionally, the authors should avoid repeating results and ensure the discussion provides meaningful interpretation rather than restating observations.

 

6. The Conclusion (page 12, lines 331-344) largely restates the results without providing a broader perspective on their significance. The authors should focus on highlighting the key contributions of their work and discuss the potential implications of their findings for viral evolution, epidemiology and vaccine development, while also acknowledging the limitations of the study.

 

7. Figures and Tables are referenced appropriately, but their descriptions are sometimes too brief. Figures 6 through 10 present statistical summaries, but the methods used to generate these statistics are not clearly explained in the figure legends.

 

8. Language issues are present throughout the manuscript and should be carefully addressed. While it is understandable that English is not the first language of the authors, there are numerous instances of incorrect verb forms and unclear expressions can obscure meaning. A thorough language revision is strongly recommended to improve clarity, readability and overall presentation.

Comments on the Quality of English Language

Language issues are present throughout the manuscript and should be carefully addressed. While it is understandable that English is not the first language of the authors, there are numerous instances of incorrect verb forms and unclear expressions can obscure meaning. A thorough language revision is strongly recommended to improve clarity, readability and overall presentation.

Author Response

Responses to Reviewers 3

Dear Reviewers:

Thank you for your letter and give some comments concerning our manuscript entitled “Genetic Divergence and Cross-Species Transmission Potential of Feline Panleukopenia Virus: Insights from NS and VP2 Protein Variations (viruses-4242030)”. Those comments are all valuable and very helpful for revising and improving my paper. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in yellow in the paper-revised. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

 

Responds to the reviewer’s major comments:

Comment 1: “The Abstract (page 1, lines 11-27) is currently difficult to interpret due to grammatical errors and unclear phrasing. It should more clearly differentiate between findings obtained from the three isolates and results derived from database analysis. A substantial rewriting is recommended to improve clarity, ensure logical flow and explicitly highlight the main contributions of the study. This will help readers quickly understand the key findings and their significance”.

Response 1:

Dear reviewer, thank you very much for pointing out this drawback in the Abstract.

We have substantially rewritten the Abstract to address your concerns. The revised version now clearly distinguishes between findings from our three FPLV isolates and results derived from the database analysis. We have also improved grammatical accuracy, enhanced logical flow, and explicitly highlighted the main contributions of our study-particularly the identification of high-frequency non-synonymous substitutions in NS1 and VP2 proteins and their implications for understanding FPLV cross-species transmission. We believe these revisions will enable readers to quickly grasp the key findings and their significance.

Details can be seen in line 11-29, page 1, in the revised manuscript.

Comment 2: The Introduction (page 1-2, lines 31-61) provides general background information but does not clearly define the specific research gap addressed by the study. Although cross-species transmission is mentioned, the rationale for focusing on the NS and VP2 proteins remains unclear. The authors should explicitly identify the knowledge gap and explain how their study addresses it, moving beyond a simple descriptive catalogue of mutations to provide a clearer scientific justification for their approach.

Response 2:

Dear reviewer, thank you for this valuable feedback.

We have substantially revised the Introduction to explicitly define the research gap and provide a clear scientific rationale for our approach. Detailed can be seen in line 50-60, page 2, in the revised manuscript.

We believe these revisions provide a compelling scientific justification for our approach and clearly position our study within the existing literature.

Comment 3: The Materials and Methods section lack sufficient detail to ensure reproducibility. In the sample collection section (page 2, lines 63-69), “faces’ swabs” should be corrected to “fecal swabs,” and the storage conditions and handling procedures need further clarification. In the PCR identification section (page 2, lines 70-77), details such as cycling conditions, reaction volumes and controls are incomplete. Similarly, in the phylogenetic analysis section (page 4, lines 120-127), the criteria for sequence inclusion and exclusion are not described. The authors should specify how the 100 sequences were selected and indicate whether any redundancy filtering was applied.

Response 3:

Dear reviewer, thank you very much for pointing out this drawback in section 2-Materials and Methods.

In sample collection, we have corrected “faces swabs” to “fecal swabs”. The samples storage conditions and handling procedures were clarified in line 74-77, section 2.1, page 2, in the revised manuscript. Details can be seen in line 84-90, section 2.2, page 2-3, in the revised manuscript.

In the phylogenetic analysis section, the criteria for sequence inclusion and exclusion are simple.  The three our FPLV isolates were separately blast in NCBI database to search the 100 highest homology parvovirus sequences, retrieved the 300 sequences from the NCBI database, removed redundant and repetitive sequences, Finally, 104 highest homology parvovirus sequences in total, together with our three FPLV isolates, were used for constructing the phylogenetic tree in this study.

We believe these revisions provide a compelling scientific justification for our approach and clearly position our study within the existing literature.

Comment 4: The Results section contains useful observations, but often lacks depth in interpretation. For instance, in the phylogenetic analysis (page 6-7, lines 180-193), the clustering of isolates is described, but the biological significance of these groupings is not explored in detail. The authors should consider discussing how these clusters relate to geographic distribution, host range or evolutionary history. In Section 3.5 (page 8, lines 199-200) and in the Discussion (page 11, lines 281 and 284), the phrases “as far as I know” or “to my knowledge” should be removed. All claims should be supported by references rather than personal statements. More generally, the discussion of mutations would benefit from additional context, particularly regarding their potential structural or functional implications. The large-scale mutation analysis presented in Sections 3.6 and 3.7 (pages 8-11) is comprehensive, but largely descriptive. The manuscript would be strengthened by more advanced analyses, such as selection pressure (dN/dS), structural mapping of mutations or statistical testing of mutation frequency. Without such analyses, the conclusions remain largely observational.

Response 4:

Dear reviewer, thank you for this valuable feedback.

In the phylogenetic analysis section, we thank the reviewer for this constructive suggestion. In fact, the phylogenetic tree constructed in this study exhibited strong characteristics of virus types and geographical regions, for CPVs clustered together, with CPV-2a group, CPV-2b group. All CPV-2a strains were further grouped into two subgroups according the geographical regions (Brazil and Turkey), the same condition also occurred in CPV-2b groups (Japan and Turkey) and FPLV groups (China, Asia, Europe and America). However, these details do not highlight the main point of the article, so I did not elaborate on them in my manuscript. instead, it merely added a concluding statement (line 210-211, page 6).

In Section 3.5 (page 8, lines 199-200) and in the Discussion (page 11, lines 281 and 284), we have removed or replaced many colloquial and subjective expressions, such as the phrases “as far as I know” or “to my knowledge”, with neutral, objective, and direct statements. Details can be seen in the revised manuscript.

In fact, selection pressure (dN/dS) is a fundamental and widely utilized metric in the field of molecular evolution. However, the scope of this study is to elucidate the genetic relationship of our three FPLV isolates and other FPLVs isolates, and the genetic difference in NS1 and VP2 protein, compared to the protype strain of FPLV. Furthermore, comprehensive understand the synonymous and non-synonymous substitutions occurred in NS1 and VP2, and then interpretation them with reference to multiple published studies.

Structural mapping of the protein is also an important method for predicting the function of mutated proteins. Some non-synonymous substitutions interpreted in this study, such as Ala91Ser, Ala300Pro, and Gly300Glu, had been mapped in many published studies. So, in this manuscript, we just citing these references to elucidate the function of these substitutions. Details can be seen in line 351-354, page 12, in the revised manuscript.

Comment 5: In the Discussion section (page 11-12, lines 267-330), the authors frequently speculate on the functional significance of mutations without direct evidence. Statements regarding the impact of mutations on host range or pathogenicity should be clearly framed as hypotheses unless experimentally validated. Additionally, the authors should avoid repeating results and ensure the discussion provides meaningful interpretation rather than restating observations.

Response 5:

Dear reviewer, we thank the reviewer for this important observation.

In fact, functional studies of FPLV and CPV genes are extremely rare. Most researchers studying non-synonymous substitutions in species-specific FPLV variants rely on structural mapping of target proteins to speculate on their functional consequences, no direct evidences.

    Although some non-synonymous substitutions in species-specific FPLV variants suggest cross-species transmission, their role in host adaptation remains unknown.

    Followed the reviewer’s suggestion, we have re-write the conclusion, details can be seen in line 370-379, page 12-13, in the revised manuscript.

Comment 6:  Figures and Tables are referenced appropriately, but their descriptions are sometimes too brief. Figures 6 through 10 present statistical summaries, but the methods used to generate these statistics are not clearly explained in the figure legends.

Response 6:

We thank the reviewer for this important observation.

In this study, the method used for statistic analysis non-synonymous and synonymous substitution of NS1 and VP2 sequences of FPLV isolates is Microsoft excel. The software for creating graphs is Sigma plot software (Version 7.0).

Comment 7: Language issues are present throughout the manuscript and should be carefully addressed. While it is understandable that English is not the first language of the authors, there are numerous instances of incorrect verb forms and unclear expressions can obscure meaning. A thorough language revision is strongly recommended to improve clarity, readability and overall presentation.

Response 7:

We thank the reviewer for this important observation.

 Following your suggestion, we have carried out a thorough language revision, removed or replaced many colloquial and subjective expressions, such as the phrases “as far as I know” or “to my knowledge”, with neutral, objective, and direct statements. We also adjust the overall logical structure of the article to improve readability.

Details can be seen in the revised manuscript.

 

Special thanks to you for your good comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscript represents a substantial improvement over the original submission. The authors have responded to each comment and have appropriately incorporated the suggested changes. Therefore, I am happy to recommend this paper for acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript shows clear and thoughtful effort in addressing the reviewer’s previous comments, leading to a notable improvement in clarity, organization and overall scientific presentation. It provides a comprehensive dataset on genetic variation in FPLV and offers a useful descriptive overview of mutation patterns in the NS1 and VP2 proteins. While the analysis remains largely descriptive, the study lays a solid foundation for future research exploring the functional and evolutionary significance of these mutations. I have no further comments.

Comments on the Quality of English Language

Language has been greatly improved.