Canine Parvovirus Asian Type 2 Variant C (CPV-2c) Detected in Côte d’Ivoire

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Canine parvovirus Asian type 2 variant c (CPV-2c) detected in Côte d’Ivoire” describes the first molecular detection and characterization of CPV-2 in Côte d’Ivoire and provides valuable epidemiological information regarding the circulation of Asian-like CPV-2c strains in West Africa. The study is well organized, the molecular analyses are appropriate, and the phylogenetic/phylogeographic approach adds relevance to the manuscript. Overall, I found the study interesting and suitable for publication after revision. However, some points should be clarified or moderated before the manuscript can be considered for acceptance.

Lines 88, 195–199, and throughout the manuscript: Several minor typographical and formatting issues should be corrected during revision. Examples include:

• “diarrohea” instead of “diarrhea”;
• “highlited” instead of “highlighted”;
• “coluor” instead of “colour/color”;
• recurrent encoding issues such as “beĴer”, “paĴern”, and “laĴer”.

Lines 87–90: Please indicate whether vaccination history was available for the animals included in the study. Even partial information would help contextualize the epidemiological findings. If these data were unavailable, this limitation should be mentioned. Clinical information is currently very limited. Please clarify whether additional clinical outcome data were available, including mortality, hospitalization, hematological findings, or evidence of coinfections.

Lines 92–99: Please specify whether appropriate positive and negative controls were included in all PCR runs.

Lines 117–149: The phylogenetic analyses are generally robust and appropriate. However, the manuscript would benefit from briefly reporting the final nucleotide substitution models selected for IQ-TREE and BEAST analyses directly in the text.

Lines 153–160 and 260–282: The manuscript would benefit from a more explicit discussion regarding the limitations associated with using partial VP2 sequences for phylogeographic inference, particularly when the analysis was further restricted to a 430 bp fragment for the African-focused dataset.

Lines 167–185: One of the main limitations of the study is the relatively small number of samples analyzed (n = 12). Although understandable for a first report, some conclusions regarding viral introduction routes and circulation dynamics appear somewhat overinterpreted considering the limited sample size and the partial VP2 sequences analyzed. I recommend moderating some statements related to viral introduction pathways and geographic dissemination.

Lines 177–190 and 251–259: The phylogeographic reconstruction is interesting and technically well performed; however, the limited availability of African CPV-2 sequences in GenBank may strongly influence the inferred ancestral origins and migration pathways. The possibility of database sampling bias should be more explicitly acknowledged in the Discussion.

Lines 238–250: The hypothesis linking commercial relationships between Vietnam and Côte d’Ivoire with viral introduction is plausible and interesting. However, in the absence of direct epidemiological evidence regarding animal movement, pet trade, or importation records, the interpretation remains speculative and the wording should be softened.

Lines 319–320: Please clarify whether owner consent was obtained or whether the samples originated exclusively from routine diagnostic submissions. Although ethical approval may not have been required, additional clarification would improve transparency.

In summary, this manuscript provides important new data regarding CPV-2 circulation in West Africa and represents a useful contribution to the molecular epidemiology of canine parvovirus. The suggested revisions mainly concern clarification of methodological limitations and moderation of some epidemiological interpretations.

Author Response

Thank you for the constructive comments. Below you can find our responses in bold.

 

Lines 88, 195–199, and throughout the manuscript: Several minor typographical and formatting issues should be corrected during revision. Examples include:

• “diarrohea” instead of “diarrhea”;
• “highlited” instead of “highlighted”;
• “coluor” instead of “colour/color”;
• recurrent encoding issues such as “beĴer”, “paĴern”, and “laĴer”.

Response: the corrections have been made accordingly although we are unsure what the reviewer means by “recurrent encoding issues such as “beĴer”, “paĴern”, and “laĴer””.

Lines 87–90: Please indicate whether vaccination history was available for the animals included in the study. Even partial information would help contextualize the epidemiological findings. If these data were unavailable, this limitation should be mentioned. Clinical information is currently very limited. Please clarify whether additional clinical outcome data were available, including mortality, hospitalization, hematological findings, or evidence of coinfections.

Response: thank you for this comment. We have added that none of the dogs were vaccinated against CPV-2 (see line 88). Additionally, we have added the clinical outcome of the dogs in Table 1.

Lines 92–99: Please specify whether appropriate positive and negative controls were included in all PCR runs.

Response: we did not have a positive control for the first samples screened but positive amplicons of the expected size were sequenced, confirmed to be CPV-2 and then used as positive controls for subsequent screenings.

Lines 117–149: The phylogenetic analyses are generally robust and appropriate. However, the manuscript would benefit from briefly reporting the final nucleotide substitution models selected for IQ-TREE and BEAST analyses directly in the text.

Response: we apologize for the missing information, which has now been added manuscript (see lines 132 to 135).

Lines 153–160 and 260–282: The manuscript would benefit from a more explicit discussion regarding the limitations associated with using partial VP2 sequences for phylogeographic inference, particularly when the analysis was further restricted to a 430 bp fragment for the African-focused dataset.

Response: we agree that the use of a partial sequence might have reduced the phylogenetic signal and thus the resolution of the study. A comment has been added to the manuscript in this regard (see lines 287 to 289).

Lines 167–185: One of the main limitations of the study is the relatively small number of samples analyzed (n = 12). Although understandable for a first report, some conclusions regarding viral introduction routes and circulation dynamics appear somewhat overinterpreted considering the limited sample size and the partial VP2 sequences analyzed. I recommend moderating some statements related to viral introduction pathways and geographic dissemination.

Response: we agree with the reviewer that the inclusion of additional strains could have revealed a more complex epidemiological scenario. Although this was not possible due to practical and financial constraints, we acknowledge this as a limitation of the study, and this aspect has now been added to the Discussion section (see lines 265 to 278).

Lines 177–190 and 251–259: The phylogeographic reconstruction is interesting and technically well performed; however, the limited availability of African CPV-2 sequences in GenBank may strongly influence the inferred ancestral origins and migration pathways. The possibility of database sampling bias should be more explicitly acknowledged in the Discussion.

Response: we agree with the reviewer’s comment. Certainly, a broader availability of African sequences would have benefited the analysis, and an increase in sequencing activity and data sharing is highly desirable in the future. We have addressed the limitations related to sampling bias and limited sequence availability in the revised manuscript (see lines 265 to 278).

Lines 238–250: The hypothesis linking commercial relationships between Vietnam and Côte d’Ivoire with viral introduction is plausible and interesting. However, in the absence of direct epidemiological evidence regarding animal movement, pet trade, or importation records, the interpretation remains speculative and the wording should be softened.

Response: we agree with the reviewer and have revised the text accordingly (see lines 249 to 251)

Lines 319–320: Please clarify whether owner consent was obtained or whether the samples originated exclusively from routine diagnostic submissions. Although ethical approval may not have been required, additional clarification would improve transparency.

Response: owner consent was obtained for all of the dogs samples. This has been added to the Informed Consent Statement (see line 343).

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

I am writing to you about the manuscript "Canine parvovirus Asian type 2 variant c (CPV-2c) detected in Côte d'Ivoire". The scientific, epidemiological and clinical relevance of this manuscript is important. However, some issues require revisions and integrations.

Firstly, the introduction section provides a background on CPV-2. However, the text presents some points which need to be clarified.

In the analysis of antigenic evolution and dynamics (Lines 54-62), the emergence of the original CPV-2 from the FPV is chronically delineated through a process of species adaptation, followed by the appearance of antigenic variants 2a, 2b and finally 2c. It should be noted that the nucleotide evolution rate of CPV-2, although it is a DNA virus, has a very high rate of nt substitutions per site. The lack of proofreading activity during viral replication in mitotically active cells favors the continuous generation of viral quasi-species. This part should be supplemented.

In Lines 70-76, the authors introduce the new "Asian CPV2c variant". The amino acid insertion of a specific mutation such as 324I or 267Y localized in specific surface loops of the viral capsid, alter the conformation of the protein domain. These topological alterations have been shown to affect hydrophobic binding affinity with the host's transferrin receptor. This altered binding not only modulates cellular tropism, increasing internalization efficiency, but at the same time modifies the antigenic profile, providing a greater immunity escape. It would be appropriate to discuss this increased viral fitness, which would further justify the replacement of pre-existing CPV-2c strains globally in some areas.

It would also be interesting if the authors better correlated the canine demographic data with the rate of accelerated urbanization of the emerging cities in the areas under analysis. The high density of susceptible hosts, presence of wildlife, combined with promiscuity and limited vaccination coverage, creates an ideal epidemiological environment to support the hyperendemicity of such virus.

The Materials and Methods section is well written. However, some inaccuracies need to be corrected. In the sampling section, table 1 shows the metadata related to the samples collected between January and September of 2025. There is no mention of the vaccination status and previous immunological history of infected puppies. It would be appropriate to specify in the main text whether these dogs had received vaccination prophylaxis against CPV-2, and if so, with which strains.

The diagnostic protocol employed (lines 92-98) seems appropriate. However, there is a considerable technical limitation in the use of Sanger sequencing. The implementation of NGS, although difficult in some areas, should be explicitly recognized as a technical limitation in the Discussion section, in relation to co-infections of viral strains.

Moving on, the authors did a brilliant job in the bioinformatics pipelines used. Only a few minor inaccuracies need to be revised. In line 131, the authors should correctly cite the use of "BEAST X". Moreover, the use of relaxed lognormal clock needs further details. Which was the specific probability model adopted? Did the author hypothesize a constant-size population model with a stable endemic situation?

In addition, in the phylogenetic analysis the selected fragment was reduced to 430 bp, resulting in a loss of the intrinsic phylogenetic signal. In viruses characterized by sustained and high mutation rates such as CPV-2, this signal degradation frequently causes the collapse of the inner branches of the phylotree. Authors should integrate this limitation into the Discussion section.

In lines 162-164, please specify whether additional negative samples were tested during the same time frame, thus providing the incidence rate. Moving on to the phylogenetic characterization (Lines 165-177), the presence of two distinct clades within the Asian lineage suggests a much more dynamic scenario. Systematic detection of mutated amino acid markers in all 12 samples serves as the molecular signature of Asian CPV-2c. On basis of these considerations, figures S1-2 captions  should be revised properly, as well as bootstrap values should be provided to support the statistical confidence of phylogenetic reconstruction.

The Discussion section outlines the retrospective and future perspectives for veterinary public health. Although the framework is valid, in line 243, it is stated that the "viral introduction through fomites" is one of the plausible vehicles. Parvoviruses are known to be naked viruses, without envelopes, and therefore very resistant to extreme pH, heat and mild solvents. However, survival on ships’ cargo seems very unlikely to start an epidemic event on a new continent.

On the other hand, the role of human movements together with the illegal trafficking of exotic fauna or the transfer of dogs from southeast asian personnel is much more plausible.

Author Response

Thank you for your constructive comments. Please find our responses in bold below 

In the analysis of antigenic evolution and dynamics (Lines 54-62), the emergence of the original CPV-2 from the FPV is chronically delineated through a process of species adaptation, followed by the appearance of antigenic variants 2a, 2b and finally 2c. It should be noted that the nucleotide evolution rate of CPV-2, although it is a DNA virus, has a very high rate of nt substitutions per site. The lack of proofreading activity during viral replication in mitotically active cells favors the continuous generation of viral quasi-species. This part should be supplemented.

Response: we agree with the reviewer that CPV-2 displays an unusually high evolutionary rate for a DNA virus and that this represents a highly interesting aspect of its biology. However, we believe that a detailed discussion of the mechanisms underlying the rapid evolution of ssDNA viruses is not within the scope of the present study, which is primarily focused on the epidemiological and phylogeographic dynamics of CPV-2c spread. Moreover, the actual causes of the elevated evolutionary rate observed in ssDNA viruses are likely multifactorial and still not completely elucidated. Therefore, we preferred to avoid introducing this topic as a separate discussion point in the Introduction.

In Lines 70-76, the authors introduce the new "Asian CPV2c variant". The amino acid insertion of a specific mutation such as 324I or 267Y localized in specific surface loops of the viral capsid, alter the conformation of the protein domain. These topological alterations have been shown to affect hydrophobic binding affinity with the host's transferrin receptor. This altered binding not only modulates cellular tropism, increasing internalization efficiency, but at the same time modifies the antigenic profile, providing a greater immunity escape. It would be appropriate to discuss this increased viral fitness, which would further justify the replacement of pre-existing CPV-2c strains globally in some areas.

Response: we thank the reviewer for this very interesting comment and fully agree that the mutations mentioned could potentially contribute to altered receptor binding, antigenic properties, and increased viral fitness, as already discussed by other authors. Again, we believe that a detailed discussion of these aspects is not within the scope of the present manuscript, which is primarily focused on the epidemiological and phylogeographic characterization of CPV-2c spread. Moreover, although these hypotheses are biologically plausible and supported by previous studies, the currently available data do not allow definitive conclusions regarding the actual phenotypic impact of these mutations in the analyzed strains. Therefore, we considered that an extensive discussion of these mechanisms could become overly speculative in the context of the present study.

It would also be interesting if the authors better correlated the canine demographic data with the rate of accelerated urbanization of the emerging cities in the areas under analysis. The high density of susceptible hosts, presence of wildlife, combined with promiscuity and limited vaccination coverage, creates an ideal epidemiological environment to support the hyperendemicity of such virus.

Response: although this is an interesting comment, unfortunately we do not have the necessary canine demographic or rate of urbanization data to make the correlation suggested.

The Materials and Methods section is well written. However, some inaccuracies need to be corrected. In the sampling section, table 1 shows the metadata related to the samples collected between January and September of 2025. There is no mention of the vaccination status and previous immunological history of infected puppies. It would be appropriate to specify in the main text whether these dogs had received vaccination prophylaxis against CPV-2, and if so, with which strains.

Response: thank you for this comment. We have added that none of the dogs were vaccinated against CPV-2 (see line 88).

The diagnostic protocol employed (lines 92-98) seems appropriate. However, there is a considerable technical limitation in the use of Sanger sequencing. The implementation of NGS, although difficult in some areas, should be explicitly recognized as a technical limitation in the Discussion section, in relation to co-infections of viral strains.

Response: we do not understand why the reviewer thinks that there are “considerable technical limitations in the use of Sanger sequencing” and why NGS should be explicitly recognized as a technical limitation in the Discussion section. Can the reviewer please clarify their statement?

Moving on, the authors did a brilliant job in the bioinformatics pipelines used. Only a few minor inaccuracies need to be revised. In line 131, the authors should correctly cite the use of "BEAST X". Moreover, the use of relaxed lognormal clock needs further details. Which was the specific probability model adopted? Did the author hypothesize a constant-size population model with a stable endemic situation?

Response: we apologize for the missing information, which has now been added to the manuscript. The population dynamics were not modelled assuming a constant population size, but using the non-parametric Skygrid model. We apologize for not clearly reporting this information in the original version of the manuscript. Both the demographic and molecular clock models were selected using the SS and PS approaches, as now specified in the manuscript (see line 135).

In addition, in the phylogenetic analysis the selected fragment was reduced to 430 bp, resulting in a loss of the intrinsic phylogenetic signal. In viruses characterized by sustained and high mutation rates such as CPV-2, this signal degradation frequently causes the collapse of the inner branches of the phylotree. Authors should integrate this limitation into the Discussion section.

Response: we agree that this represents an important limitation of the study. However, this choice was unavoidable due to the limited availability of African sequences and the lack of standardized sequenced regions among publicly available datasets. The limitations associated with this approach have now been explicitly addressed in the manuscript (see line 287 to 289).

In lines 162-164, please specify whether additional negative samples were tested during the same time frame, thus providing the incidence rate.

Response: Unfortunately, no additional negative samples collected during the same time frame were available for testing, and therefore it was not possible to estimate the incidence rate or provide a more robust assessment of infection frequency within the studied population.

Moving on to the phylogenetic characterization (Lines 165-177), the presence of two distinct clades within the Asian lineage suggests a much more dynamic scenario. Systematic detection of mutated amino acid markers in all 12 samples serves as the molecular signature of Asian CPV-2c. On basis of these considerations, figures S1-2 captions should be revised properly, as well as bootstrap values should be provided to support the statistical confidence of phylogenetic reconstruction.

Response: unfortunately, it is not clear to us what you are asking for. Can you please clarify? Please note that the bootstrap values in Figure S1 and S2 are shown as circles rather that numbers which allow for less “busy” phylogenetic trees.

The Discussion section outlines the retrospective and future perspectives for veterinary public health. Although the framework is valid, in line 243, it is stated that the "viral introduction through fomites" is one of the plausible vehicles. Parvoviruses are known to be naked viruses, without envelopes, and therefore very resistant to extreme pH, heat and mild solvents. However, survival on ships’ cargo seems very unlikely to start an epidemic event on a new continent. On the other hand, the role of human movements together with the illegal trafficking of exotic fauna or the transfer of dogs from southeast asian personnel is much more plausible.

Response: thank you for this comment. We have revised the text accordingly removing mention of fomites (see line 249) and suggesting that the movement of people/pets is a more plausible explanation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting the revised version of your manuscript entitled “Canine parvovirus Asian type 2 variant c (CPV-2c) detected in Côte d’Ivoire”.

The revised manuscript has been substantially improved and the majority of the reviewers’ comments have been adequately addressed. In particular, the inclusion of additional clinical information, clarification of the phylogenetic and phylogeographic methodologies, and the expanded discussion of study limitations have strengthened the scientific rigor and interpretation of the findings. The revised text also provides a more balanced discussion regarding the potential introduction and dissemination pathways of the detected strains. Before the manuscript can be considered for final acceptance, a few minor issues should still be addressed. Specifically, the Materials and Methods section would benefit from a brief clarification indicating that the initial PCR screenings were performed without an available positive control (i.e., effectively in a blinded manner), and that positive amplicons were subsequently confirmed by sequencing and then used as positive controls for later analyses. This information is currently provided in the response to reviewers but should also be incorporated into the manuscript for transparency and reproducibility. In addition, the manuscript should be carefully proofread to correct the remaining formatting and character-encoding issues present throughout the text, as well as any minor language inconsistencies. Subject to satisfactory revision of these remaining points, the manuscript will be suitable for publication.

Kind regards

Author Response

Thank you for your further comments. Responses are provided below.

Comment: Before the manuscript can be considered for final acceptance, a few minor issues should still be addressed. Specifically, the Materials and Methods section would benefit from a brief clarification indicating that the initial PCR screenings were performed without an available positive control (i.e., effectively in a blinded manner), and that positive amplicons were subsequently confirmed by sequencing and then used as positive controls for later analyses. 

Response: the text has been revised accordingly (see lines 100-102)

 

Comment: the manuscript should be carefully proofread to correct the remaining formatting and character-encoding issues present throughout the text, as well as any minor language inconsistencies. 

Response: the manuscript has been proofread once again and minor inconsistencies have been corrected. 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is acceptable in the present form.

Author Response

Thank you for agreeing that our manuscript is acceptable for publication.