Review Reports - Molecular Characterization of Helicase and Nuclease Domains in PPV5 NS1 from Mexican Full-Length Sequence

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Since its first discovery in the United States in 2013, porcine parvovirus 5 (PPV5) has been reported in many countries around the world, turning it into a global threat to the swine industry. In this study, Araiza-Hernández and co-workers performed structural modeling for the PPV5 NS1 protein. Based on the model and sequence, they then compared the solvent-accessible surface area (SASA) and electrostatic potential of PPV5 NS1 helicase domain with the homologous proteins, especially around the Walker A, Walker B, B’ motif, C motif, and Box VII regions. In addition, they also predicted the residues critical for DNA binding and cleavage by the nuclease domain of PPV5 NS1. As mentioned in the manuscript, sequence and modeling of PPV5 NS1 has been reported in other journal, which significantly lowered the novelty of this study. The sequence of PPV5 NS1 is very different from the homologous proteins, more evidences, such as in vitro DNA binding and cleavage assays by wild-type and mutant proteins of PPV5 NS1 nuclease domain, will be needed to confirm the functional importance of the predicted residues and to guarantee the publication of this manuscript. Other issues are listed below,

1) It is good to mention that AF stands for the arginine finger in the legend of figure 6.

2) Was ATP included in the models shown in figure 6? As demonstrated by Rep proteins or NS1 protein from human B19V virus, the assembly of the SF3 helicase domain can be significantly altered by the binding of ATP.

3) It is helpful to shown structure-based sequence alignment of PPV5 and homologous proteins in figure 8.

4) The labels of the conserved amino acids are difficult to follow in Figures 9 and 10.

Author Response

GENERAL COMMENT.

GENERAL ANSWER :

We carefully read the manuscript in order to clarify the reviewer concern regarding the novelty of the current work. We think that each manuscript scope might be not explained in detail, or that some ideas might be lost in the translation. Also, we were extremely careful not to fall into self-plagiarism throughout the manuscript by using previously reported information, but that might create an impression that nothing was added in the present work, as the reviewer commented. For instance, the ribbon and sphere models portrayed in this work (Figure 1, former Figure 6), are merely used to help in locating the motifs since in the electrostatic potential models that cannot be differentiated. We pointed out because we believe that might give the reviewer lack of novelty impression, since it is true, the modeling of helicase was done previously, but in this work is used as comparative only. The models we are presenting and discussing for the first time are those based on the electrostatic potential. This is indicated in the Figure 1 legend. Also, the previous report the aa prediction and modeling were focused the helicase domain while in this work that was done for the nuclease domain. Again, to our knowledge there are not models in the viruses we are presenting.

To clarify in the edited manuscript:

In the introduction section (Pages 1-2, lines 80-92), we relate in detail what exactly has been already done in the previous report to point out the differences with what the present work. As noted, it is acknowledged that modeling and prediction amino acid differences in the PPV5 helicase domain were reported previously throughout the text, but such findings were the reason to explore the SASA values and the electrostatic potential of PPV5 since major helicase functions are achieved through electrostatic interactions. Therefore, we justify our studies under two premises: 1. Electrostatic measurement aids in understanding the stability and union of biomolecules, modifications at this level might suggest a disfunction (Sun et al., 2022), and 2. SASA values provide information regarding aa exposition to the surroundings which is proportional to the degree of interaction of proteins with that settings (Durham 2009). In the helicase context, we wanted to explore structural differences based on electrostatic potential and SASA since the motifs that previously showed amino acid differences at the ATP binding pocket. To our knowledge, there are neither models regarding the electrostatic potential nor SASA assessment for PPV5 and the pathogenic viruses (PPV1 or CPV) that we selected in our comparative approach.
Also, we work in each section to make clear what previous report information was used to discuss our findings, emphasizing in what is first documented in this study, avoiding mixing information that may create confusion. We hope the edits are sufficient to be precise enough to discriminate previously reported from the current study information.
In the Material and Methods section, we provide more details concerning case selection to show the analyses were performed using four PPV5 sequences. Again, we were so concerned about not to give the impression of self-plagiarism that we were not presenting all information that was done in this study. We were so focused on the full-length PPV5 that we did not show the whole picture that leads to the final depiction. In the edited version this information is provided, indicating that all evaluations were performed in the previously reported PPV5 sequences, and in some cases using worldwide PPV5 (location and type of amino acids in active sites of helicase motifs and nuclease, probability of ATP binding in Walker A motif and Selection Pressure analysis). The latter two analyses were added to provide more insight. Also, a detailed description of the molecular analysis is provided, and a justification for the selection of templates since most models for SF3 helicases are based in AAV, SV40 and papilloma virus whereas for nuclease domain MVM, HBoV1, and AAV5 have been portrayed.
In the result section, a 3D model of the ATP-binding pockets with an integrated ATP molecule in the cleft and the probability of ATP binding were added to have more elements to sustain our discussion since functional assays cannot be implemented at the moment.
In the discussion section, we provided an orderly approach, providing first the main difficulties and limitations research in novel PPVs is facing to justify a bioinformatic based study. Then, a detailed discussion by helicase motif based on well-documented information available for ATP binding sites. We hope the new version will be more in-depth than the previous one, allowing to sustain our suggested notions.

PARTICULAR COMMENTS

The sequence of PPV5 NS1 is very different from the homologous proteins, more evidences, such as in vitro DNA binding and cleavage assays by wild-type and mutant proteins of PPV5 NS1 nuclease domain, will be needed to confirm the functional importance of the predicted residues and to guarantee the publication of this manuscript. Other issues are listed below.

ANSWER:

In the discussion section, it was mentioned that functional assays are necessary to sustain our findings, listing some assay examples with references. However, isolation and propagation of novel PPVs have been elusive because they have shown slow growth which makes difficult their propagation. Another issue that complicate the scenario is that emerging PPVs often show high rate of interspecies coinfection or they are found in coinfection with other pathogenic viruses such as porcine circovirues and PRRSV, among others. That precludes the isolation of pure strains. To our knowledge, the isolation and propagation of PPV5 have recently reported (Anoyatbekova, A.; Komina, A.; Vlasova, N.; Kononova, E.; Gulyukin, A.; Krasnikov, N.; Yuzhakov, A. Molecular Detection of Porcine Parvovirus 5 in Domestic Pigs in Russia and Propagation of Field Isolates in Primary Porcine Testicular Cells. Vet. Sci. 2025, 12, 535, doi:10.3390/vetsci12060535).

Therefore, the propagation and titration could be successfully attempted since it is the first step to implement in vivo studies such as experimental infections and functional in vitro tests. However, these tasks will face the same difficulties above mentioned. All of the above have delayed the study of the pathogenic potential in novel PPVs, and the reason we opted for approaching studying pathogenic potential by means of bioinformatic tools despite their limitations using paraffin embedded tissues from archival. We anticipate that the success regarding PPV5 propagation will pave the way for functional in vitro test as well as for experimental infections, but not in the short time.

Again, we reiterate that functional tests are needed as referred in the discussion and conclusion sections. Then, in the edited manuscript amendments were made in those sections in order to emphasize the aforementioned limiting possibilities to pursue functional tests, providing more grounds for suggesting likely functional effects on the NS1 functions based on the references.

Comments 1: It is good to mention that AF stands for the arginine finger in the legend of figure 6.

Response 1: This omission was attended in the edited manuscript. Also, we used the acronym throughout the body text.

Comments 2: Was ATP included in the models shown in figure 6? As demonstrated by Rep proteins or NS1 protein from human B19V virus, the assembly of the SF3 helicase domain can be significantly altered by the binding of ATP.

Response 2: ATP molecule is not included in that figure. In the edited manuscript, a new figure (Figure 3) is added showing the ATP-binding pocket with an ATP integrated in the cleft. It was attempted in the figure 1 (formerly Figure 6), but ATP was not easily appreciated in all the PPV models. We thought that it will be more difficult to grasp any difference in the hexamer model because it is a busy illustration anyways. Then, we decided to create an ATP binding pocket model with an ATP since we provided one in our graphical abstract where differences in charges were clearly visualized. We decided that figure is more illustrative, offering conformational differences. We are thankful for the reviewer’s suggestion.

Comments 3: It is helpful to shown structure-based sequence alignment of PPV5 and homologous proteins in figure 8.

Response 3: In the former figure 8 (Figure 4), the nuclease domain amino acid alignment for the Mexican PPV5 sequences and their homologous PPV1 and CPV, indicating the location of critical functional amino acid. We believe that in showing those important residues we are providing the structure in a lineal sequence, but perhaps we are not getting at the reviewer suggestion. On the other hand, given that we are presenting all analysis we did, but did not either provide or discuss, in the edited manuscript we added 15 worldwide PPV5 sequences we have been utilizing in our analysis. This addition allows identifying that PPV5 share same amino acid sequences, which is discussed further.

Comments 4: The labels of the conserved amino acids are difficult to follow in Figures 9 and 10.

Response 4: We opted for the licorice representation for active site functional amino acids because in our opinion this format better illustrates the interaction with Mg2+, but as the reviewer noted, it is not be easy to localize them. In the edited manuscript, labels were added to attend the reviewer concern.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes the molecular characteristics of helicase and nuclease domains in NS1 of a Mexican PPV5 sequence. The authors compared the NS1 of PPV5 with PPV1 and CPV and claimed that the sequence differences may have functional effects. However, this study did not provide any validation results of the functions. More importantly, the solely computational predictions are over-interpreted and do not provide any new mechanistic understanding on PPV5 NS1 functions.

Major concerns:

The authors claimed that PPV5 are more prevalent in Mexico than in other countries. However, the authors only determined one PPV5 sequence in this study. This amount of data is definitely not enough to support any conclusion. Multiple Mexican PPV5 sequences should be analyzed.
The authors compared the NS1 of PPV5 with PPV1 and CPV. The rational of the study design is questionable. CPV is not even a swine pathogen. The authors should compare PPV5 with PPV1 and other PPVs.
The authors claimed several findings in this study. However, none of the predictions were validated by any experimental analysis.
Figure 8. The description of conserved amino acids in the nuclease domain adds nothing new. The authors should provide real evidence for their relationships with functions.
Figures 1-5 are actually tables.

Author Response

GENERAL COMMENT

GENERAL ANSWER:

Research in novel PPVs faces an incredible limitation, their isolation and propagation have been elusive, precluding the implementation of in vivo and in vitro experiments to study its pathogenic potential. Therefore, the functional assays that are necessary to validate our findings, as the reviewer correctly mentioned, cannot be performed due to the lack of strain repositories. In the manuscript reviewed, the need for corroboration with functional assays were mentioned, providing references, but the current problematic just mentioned did not. In the edited manuscript, we discuss that to offer a justification.

It is acknowledged that isolation and propagation of novel PPVs is difficult because they have shown slow growth which makes their propagation in cell cultures difficult following standard procedures. Another issue that complicate the scenario is that emerging PPVs often show high rate of interspecies coinfection, or they are found in coinfection with other pathogenic viruses such as porcine circovirues and PRRSV, among others. That precludes the isolation of pure strains. To our knowledge, the isolation and propagation of PPV5 have recently reported (Anoyatbekova, A.; Komina, A.; Vlasova, N.; Kononova, E.; Gulyukin, A.; Krasnikov, N.; Yuzhakov, A. Molecular Detection of Porcine Parvovirus 5 in Domestic Pigs in Russia and Propagation of Field Isolates in Primary Porcine Testicular Cells. Vet. Sci. 2025, 12, 535, doi:10.3390/vetsci12060535). However, despite propagation and titration is the first step to implement in vivo studies such as experimental infections and functional in vitro tests, these tasks will face the same difficulties above mentioned. Moreover, propagation and titration in primary cell cultures will take an enormous effort and time. All of the above have delayed the study of the pathogenic potential in novel PPVs, and the reason we opted for approaching studying pathogenic potential by means of bioinformatic tools despite their limitations using paraffin embedded tissues from archival. We anticipate that the success regarding PPV5 propagation will pave the way for functional in vitro test as well as for experimental infections, but not in the short time.

To clarify in the edited manuscript:

In the introduction section (Pages 1-2, lines 80-92), we relate in detail what exactly has been already done in the previous report to point out the differences with what the present work. As noted, it is acknowledged that modeling and prediction amino acid differences in the PPV5 helicase domain were reported previously throughout the text, but such findings were the reason to explore the SASA values and the electrostatic potential of PPV5 since major helicase functions are achieved through electrostatic interactions. Therefore, we justify our studies under two premises: 1. Electrostatic measurement aids in understanding the stability and union of biomolecules, modifications at this level might suggest a disfunction (Sun et al., 2022), and 2. SASA values provide information regarding aa exposition to the surroundings which is proportional to the degree of interaction of proteins with that settings (Durham 2009). In the helicase context, we wanted to explore structural differences based on electrostatic potential and SASA since the motifs that previously showed amino acid differences at the ATP binding pocket. To our knowledge, there are neither models regarding the electrostatic potential nor SASA assessment for PPV5 and the pathogenic viruses (PPV1 or CPV) that we selected in our comparative approach.
Also, we work in each section to make clear what previous report information was used to discuss our findings, emphasizing in what is first documented in this study, avoiding mixing information that may create confusion. We hope the edits are sufficient to be precise enough to discriminate previously reported from the current study information.
In the Material and Methods section, we provide more details concerning case selection to show the analyses were performed using four PPV5 sequences. Also, we are presenting all information that was done in this study because were so focused in the full-length PPV5 that we did not show the whole work that was done to create the final depiction. In the edited version this information is provided, indicating that all evaluations were performed in the previously reported PPV5 sequences, and in some cases using worldwide PPV5 (location and type of amino acids in active sites of helicase motifs and nuclease, probability of ATP binding in Walker A motif and Selection Pressure analysis). The latter two analyses were added to provide more insight. Also, a detailed description of the molecular analysis is provided, and a justification for the selection of templates since most models for SF3 helicases are based in AAV, SV40 and papilloma virus whereas for nuclease domain MVM, HBoV1, and AAV5 have been portrayed.
In the result section, a 3D model of the ATP-binding pockets with an integrated ATP molecule in the cleft and the probability of ATP binding were added to have more elements to sustain our discussion since functional assays cannot be implemented at the moment.
In the discussion section, we provided an orderly approach, providing first the main difficulties and limitations research in novel PPVs is facing to justify a bioinformatic based study. Then, a detailed discussion by helicase motif based on well-documented information available for ATP binding sites. We hope the new version will be more in-depth than the previous one, allowing to sustain our suggested notions.

Major concerns:

Comments 1: The authors claimed that PPV5 are more prevalent in Mexico than in other countries. However, the authors only determined one PPV5 sequence in this study. This amount of data is definitely not enough to support any conclusion. Multiple Mexican PPV5 sequences should be analyzed.

Response 1: As mentioned in the general answer, the analyses were performed in four Mexican PPV5 sequences (three of them previously reported), we also used 15 worldwide PPV5 sequences and provided reasons we omitted in the revised manuscript. As we are describing in the edited manuscript, the analyses revealed similar results, thus, the 3D models we are presented are based in the full-length PPV5 sequence. We hope the amendments and additions we have made will be helpful in the edited manuscript review.

Concerning the PPV5 prevalence in Mexico, it is true and it is published. Our samples are paraffin-embedded, because it is retrospective and we are currently working to obtain more sequences. We have had excellent results in amplifying short products from ADN extracted from paraffin-embedded tissues, but we are having problems with long amplicons of PPV5. We think it is related to DNA quality, but we are working hard on that. We totally understand the reviewer comment, but that is the situation. We have amplified PPV5 using nested PCR and qPCR, but the four segments to complete the ORF1 has been elusive, but we are working to increase the number of full-length PPV5 sequences to analyse. Right now, we are presenting 3Dmodels to aid in our understanding regarding PPV5 pathogenic potential. We modify our discussion making an orderly in depth description based on what it is well-documented for helicases and nucleases. We hope our improved approach will support the notions we are discussing.

Comments 2: The authors compared the NS1 of PPV5 with PPV1 and CPV. The rational of the study design is questionable. CPV is not even a swine pathogen. The authors should compare PPV5 with PPV1 and other PPVs.

Response 2: The rational for the comparation is based on depicting differences with pathogenic PPVs. The pathogenic potential of novel PPVs is unknown, thus we decided to compared with PPV1 since it is the only pathogenic PPV in porcine, including another well-documented pathogenic PPV even though affects other species, provides more information to discuss the findings based on what is published. In our opinion, comparisons among family members is acceptable, even if they affect different animal species, because they share similar phylogeny and the NS1 is a well conserved protein. Also, the information regarding novel PPVs is scarce, and SF3 helicases are not extensively evaluated. As commented, the main problem of novel PPV research is the unavailability of performing functional assay. This report is providing insight regarding functional activity of helicase and nuclease domain on a structural basis.

Comments 3: The authors claimed several findings in this study. However, none of the predictions were validated by any experimental analysis.

Response 3: This comment was discussed in the general answer since it is mentioned also in the general comment.

Comments 4: Figure 8. The description of conserved amino acids in the nuclease domain adds nothing new. The authors should provide real evidence for their relationships with functions.

Response 4: The objective of the former figure 8 (Figure 4), is to show the nuclease domain amino acid alignment for the Mexican PPV5 sequences and their homologous PPV1 and CPV, indicating the location of critical functional amino acid. We believe that in showing The presence of those important residues provides the structure in a lineal sequence to classify the type of nuclease. Perhaps we are not presenting something new in this figure, but the amino acid sequence is the basis to construct the 3D model in which we discussed the potential functional differences with pathogenic PPVs. On the other hand, given that we are presenting all analysis we did, in the edited manuscript we added 15 worldwide PPV5 sequences we have been utilizing in our analysis. This addition allows identifying that PPV5 published sequences share same amino acid sequences, therefore the 3D models for the nuclease domain based on our sequences are representative of the viral species.

Comments 5: Figures 1-5 are actually tables.

Response 5: We totally agree with the reviewer, in the edited manuscript, they were modified and cited as tables.

Reviewer 3 Report

Comments and Suggestions for Authors

I reviewed the manuscript entitled “Molecular characterization of helicase and nuclease domains in PPV5 NS1 from a Mexican full‑length sequence.” In this study, the authors perform a bioinformatic and structural characterization of the helicase and nuclease domains of PPV5 NS1, comparing them with porcine parvovirus 1 (PPV1) and canine parvovirus (CPV). Based on differences in electrostatic potential and Mg²⁺ interactions, the authors suggest potential functional divergence between PPV5 and pathogenic parvoviruses.

While the topic is relevant and the analyses are technically sound, I believe the manuscript requires substantial improvement before it can support the conclusions drawn.

Major concerns:

Insufficient description and justification of bioinformatic analyses
Given the entirely bioinformatic nature of this study, additional methodological detail is required. A clearer explanation of the modeling assumptions, comparative framework, and interpretation limits would help readers better understand the biological relevance of the structural findings.
Limited evolutionary context and sequence representation
A major limitation of the study is the very small number of PPV5 sequences used for comparison, particularly when functional interpretations are proposed. The conclusions would be significantly strengthened by incorporating a broader set of PPV5 sequences representing the genetic diversity of this virus in nature.
Lack of evolutionary analysis of functional domains
I strongly suggest expanding the study to include an evolutionary framework. Analyses based on nucleotide sequences, not only amino acid properties, would allow the authors to address key questions such as:

Are the helicase and nuclease domains highly conserved among PPV5 isolates?

Are specific residues evolving under purifying or positive selection?

Are the previously reported Mexican mutations fixed, recurrent, or lineage‑specific?

How do these mutations compare with those present in PPV5 isolates from other geographic regions?

Codon‑based analyses of selection would provide essential context and help distinguish biologically meaningful changes from neutral variation.

Author Response

GENERAL COMMENT

I reviewed the manuscript entitled “Molecular characterization of helicase and nuclease domains in PPV5 NS1 from a Mexican full‑length sequence.” In this study, the authors perform a bioinformatic and structural characterization of the helicase and nuclease domains of PPV5 NS1, comparing them with porcine parvovirus 1 (PPV1) and canine parvovirus (CPV). Based on differences in electrostatic potential and Mg²⁺ interactions, the authors suggest potential functional divergence between PPV5 and pathogenic parvoviruses.

While the topic is relevant and the analyses are technically sound, I believe the manuscript requires substantial improvement before it can support the conclusions drawn.

GENERAL ANSWER

Thanks for your comments and suggestions, all were taken into account to make a better manuscript. Given the limitations on novel PPVs research, we would like to provide a general description concerning the editions we are presenting . We hope our changes are sufficient to improve the manuscript as the reviewer requested.

These are the main editions:

- In the introduction section (Pages 1-2, lines 80-92), we relate in detail what exactly has been already done in the previous report to point out the differences with what the present work. As noted, it is acknowledged that modeling and prediction amino acid differences in the PPV5 helicase domain were reported previously throughout the text, but such findings were the reason to explore the SASA values and the electrostatic potential of PPV5 since major helicase functions are achieved through electrostatic interactions. Therefore, we justify our studies under two premises: 1. Electrostatic measurement aids in understanding the stability and union of biomolecules, modifications at this level might suggest a disfunction (Sun et al., 2022), and 2. SASA values provide information regarding aa exposition to the surroundings which is proportional to the degree of interaction of proteins with that settings (Durham 2009). In the helicase context, we wanted to explore structural differences based on electrostatic potential and SASA since the motifs that previously showed amino acid differences at the ATP binding pocket. To our knowledge, there are neither models regarding the electrostatic potential nor SASA assessment for PPV5 and the pathogenic viruses (PPV1 or CPV) that we selected in our comparative approach.

- Also, we work in each section to make clear what information of our previous report was used to discuss our findings, emphasizing in what is first documented in this study, avoiding mixing information that may create confusion. We hope the edits are sufficient to be precise enough to discriminate our previously reported from the current study information.

- In the Material and Methods section, we provide more details concerning case selection to show the analyses were performed using four PPV5 sequences. Also, we are presenting all information that was done in this study because we were so focused in the full-length PPV5 that we did not show the whole work that was done to create the final depiction. In the edited version this information is provided, indicating that all evaluations were performed in the previously reported PPV5 sequences, and in some cases using worldwide PPV5 (location and type of amino acids in active sites of helicase motifs and nuclease, probability of ATP binding in Walker A motif and Selection Pressure analysis). The latter two analyses were added to provide more insight. Also, a detailed description of the molecular analysis is provided, and a justification for the selection of templates since most models for SF3 helicases are based in AAV, SV40 and papilloma virus whereas for nuclease domain MVM, HBoV1, and AAV5 have been portrayed.

- In the result section, a 3D model of the ATP-binding pockets with an integrated ATP molecule in the cleft and the probability of ATP binding were added to have more elements to sustain our discussion since functional assays cannot be implemented at the moment.

- In the discussion section, we provided an orderly approach, providing first the main difficulties and limitations research in novel PPVs is facing to justify a bioinformatic based study. Then, a detailed discussion by helicase motif based on well-documented available information for ATP binding sites. We hope the new version will be more in-depth than the previous one, allowing to sustain our suggested notions.

Major concerns:

Comments 1: Insufficient description and justification of bioinformatic analyses
Given the entirely bioinformatic nature of this study, additional methodological detail is required. A clearer explanation of the modeling assumptions, comparative framework, and interpretation limits would help readers better understand the biological relevance of the structural findings.

Response 1: As mentioned in the general answers we provides more information about the bioinformatic analysis. We hope the additions are sufficient to attend the reviewer request.

Comments 2: Limited evolutionary context and sequence representation
A major limitation of the study is the very small number of PPV5 sequences used for comparison, particularly when functional interpretations are proposed. The conclusions would be significantly strengthened by incorporating a broader set of PPV5 sequences representing the genetic diversity of this virus in nature.

Response 2: We totally agree that the number is limited, we are working on obtaining more full-length PPV5 to perform an in-depth phylogenetic analysis. However, we think the preliminary molecular findings presented herein, are representative of PPV5 species and will provide some insights concerning PPV5 NS1 which exhibited electrostatic-related differences compared to pathogenic PPV.

Comments 3: Lack of evolutionary analysis of functional domains
I strongly suggest expanding the study to include an evolutionary framework. Analyses based on nucleotide sequences, not only amino acid properties, would allow the authors to address key questions such as:

Are the helicase and nuclease domains highly conserved among PPV5 isolates?

Are specific residues evolving under purifying or positive selection?

Are the previously reported Mexican mutations fixed, recurrent, or lineage‑specific?

How do these mutations compare with those present in PPV5 isolates from other geographic regions?

Codon‑based analyses of selection would provide essential context and help distinguish biologically meaningful changes from neutral variation.

Response 3: Thanks for your suggestion, as noted in the general answer, this request was attended. The analysis that was published previously, was done based on amino acid sequence because it is the classification criteria by ICTV. We are currently working to obtain more full-length PPV5 to study Mexican PPV5 phylogeny. Because the evolutionary analysis did not reveal positive or purifying selection, no further analyses were performed. However, a revisited revision on the amino acid alignment allows to see that the Mexican and worldwide PPV5 exhibited the same amino acid substitutions in the helicase motifs and nuclease domain that we are portraying in our electrostatic analyses. Therefore, they are not lineage specific, and as it is mentioned in the closing of discussion are present in other geographical regions. We need to work harder to increase our PPV5 sequence to answer more questions.

Again, we hope our edited manuscript showed sufficient improvements for the review.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all the concerns. Congratulations on the great job . In the opinion of this reviewer, the manuscript is now acceptable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been well revised.