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Open AccessArticle

Peer-Review Record

Molecular Analysis of the Complete Genome of a Simian Foamy Virus Infecting Hylobates pileatus (pileated gibbon) Reveals Ancient Co-Evolution with Lesser Apes

Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Viruses 2019, 11(7), 605; https://doi.org/10.3390/v11070605
Received: 1 April 2019 / Revised: 27 June 2019 / Accepted: 30 June 2019 / Published: 3 July 2019
(This article belongs to the Special Issue Spumaretroviruses)

Round 1

Reviewer 1 Report

Shankar et al. obtained the full length genetic sequence from an SFV strain isolated from pileated gibbon. They compared this sequence to those of other SFV species, analyzed the presence of functional motifs in genes and proteins and studied the evolutionary history of this strain. This thorough presentation of the first SFVhpi sequence represents significant knowledge for further basic virology and SFV diagnosis. The paper should be enhanced by a better description of recombination analysis, a study of env gene variability, rigorous checking of functional motifs, inclusion of recently obtained SFV sequences, adequate citation of the bibliography, and use of a more fluent writing style.

Broad comments

Line 35: “the functional SFV genome is double stranded DNA rather than single stranded-DNA”. This is an overstatement, as controversial data have been published. The two cited reviews state that DNA genome is functional but do not mention data on the relative infectivity of DNA and RNA genomes.

Lines 34 and 37: two redundant sentences are separated by a third one. Please edit this paragraph and the whole manuscript for fluency.

Lines 41-43: For the description of SFV infection in NHPs, the authors cite only their papers, but neither a review, nor major papers from other teams. Lines 43-51: For the description of SFV infection in humans, the authors cite their research papers and reviews, but no research papers from other teams. In addition, Ref 22 is erroneous as it described SFV infection in macaques and not in humans. To avoid skewing towards self-citation, the authors have to cite the most recent review(s) and research papers from all teams if published after the review.

Lines 50-53: Please cite more recent reviews for the description of in vitro cytopathic effect and of SFV infection in NHPs. The authors should add the two papers that search for medical consequences in humans, published by Boneva et al. in 2007 and Buseyne et al. in 2018.

Lines 68-75: The authors provide the list of isolates with full length sequences provided in ref 28. Additional complete sequences are available in Genbank, including SFV from Papio Anubis, and from Brachyteles arachnoides. These should be used in the analysis.

Lines 100-102: The treatment of PBMCs need to be described. Where the cells fresh? Frozen? Stimulated with a mitogen? For how long? In what medium? What was the time lapse between coculture initiation and ECP appearance?

Lines 107-109: This sentence describes the first step of the process and is nevertheless placed at the end of the paragraph. Please described the work in a logical order.

Tables 1 to 3: Why do the authors consider SFV from only two chimpanzee subspecies, while three have been described?

Description of viral promoters: The IP is described in the middle of the paragraph describing Env, in accordance to its location, but not to the biological function. Please describe IP in a paragraph following the description of LTR. Undescribed elements are: Tas responsive elements, splice donor site, the lack of central ppt, dimerization sequence, Poly A.

Lines 301-302: the location of the ER retention signal is erroneous.

Lines 295-302: Missing elements in the Env description are: The hydrophobic region in LP, the RBD in SU, and fusion peptide in TM.

Overall, the authors should refer to two papers (Schulze et al. 2011, and Rua et al., 2012) that had performed similar analysis that will help them to provide a comprehensive description of the new sequence.

Lines 339-346: Analyses of recombination. The authors should describe the sequences used for recombination analysis and the rational for their choice. We suggest that they expand the number of sequences. Why do the author limit their analysis to the concatamer? This strategy is expected to decrease the probability of detecting a recombination event.

SFV recombination has been described in two contexts. Some recombinant SFV infect a single or few NHPs/ Their parental strains are identified and prevalent in the studied NHP population (or their prey). The recombination is probably a recent event. The second context is an ancient recombination event that may have generated the bimorphic env genes found in feline FV and in SFV infecting chimpanzee, macaques, gorilla and AGM. The parental strains are unknown. This genetic diversity is functionally relevant as this region is targeted by neutralizing antibodies (Zemba et al., 2000; Lambert et al., 2018). Furthermore, env swapping is an important event in the biology and evolution of the retroviruses. The env gene fragments described as parental/recombinant in Galvin et al. (2013) or conserved/variant by Richard et al. (2015) should be aligned with those of SFV species comprising two genotypes. These alignment and phylogenic analysis will allow the authors to describe whether the SFVhpi env fragments are more related to one or the other 2 env genotypes from apes SFV.

Lines 450-451: “Comparison of LTR in the host and those obtained by culture are needed.” This sentence is erroneous, as the comparison has been reported in reference 56.

Specific comments

Line 27: “fills an important knowledge gap in SFV biology” sounds like an overstatement as no functional studies are presented in the paper. Please rephrase.

Lines 60-61: “the generation of database” is useless.

Lines 84-85: “The researchers were unable to obtain viral sequence” sounds awkward. First, it does not describe the experiment, but the people who carried the test; secondly word use deflates the contribution of the researchers. Please rephrase.


Author Response

Comments and Suggestions for Authors

Shankar et al. obtained the full length genetic sequence from an SFV strain isolated from pileated gibbon. They compared this sequence to those of other SFV species, analyzed the presence of functional motifs in genes and proteins and studied the evolutionary history of this strain. This thorough presentation of the first SFVhpi sequence represents significant knowledge for further basic virology and SFV diagnosis. The paper should be enhanced by a better description of recombination analysis, a study of env gene variability, rigorous checking of functional motifs, inclusion of recently obtained SFV sequences, adequate citation of the bibliography, and use of a more fluent writing style.

Broad comments

Line 35: “the functional SFV genome is double stranded DNA rather than single stranded-DNA”. This is an overstatement, as controversial data have been published. The two cited reviews state that DNA genome is functional but do not mention data on the relative infectivity of DNA and RNA genomes.

The evidence presented in Yu SF, Sullivan MD, Linial ML. Evidence that the human foamy virus genome is DNA. J Virol. 1999;73(2):1565–1572.  (cited in the review by Pinto-Santini et al.) supports this fact that the particle-associated “DNA is sufficient for new rounds of replication” (Yu et al) . We do not discuss relative infectivity of DNA and RNA genomes, rather, we just state that the functional genome is double stranded DNA. Nonetheless, we have removed the word “functional” from this sentence.

Lines 34 and 37: two redundant sentences are separated by a third one. Please edit this paragraph and the whole manuscript for fluency.

We have checked these sentences and cannot find the redundancies the reviewer cites. We have addressed areas we feel are appropriate and as suggested elsewhere by this and the other reviewers.

Lines 41-43: For the description of SFV infection in NHPs, the authors cite only their papers, but neither a review, nor major papers from other teams. Lines 43-51: For the description of SFV infection in humans, the authors cite their research papers and reviews, but no research papers from other teams. In addition, Ref 22 is erroneous as it described SFV infection in macaques and not in humans. To avoid skewing towards self-citation, the authors have to cite the most recent review(s) and research papers from all teams if published after the review.

The majority of work on SFV co-evolution has come from our team. Hence, those articles are naturally more citated. However, we now cite other SFV co-evolution articles, but not exhaustively, and include or substitute review articles as suggested.

Lines 50-53: Please cite more recent reviews for the description of in vitro cytopathic effect and of SFV infection in NHPs. The authors should add the two papers that search for medical consequences in humans, published by Boneva et al. in 2007 and Buseyne et al. in 2018.

We have added the relevant citations as suggested.

Lines 68-75: The authors provide the list of isolates with full length sequences provided in ref 28. Additional complete sequences are available in Genbank, including SFV from Papio Anubis, and from Brachyteles arachnoides. These should be used in the analysis.

Since we already have four representatives for SFV from New World monkeys, including a spider monkey species (Ateles geoffroyi), as well as representatives from other Old World monkeys, to provide a diverse data set for comparison, we feel that addition of these additional sequences to the analysis for the purpose of this manuscript, will not change the FV phylogeny or recombination results given the co-evolutionary history of FVs. For example, we also have a complete baboon SFV genome (GenBank accession number MF472626) that we included in the analyses and which did not change the results presented in our manuscript. We removed the baboon SFV from the paper since we plan to publish that sequence in detail elsewhere.

Lines 100-102: The treatment of PBMCs need to be described. Where the cells fresh? Frozen? Stimulated with a mitogen? For how long? In what medium? What was the time lapse between coculture initiation and ECP appearance?

Although this information is available in the cited reference, we briefly describe the culture details.

Lines 107-109: This sentence describes the first step of the process and is nevertheless placed at the end of the paragraph. Please described the work in a logical order.

We have changed the order as suggested.

Tables 1 to 3: Why do the authors consider SFV from only two chimpanzee subspecies, while three have been described?

We have used representative sequences from each group of primates for the purpose of comparative phylogeny rather than using all available sequences to minimize the computationally intensive phylogenetic analyses. We feel this approach provides sufficient molecular information for our study.

Description of viral promoters: The IP is described in the middle of the paragraph describing Env, in accordance to its location, but not to the biological function. Please describe IP in a paragraph following the description of LTR. Undescribed elements are: Tas responsive elements, splice donor site, the lack of central ppt, dimerization sequence, Poly A.

We have moved the paragraph as suggested. We also now add on page 8 the locations of the additional elements noted when they were identifiable.

Lines 301-302: the location of the ER retention signal is erroneous.

We provided the location of the ER RS using the TM codon positions and not the entire Env coding region. We have now added the Env codon positions to avoid confusion.

Lines 295-302: Missing elements in the Env description are: The hydrophobic region in LP, the RBD in SU, and fusion peptide in TM.

Overall, the authors should refer to two papers (Schulze et al. 2011, and Rua et al., 2012) that had performed similar analysis that will help them to provide a comprehensive description of the new sequence.

Thanks for the suggestion. We used both papers for identifying motifs in our original submission. However, neither the Schulze nor the Rua articles show these additional motifs. Rua describes the hydrophobic tryptophan (W) residues being in the first 15 amino acids, which differs from that of Lindeman and Goepfert CTMI 2003 chapter) which shows them at pos 86/86 of LP (SP). The WXXW motif in SFVhpi is also within the first 15 amino acids at positions 10-13. In comparison to PFV we located a putative RBD in SU and the fusion peptide in TM. We have added this information to the results.

Lines 339-346: Analyses of recombination. The authors should describe the sequences used for recombination analysis and the rational for their choice. We suggest that they expand the number of sequences. Why do the author limit their analysis to the concatamer? This strategy is expected to decrease the probability of detecting a recombination event.

SFV recombination has been described in two contexts. Some recombinant SFV infect a single or few NHPs/ Their parental strains are identified and prevalent in the studied NHP population (or their prey). The recombination is probably a recent event. The second context is an ancient recombination event that may have generated the bimorphic env genes found in feline FV and in SFV infecting chimpanzee, macaques, gorilla and AGM. The parental strains are unknown. This genetic diversity is functionally relevant as this region is targeted by neutralizing antibodies (Zemba et al., 2000; Lambert et al., 2018). Furthermore, env swapping is an important event in the biology and evolution of the retroviruses. The env gene fragments described as parental/recombinant in Galvin et al. (2013) or conserved/variant by Richard et al. (2015) should be aligned with those of SFV species comprising two genotypes. These alignment and phylogenic analysis will allow the authors to describe whether the SFVhpi env fragments are more related to one or the other 2 env genotypes from apes SFV.

Please see lines 154-162 of Methods where we describe the sequences used and the rationale for using a concatamer. Briefly, we used the concatamer in order to maximize the molecular information across the coding regions with the most similarity to other SFV, while also ensuring a high quality alignment. This approach is common in the analysis of slow evolving cell-associated retroviruses. Using the entire genome creates alignment challenges because of highly divergent regions of the genome, which don’t align well and are then gap-stripped using SimPlot to evaluate recombination. Furthermore, recombination occurs in areas of homology and not high divergence and has mostly been reported in env, gag, and RT of closely related species. We have completed the additional recombination analyses using the sequences from the Galvin and Richard articles as suggested and have added this information to the paper. These analyses still did not show recombination in env for SFVhpi.

 

Lines 450-451: “Comparison of LTR in the host and those obtained by culture are needed.” This sentence is erroneous, as the comparison has been reported in reference 56.

Sorry for any confusion. Our statement refers to the current study where these comparisons of the SFVhpi LTRs are still needed but which are beyond the scope of the current study.

Specific comments

Line 27: “fills an important knowledge gap in SFV biology” sounds like an overstatement as no functional studies are presented in the paper. Please rephrase.

We used biology in a general sense, since the term encompasses a wide array of information. We have changed it to genetics.

Lines 60-61: “the generation of database” is useless.

We have re-phrased the sentence to read “The design of molecular methods for the accurate identification of SFV infection requires a database containing representative sequences from divergent SFV lineages”.

Lines 84-85: “The researchers were unable to obtain viral sequence” sounds awkward. First, it does not describe the experiment, but the people who carried the test; secondly word use deflates the contribution of the researchers. Please rephrase.

We have re-phrased the sentence as “A recent study reported a high seroprevalence of SFV in gibbons from Cambodia although the seropositive samples were not SFV PCR positive”.

Reviewer 2 Report

Dear authors,

it was a pleasure to read this manuscript, the work is solid and the data are well presented.

My only suggestion would be to alter the sentence about "disease" in the abstract. I dont think the full Genome is mainly of interest to detect potential disease causing properties, this full genome study is interesting as such. 

Author Response

Comments and Suggestions for Authors

Dear authors,

it was a pleasure to read this manuscript, the work is solid and the data are well presented.

My only suggestion would be to alter the sentence about "disease" in the abstract. I dont think the full Genome is mainly of interest to detect potential disease causing properties, this full genome study is interesting as such. 

This sentence has been modified to read, “SFVs can zoonotically infect humans but very few complete SFV genomes are available, hampering the design of diagnostic assays”.

Reviewer 3 Report

The manuscript by Shankar et al describes the first complete genome analysis simian foamy virus from pileated gibbon ape (Hylobates pileatus) inferring the co-evolution of host and virus.  The work presented is straightforward. It is based on sequencing and sequence analysis to determine host virus evolution as well as sequence comparisons with other foamy virus strains and description of putative motifs based on sequence.  Although this paper describes the first complete sequence foamy virus from Hylobates pileatusthe main finding of co-evolution of foamy virus with the host is not new or novel except strengthen the previous studies.  Additional new foamy virus genome sequence, however, provide information for further understanding the epidemiology and evolutionary history of foamy viruses. 

 

Other points

 

1.    Sequence analysis show that the genome is the longest of foamy viruses sequenced thus far due the a much longer LTR.  However, caution should be taken because older sequence analyses show insertion and deletion in the LTR within the prototypye foamy virus even at times showing differences 5’ LTR from the 3’ LTR.

 

2.    The genome is sequenced after growing the virus in cf2th cell line, which would be under no host influence.  Would this lead to sequence variation that would impede on the conclusion of co-evolving with the host?

 

 

3.    The sentence page 1, lines 37-38 “To initiate infection, FVs use multiple promoters, a trait seen in complex DNA viruses, but not in other retroviruses [3].”  is awkward and needs re-writing.  


Author Response

Comments and Suggestions for Authors

The manuscript by Shankar et al describes the first complete genome analysis simian foamy virus from pileated gibbon ape (Hylobates pileatus) inferring the co-evolution of host and virus.  The work presented is straightforward. It is based on sequencing and sequence analysis to determine host virus evolution as well as sequence comparisons with other foamy virus strains and description of putative motifs based on sequence.  Although this paper describes the first complete sequence foamy virus from Hylobates pileatus the main finding of co-evolution of foamy virus with the host is not new or novel except strengthen the previous studies.  Additional new foamy virus genome sequence, however, provide information for further understanding the epidemiology and evolutionary history of foamy viruses. 

 

Other points

Sequence analysis show that the genome is the longest of foamy viruses sequenced thus far due the a much longer LTR.  However, caution should be taken because older sequence analyses show insertion and deletion in the LTR within the prototypye foamy virus even at times showing differences 5’ LTR from the 3’ LTR.

We have confirmed the length of the LTR using SFVhpi-specific primers and PCR with traditional Sanger sequencing and have added this information to the manuscript.

 2.    The genome is sequenced after growing the virus in cf2th cell line, which would be under no host influence.  Would this lead to sequence variation that would impede on the conclusion of co-evolving with the host?

Foamy viruses are extremely stable in vivo and in vitro. Our phylogenetic and recombination analyses show that culturing in Cf2th cells does not have an effect on the way SFVhpi sequence clusters in relation to FVs from other species. The position of SFVhpi in the tree constructed from viral sequence alignments almost exactly mirrors that of the phylogeny from host mitochondrial sequences, indicating co-evolution. In addition, the pol sequence from the PBMC and Cf2Th DNAs, although short (425-bp) are 100% identical supporting the stability. To adequately test this hypothesis would require obtaining the complete genome from the PBMC DNA, which is beyond the scope of our study.

 3.    The sentence page 1, lines 37-38 “To initiate infection, FVs use multiple promoters, a trait seen in complex DNA viruses, but not in other retroviruses [3].”  is awkward and needs re-writing.  

This sentence has been re-worded to read, “FVs also differ from other retroviruses in how they initiate infection. Like complex DNA viruses, they use multiple promoters [4]."

Round 2

Reviewer 1 Report

See attached file


Comments and Suggestions for Authors

Shankar et al. obtained the full length genetic sequence from an SFV strain isolated from pileated gibbon. They compared this sequence to those of other SFV species, analyzed the presence of functional motifs in genes and proteins and studied the evolutionary history of this strain. This thorough presentation of the first SFVhpi sequence represents significant knowledge for further basic virology and SFV diagnosis. The paper should be enhanced by a better description of recombination analysis, a study of env gene variability, rigorous checking of functional motifs, inclusion of recently obtained SFV sequences, adequate citation of the bibliography, and use of a more fluent writing style.

Second review: Shankar et al. improved their paper. Some elements of their rebuttal are still not convincing.

 

Broad comments

Line 35: “the functional SFV genome is double stranded DNA rather than single stranded-DNA”. This is an overstatement, as controversial data have been published. The two cited reviews state that DNA genome is functional but do not mention data on the relative infectivity of DNA and RNA genomes.

The evidence presented in Yu SF, Sullivan MD, Linial ML. Evidence that the human foamy virus genome is DNA. J Virol. 1999;73(2):1565–1572.  (cited in the review by Pinto-Santini et al.) supports this fact that the particle-associated “DNA is sufficient for new rounds of replication” (Yu et al) . We do not discuss relative infectivity of DNA and RNA genomes, rather, we just state that the functional genome is double stranded DNA. Nonetheless, we have removed the word “functional” from this sentence.

SFV genome can be DNA or RNA. The data from Yu et al. have not been confirmed by others.

 

Lines 34 and 37: two redundant sentences are separated by a third one. Please edit this paragraph and the whole manuscript for fluency.

We have checked these sentences and cannot find the redundancies the reviewer cites. We have addressed areas we feel are appropriate and as suggested elsewhere by this and the other reviewers.

Here they are:

For example, FVs use two promoters for gene expression,one in the long terminal repeat (LTR) and one in the envelope (env) gene. In addition, FVs complete reverse transcription within the virion before infection of a new host cell, such that the functional SFV genome is double-stranded DNA rather than single-stranded RNA [1, 2]. To initiate infection, FVs use multiple promoters, a trait seen in complex DNA viruses, but not in other retroviruses [3].

Lines 41-43: For the description of SFV infection in NHPs, the authors cite only their papers, but neither a review, nor major papers from other teams. Lines 43-51: For the description of SFV infection in humans, the authors cite their research papers and reviews, but no research papers from other teams. In addition, Ref 22 is erroneous as it described SFV infection in macaques and not in humans. To avoid skewing towards self-citation, the authors have to cite the most recent review(s) and research papers from all teams if published after the review.

The majority of work on SFV co-evolution has come from our team. Hence, those articles are naturally more citated. However, we now cite other SFV co-evolution articles, but not exhaustively, and include or substitute review articles as suggested.

There are no change in the bibliography. The authors have not address the whole comment.

Lines 50-53: Please cite more recent reviews for the description of in vitro cytopathic effect and of SFV infection in NHPs. The authors should add the two papers that search for medical consequences in humans, published by Boneva et al. in 2007 and Buseyne et al. in 2018.

We have added the relevant citations as suggested.

One citation is missing

Lines 68-75: The authors provide the list of isolates with full length sequences provided in ref 28. Additional complete sequences are available in Genbank, including SFV from Papio Anubis, and from Brachyteles arachnoides. These should be used in the analysis.

Since we already have four representatives for SFV from New World monkeys, including a spider monkey species (Ateles geoffroyi), as well as representatives from other Old World monkeys, to provide a diverse data set for comparison, we feel that addition of these additional sequences to the analysis for the purpose of this manuscript, will not change the FV phylogeny or recombination results given the co-evolutionary history of FVs. For example, we also have a complete baboon SFV genome (GenBank accession number MF472626) that we included in the analyses and which did not change the results presented in our manuscript. We removed the baboon SFV from the paper since we plan to publish that sequence in detail elsewhere.

My comment was on the citation of available full length SFV sequence, not on the phylogeny or recombination analysis.  The text can be easily updated.

Lines 100-102: The treatment of PBMCs need to be described. Where the cells fresh? Frozen? Stimulated with a mitogen? For how long? In what medium? What was the time lapse between coculture initiation and ECP appearance?

Although this information is available in the cited reference, we briefly describe the culture details.

OK

Lines 107-109: This sentence describes the first step of the process and is nevertheless placed at the end of the paragraph. Please described the work in a logical order.

We have changed the order as suggested.

OK

Tables 1 to 3: Why do the authors consider SFV from only two chimpanzee subspecies, while three have been described?

We have used representative sequences from each group of primates for the purpose of comparative phylogeny rather than using all available sequences to minimize the computationally intensive phylogenetic analyses. We feel this approach provides sufficient molecular information for our study.

Description of viral promoters: The IP is described in the middle of the paragraph describing Env, in accordance to its location, but not to the biological function. Please describe IP in a paragraph following the description of LTR. Undescribed elements are: Tas responsive elements, splice donor site, the lack of central ppt, dimerization sequence, Poly A.

We have moved the paragraph as suggested. We also now add on page 8 the locations of the additional elements noted when they were identifiable.

OK

Lines 301-302: the location of the ER retention signal is erroneous.

We provided the location of the ER RS using the TM codon positions and not the entire Env coding region. We have now added the Env codon positions to avoid confusion.

OK

Lines 295-302: Missing elements in the Env description are: The hydrophobic region in LP, the RBD in SU, and fusion peptide in TM.

Overall, the authors should refer to two papers (Schulze et al. 2011, and Rua et al., 2012) that had performed similar analysis that will help them to provide a comprehensive description of the new sequence.

Thanks for the suggestion. We used both papers for identifying motifs in our original submission. However, neither the Schulze nor the Rua articles show these additional motifs. Rua describes the hydrophobic tryptophan (W) residues being in the first 15 amino acids, which differs from that of Lindeman and Goepfert CTMI 2003 chapter) which shows them at pos 86/86 of LP (SP). The WXXW motif in SFVhpi is also within the first 15 amino acids at positions 10-13. In comparison to PFV we located a putative RBD in SU and the fusion peptide in TM. We have added this information to the results.

I do agree that the two papers (Schulze et al. 2011, and Rua et al., 2012) do not describe all motifs. I proposed them as model to describe new sequences.

Lines 339-346: Analyses of recombination. The authors should describe the sequences used for recombination analysis and the rational for their choice. We suggest that they expand the number of sequences. Why do the author limit their analysis to the concatamer? This strategy is expected to decrease the probability of detecting a recombination event.

SFV recombination has been described in two contexts. Some recombinant SFV infect a single or few NHPs/ Their parental strains are identified and prevalent in the studied NHP population (or their prey). The recombination is probably a recent event. The second context is an ancient recombination event that may have generated the bimorphic env genes found in feline FV and in SFV infecting chimpanzee, macaques, gorilla and AGM. The parental strains are unknown. This genetic diversity is functionally relevant as this region is targeted by neutralizing antibodies (Zemba et al., 2000; Lambert et al., 2018). Furthermore, env swapping is an important event in the biology and evolution of the retroviruses. The env gene fragments described as parental/recombinant in Galvin et al. (2013) or conserved/variant by Richard et al. (2015) should be aligned with those of SFV species comprising two genotypes. These alignment and phylogenic analysis will allow the authors to describe whether the SFVhpi env fragments are more related to one or the other 2 env genotypes from apes SFV.

Please see lines 154-162 of Methods where we describe the sequences used and the rationale for using a concatamer. Briefly, we used the concatamer in order to maximize the molecular information across the coding regions with the most similarity to other SFV, while also ensuring a high quality alignment. This approach is common in the analysis of slow evolving cell-associated retroviruses. Using the entire genome creates alignment challenges because of highly divergent regions of the genome, which don’t align well and are then gap-stripped using SimPlot to evaluate recombination. Furthermore, recombination occurs in areas of homology and not high divergence and has mostly been reported in env, gag, and RT of closely related species. We have completed the additional recombination analyses using the sequences from the Galvin and Richard articles as suggested and have added this information to the paper. These analyses still did not show recombination in env for SFVhpi.

 Ok

Lines 450-451: “Comparison of LTR in the host and those obtained by culture are needed.” This sentence is erroneous, as the comparison has been reported in reference 56.

Sorry for any confusion. Our statement refers to the current study where these comparisons of the SFVhpi LTRs are still needed but which are beyond the scope of the current study.

OK

Specific comments

Line 27: “fills an important knowledge gap in SFV biology” sounds like an overstatement as no functional studies are presented in the paper. Please rephrase.

We used biology in a general sense, since the term encompasses a wide array of information. We have changed it to genetics.

OK

Lines 60-61: “the generation of database” is useless.

We have re-phrased the sentence to read “The design of molecular methods for the accurate identification of SFV infection requires a database containing representative sequences from divergent SFV lineages”.

OK

Lines 84-85: “The researchers were unable to obtain viral sequence” sounds awkward. First, it does not describe the experiment, but the people who carried the test; secondly word use deflates the contribution of the researchers. Please rephrase.

We have re-phrased the sentence as “A recent study reported a high seroprevalence of SFV in gibbons from Cambodia although the seropositive samples were not SFV PCR positive”.

 


Comments for author File: Comments.pdf

Author Response

Reviewer 1:

Comments and Suggestions for Authors

 

Shankar et al. obtained the full length genetic sequence from an SFV strain isolated from

pileated gibbon. They compared this sequence to those of other SFV species, analyzed the

presence of functional motifs in genes and proteins and studied the evolutionary history of

this strain. This thorough presentation of the first SFVhpi sequence represents significant

knowledge for further basic virology and SFV diagnosis. The paper should be enhanced by a

better description of recombination analysis, a study of env gene variability, rigorous

checking of functional motifs, inclusion of recently obtained SFV sequences, adequate

citation of the bibliography, and use of a more fluent writing style.

Second review: Shankar et al. improved their paper. Some elements of their rebuttal are still not

convincing.

Thanks for agreeing that the manuscript was improved. We hope the new revisions are satisfactory.

 

Broad comments

Line 35: “the functional SFV genome is double stranded DNA rather than single stranded-

DNA”. This is an overstatement, as controversial data have been published. The two cited

reviews state that DNA genome is functional but do not mention data on the relative

infectivity of DNA and RNA genomes.

The evidence presented in Yu SF, Sullivan MD, Linial ML. Evidence that the human foamy

virus genome is DNA. J Virol. 1999;73(2):1565–1572. (cited in the review by Pinto-Santini

et al.) supports this fact that the particle-associated “DNA is sufficient for new rounds of

replication” (Yu et al) . We do not discuss relative infectivity of DNA and RNA genomes,

rather, we just state that the functional genome is double stranded DNA. Nonetheless, we

have removed the word “functional” from this sentence.

SFV genome can be DNA or RNA. The data from Yu et al. have not been confirmed by

others.

We have changed this sentence to state the genome can be DNA or RNA.

 

Lines 34 and 37: two redundant sentences are separated by a third one. Please edit this

paragraph and the whole manuscript for fluency.

We have checked these sentences and cannot find the redundancies the reviewer cites. We

have addressed areas we feel are appropriate and as suggested elsewhere by this and the other

reviewers.

Here they are:

For example, FVs use two promoters for gene expression,one in the long terminal repeat

(LTR) and one in the envelope (env) gene. In addition, FVs complete reverse transcription

within the virion before infection of a new host cell, such that the functional SFV genome is

double-stranded DNA rather than single-stranded RNA [1, 2]. To initiate infection, FVs use

multiple promoters, a trait seen in complex DNA viruses, but not in other retroviruses [3].

 

Thanks. We have removed the redundant multiple promoters information.

 

Lines 41-43: For the description of SFV infection in NHPs, the authors cite only their papers,

but neither a review, nor major papers from other teams. Lines 43-51: For the description of

SFV infection in humans, the authors cite their research papers and reviews, but no research

papers from other teams. In addition, Ref 22 is erroneous as it described SFV infection in

macaques and not in humans. To avoid skewing towards self-citation, the authors have to cite

the most recent review(s) and research papers from all teams if published after the review.

The majority of work on SFV co-evolution has come from our team. Hence, those articles are

naturally more citated. However, we now cite other SFV co-evolution articles, but not

exhaustively, and include or substitute review articles as suggested.

There are no change in the bibliography. The authors have not address the whole comment.

Thanks. This was an Endnote formatting issue. The original manuscript had 63 references whereas the revision has 76.

 

Lines 50-53: Please cite more recent reviews for the description of in vitro cytopathic effect

and of SFV infection in NHPs. The authors should add the two papers that search for medical

consequences in humans, published by Boneva et al. in 2007 and Buseyne et al. in 2018.

We have added the relevant citations as suggested.

One citation is missing

This may also have been an Endnote formatting issue. Both citations were included in the first revision as references 31, and 32.

 

Lines 68-75: The authors provide the list of isolates with full length sequences provided in ref

28. Additional complete sequences are available in Genbank, including SFV from Papio

Anubis, and from Brachyteles arachnoides. These should be used in the analysis.

Since we already have four representatives for SFV from New World monkeys, including a

spider monkey species (Ateles geoffroyi), as well as representatives from other Old World

monkeys, to provide a diverse data set for comparison, we feel that addition of these

additional sequences to the analysis for the purpose of this manuscript, will not change the FV

phylogeny or recombination results given the co-evolutionary history of FVs. For example,

we also have a complete baboon SFV genome (GenBank accession number MF472626) that

we included in the analyses and which did not change the results presented in our manuscript.

We removed the baboon SFV from the paper since we plan to publish that sequence in detail

elsewhere.

My comment was on the citation of available full length SFV sequence, not on the phylogeny

or recombination analysis. The text can be easily updated.

We are confused by the reviewer’s new request. Clearly, they requested these additional SFVs be included in the “analysis”. There is no need to cite these additional SFVs if they were not included in the analysis.

 

Lines 100-102: The treatment of PBMCs need to be described. Where the cells fresh? Frozen?

Stimulated with a mitogen? For how long? In what medium? What was the time lapse

between coculture initiation and ECP appearance?

Although this information is available in the cited reference, we briefly describe the culture

details.

OK

 

Lines 107-109: This sentence describes the first step of the process and is nevertheless placed

at the end of the paragraph. Please described the work in a logical order.

We have changed the order as suggested.

OK

 

Tables 1 to 3: Why do the authors consider SFV from only two chimpanzee subspecies, while

three have been described?

We have used representative sequences from each group of primates for the purpose of

comparative phylogeny rather than using all available sequences to minimize the

computationally intensive phylogenetic analyses. We feel this approach provides sufficient

molecular information for our study.

Description of viral promoters: The IP is described in the middle of the paragraph describing

Env, in accordance to its location, but not to the biological function. Please describe IP in a

paragraph following the description of LTR. Undescribed elements are: Tas responsive

elements, splice donor site, the lack of central ppt, dimerization sequence, Poly A.

We have moved the paragraph as suggested. We also now add on page 8 the locations of the

additional elements noted when they were identifiable.

OK

 

Lines 301-302: the location of the ER retention signal is erroneous.

We provided the location of the ER RS using the TM codon positions and not the entire Env

coding region. We have now added the Env codon positions to avoid confusion.

OK

 

Lines 295-302: Missing elements in the Env description are: The hydrophobic region in LP,

the RBD in SU, and fusion peptide in TM.

Overall, the authors should refer to two papers (Schulze et al. 2011, and Rua et al., 2012) that

had performed similar analysis that will help them to provide a comprehensive description of

the new sequence.

Thanks for the suggestion. We used both papers for identifying motifs in our original

submission. However, neither the Schulze nor the Rua articles show these additional motifs.

Rua describes the hydrophobic tryptophan (W) residues being in the first 15 amino acids,

which differs from that of Lindeman and Goepfert CTMI 2003 chapter) which shows them at

pos 86/86 of LP (SP). The WXXW motif in SFVhpi is also within the first 15 amino acids at

positions 10-13. In comparison to PFV we located a putative RBD in SU and the fusion

peptide in TM. We have added this information to the results.

I do agree that the two papers (Schulze et al. 2011, and Rua et al., 2012) do not describe all

motifs. I proposed them as model to describe new sequences.

Thanks. We have already made the changes in the first revision.

 

Lines 339-346: Analyses of recombination. The authors should describe the sequences used

for recombination analysis and the rational for their choice. We suggest that they expand the

number of sequences. Why do the author limit their analysis to the concatamer? This strategy

is expected to decrease the probability of detecting a recombination event.

SFV recombination has been described in two contexts. Some recombinant SFV infect a

single or few NHPs/ Their parental strains are identified and prevalent in the studied NHP

population (or their prey). The recombination is probably a recent event. The second context

is an ancient recombination event that may have generated the bimorphic env genes found in

feline FV and in SFV infecting chimpanzee, macaques, gorilla and AGM. The parental strains

are unknown. This genetic diversity is functionally relevant as this region is targeted by

neutralizing antibodies (Zemba et al., 2000; Lambert et al., 2018). Furthermore, env swapping

is an important event in the biology and evolution of the retroviruses. The env gene fragments

described as parental/recombinant in Galvin et al. (2013) or conserved/variant by Richard et

al. (2015) should be aligned with those of SFV species comprising two genotypes. These

alignment and phylogenic analysis will allow the authors to describe whether the SFVhpi env

fragments are more related to one or the other 2 env genotypes from apes SFV.

Please see lines 154-162 of Methods where we describe the sequences used and the rationale

for using a concatamer. Briefly, we used the concatamer in order to maximize the molecular

information across the coding regions with the most similarity to other SFV, while also

ensuring a high quality alignment. This approach is common in the analysis of slow evolving

cell-associated retroviruses. Using the entire genome creates alignment challenges because of

highly divergent regions of the genome, which don’t align well and are then gap-stripped

using SimPlot to evaluate recombination. Furthermore, recombination occurs in areas of

homology and not high divergence and has mostly been reported in env, gag, and RT of

closely related species. We have completed the additional recombination analyses using the

sequences from the Galvin and Richard articles as suggested and have added this information

to the paper. These analyses still did not show recombination in env for SFVhpi.

Ok

 

Lines 450-451: “Comparison of LTR in the host and those obtained by culture are needed.”

This sentence is erroneous, as the comparison has been reported in reference 56.

Sorry for any confusion. Our statement refers to the current study where these comparisons of

the SFVhpi LTRs are still needed but which are beyond the scope of the current study.

OK

 

Specific comments

Line 27: “fills an important knowledge gap in SFV biology” sounds like an overstatement as

no functional studies are presented in the paper. Please rephrase.

We used biology in a general sense, since the term encompasses a wide array of information.

We have changed it to genetics.

OK

 

Lines 60-61: “the generation of database” is useless.

We have re-phrased the sentence to read “The design of molecular methods for the accurate

identification of SFV infection requires a database containing representative sequences from

divergent SFV lineages”.

OK

 

Lines 84-85: “The researchers were unable to obtain viral sequence” sounds awkward. First, it

does not describe the experiment, but the people who carried the test; secondly word use

deflates the contribution of the researchers. Please rephrase.

We have re-phrased the sentence as “A recent study reported a high seroprevalence of SFV in

gibbons from Cambodia although the seropositive samples were not SFV PCR positive”.

The reviewer did not provide comments on our revision.

 

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