PONE-D-20-07350

“Effect of bovine leukemia virus (BLV) infection on bovine mammary epithelial cells transcriptome profile.”

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This group published two BLV-related manuscript in 2018 (Stable infection of a bovine mammary epithelial cell line (MAC-T) with bovine leukemia virus (BLV).), and in 2019 (Effect of bovine leukemia virus on bovine mammary epithelial cells.). This work is a continuation of the previous studies while RNAseq was performed to observe the impact of BLV infection on the transcriptome of bovine mammary epithelial cells.

The authors are encouraged to revise this manuscript so that the readers can understand and follow your manuscript well. Each figure should have its own detailed figure legend. You do not have to leave the Figures in the end of this manuscript while put the title of figure inside of the main text. Large table such as Table-2 can be moved to the supplementary Tables.

This manuscript has two parts: the part-1 is about the RNAseq, and the part-2 is related to the BLV sequencing. It is my opinion that the authors may consider to remove the part-2 totally from this manuscript. You obtained a viral sequence of 8419 pb that represents 96.6% of the complete genome, and this is not enough for BLV as a retrovirus. Then, you use the sequence with 96.8% of coverage to perform phylogenetic comparison, and it is not valid to have the statement (showed a 99% homology with reported Japanese viral genomes).

Please focus on the part-1 of your research, and come up with more specific take-home messages from the analysis of the RNAseq (more information that what you have now: BLV infected MAC-T cells have an altered expression of INF I signal pathway and genes involved in defense response to virus, as well as a collagen catabolic process and some protooncogenes and tumor suppressor genes).

Reviewer #2: Title- just suggestion - to add that the transcriptomic analysis was done with the RNAseq method would be more informative

Abstract - line 21 - should be: the RNA seq data

Materials and methods

- Preparation of experimental material should be more precisely written- how the biological replicate of the experiment were prepared? Were the cultures of MAC-T and MAC-T BLV conducted and harvested in parallel? Was total RNA isolated from each repetition at the same time? These questions will be reflected in the comments on the results.

- The RNA seq data were prepared by the sequencing platform - where? any company name? If whole bioinformatic analysis were done by Authors or any Center for Informatics?

As far as I know for PLOS ONE Authors contributions is required

Results

Fig.1 and line 87-89 - will be more informative which program was used for compute the heat map ans some datails for scale bar is missing.

It is true that hierarchical analysis revealed that MAC-T samples segregate differently than MAC-T BLV but also showes that intra-group variability is high, only mactblv1 and mactblv2 are similar. In the results section RIN data are missing. Here the way of preparation of experimental material could be significant. The number of 352 genes differentially expressed might be real but there is some kind of doubt that intra-group variability can affect this. Please comment and explain...

Fig.2 and line 92-95

Will be more informative when samples on the PCA plot have the same, corresponding name as in the heat map as mactblv1....It seems that the top one red point probably showed differential RNA-seq quality? Please comment...

There is an information that the genetic variation between samples is related to ribosomal complex development

and ribosomal RNA metabolism and process- commentary on these results is not visible in the discussion. Why?

Line 97-98, tab.2 and 138 -the data should be compatible fold change or log2FC?

Line 123-126 and Figure 5 - Is not the aim of the study but if BLV sequence obtained from MAC-T BLV has been already phylogenetically characterised, it would be interesting to classify it in terms of genotypes. Moreover, the tree could also contain sequences from the United States. On a tree there is a mistake - the sequence LC164083 is not a Sheep but come from FLK-BLV cells.

Discussion and conclussion

Line 145 West Nile

It is surprising that Authors do not discuss their results with thealready mentioned papers Arainga et al and Brym, Kaminski; for example the CXCL8 proteins was also mentioned as DEG at Brym paper. The authors themselves have already done some expressive RTqPCR tests on this line - whether there was any reflection of the results in RNAseq? - for example TLR9.

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Reviewer #1: Yes: Chengming Wang

Reviewer #2: No

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“Effect of bovine leukemia virus (BLV) infection on bovine mammary epithelial cells RNA-seq transcriptome profile.”

PONE-D-20-07350R1

Dear Dr. Ceriani,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Maria del Mar Ortega-Villaizan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The reviewers comments have been adressed. Just check one more correction by Reviewer 2. The manuscript is ready for publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Appreciate the efforts from the authors to revise this manuscript. A great job by the authors to address this reviewer's comments.

Reviewer #2: Please check - line 59 - passage 35 and line 214 - 35 days after infection - whether this is compatible with each other?

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Chengming Wang

Reviewer #2: No