Dear Dr. Lail,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: No

Reviewer #7: No

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The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

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Reviewer #1: This manuscript outlines an interesting protocol for testing milk products for the presence of H5N1 in cattle. It is not clear from the data presented what the sensitivity and specificity of the technique might be. This information is needed to assess the value of the protocol from a diagnostic perspective, Please clarify the statement '....Human infections have also been observed in poultry workers after presumed spillover of HPAI into poultry from cattle, underscoring the zoonotic potential of this outbreak (9).- currently it does not make sense and is not supported by the reference https://www.cdc.gov/mmwr/volumes/73/wr/mm7334a1.htm. As presented, this study does not fully align with a 'One Health approach' which is usually considered to be a transdisciplinary integrated approach to understanding a complex problem vs a focused study on a technique applied to detecting a potential pathogen which is zoonotic. There is mention in the conclusions about further sampling of pig farms to detect AIVs but no real consideration of how such data could be used in epidemiological studies and risk assessments. Can this be further discussed ?

Reviewer #2: The manuscript entitled “Pasteurized retail dairy enables genomic surveillance of H5N1 avian influenza virus in United States cattle” is a comprehensive short study of the detection and molecular characterization of HPAIV H5N1 in retail milk in the USA. The study is descriptive and well written, it brings in a new protocol for NGS of retail milk and confirms a surveillance perspective: looking at bulk milk rather than at individual cattle heads would allow for a faster, cheaper and more global detection and characterization of dairy cattle HPAIV H5N1. I just have the following questions/suggestions:

- There seem to be discrepancies between numbers in the text and tables. While it is clear that the authors have tested 66 cartons in 2 months, positives are described as coming from 5 states in the abstract/text but seem to come from just 3 in Table 2. While 18 cartons are positive according to table S3, 10 are positive in Table 2 and 13 described as positive in the text. Please clarify. The Ct value indicated for carton 28 (68.48) also seems off.

- How did you set your thresholds? Both for the 20x sequence coverage chosen and for the 10% frequency for the variants search.

- Mammalian adaptation markers are known. It is a pity that the authors did not screen their sequence data for those (irrespective of the variants frequency actually, it would be quite informative in a One Health perspective as stressed in the Introduction and Discussion sections).

- Figure 2: the tree is not very informative. What do divergence percentages refer to? What is the scale? The Figures legends are very short and would deserve more info.

Reviewer #3: The manuscript presents a novel and impactful approach to H5N1 surveillance using retail dairy products, aligning with the One Health framework and providing valuable insights into the spread of the virus in U.S. dairy cattle. However, the study has some limitations, including sample representativeness, phylogenetic challenges, and technical difficulties in sequencing low-abundance viral RNA. Addressing these limitations in future work could enhance the utility of retail dairy surveillance for monitoring zoonotic pathogens. Overall, the study is a significant contribution to the field of genomic epidemiology and has important implications for public health and food security.

Authors may improve the discussion section by discussing the global implications of the findings and how the approach could be adapted for use in other regions or for monitoring other zoonotic pathogens. Also, they have to clearly articulate the limitations of using retail dairy for surveillance, such as sample representativeness, phylogenetic challenges, and potential for false negatives.

Reviewer #4: I think this study fills an important gap In One Health approach to an outbreak response considering the challanges to cooperate with farm facilities which are the sourse of an ongoing infections locally and nationally. The study shows efficient traceability of a viral disease in readlily available milk samples using their resources in the lab facility. They not only detected viral RNA in milk samples using RT-QPCR but also developed a method to amplify and sequence the genome of the virus to discover mutations and understand dispersal of the virus. Although the method is not perfect, the authors discuss potential limitations of their study. Their methodology could be applied to sample dairy products rather than just milk in future studies.

Reviewer #5: Thank you for wonderful research and data which you present on this paper. I understand how difficult it is to collect the data and summarize it as it might be difficult to track whether the sequence is from vRNA of single dairy milk or from vRNA of some dairy milks.

Therefore, I have some questions and recommendations regarding this paper:

1. Paragraph 6, you don’t need to mentioned “the home state of the researchers on this study”, instead you might need to give more other interesting point about Wisconsin besides “the second- largest diary producing state in the US”.

2. It is better to put a reference about the existing workflow from your work in SARS-CoV-2 sequencing, as you mentioned it in introduction.

3. It is better to give reference to “OHHLEP’s 4 Cs” strategy which you also mentioned it in introduction.

4. You mentioned in “sample collections” about the dairy products which you took as sample, can you explain about the percentage of each sample taken? How many percent of whole milk did you take and how many percent low-fat wilk did you take? Etc.

5. It is unclear about how you finally only mentioned about samples from 10 sample cartons from 66 samples which you previously mentioned.

6. Why did the observation only do in cartons 21,24, 46,63 and 65? Because we could see that segments’ coverage on cartons 5 and 6 are also over than 90%.

7. The discussion part is more like simple introduction rather than a discussion of result which you have.

Reviewer #6: Summary:

This study describes detection and characterization of H5N1 viral RNA from commercial pasteurized dairy products. This is very timely as the H5N1 outbreak in wild and agricultural animals and resulting spillover infections in humans are of increasing and high concern. This study finds that H5N1 can be detected and sequenced from commercial milk, showing a readily accessible data source for genomic surveillance efforts. While results described are somewhat limited and figures could use improvement, especially figure 2, the interest and timeliness of the study are high. Further, the efforts towards methodological and data openness and utilizing tools developed in other outbreaks (SARS-CoV-2) demonstrates an important framework for the H5N1 and future outbreak responses.

Major Comments:

The abstract could use a more comprehensive summary of results – e.g. qPCR results, more description of genome coverage (significant correlation with qPCR result?) and/or phylogenetic clustering of resulting sequences.

A Spearman correlation should be performed to support the following claim “The method yielded an overall average sequencing depth of 8,176x across all samples, with individual samples varying from 325x to 29,256x. This correlated with the input vRNA amount, with less input vRNA yielding lower depth in sequencing.”

The quality of the Fig. 2 phylogeny needs to be improved for readability/interpretation. If a tree at higher resolution with less node crowding cannot be made in Auspice, recommend downloading the tree and metadata files from Auspice and re-creating the tree. Several tree tips in Fig. 2 are white/gray – do these not have location metadata? Besides trait inference, did sequenced samples cluster with other specimens from the state? It appears possible for Michigan samples but not Colorado but given the low quality/crowding in the tree is difficult to see. A description of clades that sequenced samples fall into may help.

Further description of sub-consensus mutations could be informative as the samples are pooled from many animals and detection of low-frequency mutations could indicate whether H5N1 was derived from one or many individuals.

Minor Comments:

The caveat that the H5 qPCR standard curve was dsDNA, which is not ideal for RNA virus absolute quantification should be mentioned.

“resulting in viral titers in milk of at least 107 TCID50 per mL (10).” should be “milk titers up to 107 TCID50 per mL”. Could also cite https://www.nature.com/articles/s41586-024-07849-4

Cited preprint 13 is now published and should be updated, other preprints should be checked for publication status

The last paragraph of the introduction contains text more appropriate in the results and discussion sections, consider revising.

Was the Ct cutoff of 39 based on limit of detection or other assay testing?

Table 3 could be converted to a heatmap to improve readability

Mutation PB2:M631L is common in H5N1 sequences from dairy cattle. Was this mutation not prevalent during the screening time described here? Why this mutation was not observed should be explained.

Putative reasons for repeated sampling of rare mutants could be discussed (PB1:171, PA:24). Are the sequences used in “% of nextstrain H5N1 cattle outbreak sequences with mutation” largely from a different source, e.g. could these be mutations associated with milk? A brief description of the referenced nextstrain sequences would be informative : # of sequences, collection dates and locations.

In S3 table, dholab_carton_0028 result is 68.48, is this an error (50 cycles reported in methods)?

Reviewer #7: Summary

The manuscript by Lail et al entitled, “Pasteurized retail dairy enables genomic surveillance of H5N1 avian influenza virus in United States cattle” reports the detection and whole genome sequencing of H5N1 high pathogenicity avian influenza viruses in pasteurized milk purchased in Madison, WI. The authors reported 18/67 cartons that were positive by qRT-PCR and 10 nearly complete genomes that were submitted to a public database. A tiled amplicon approach was used by the investigators to achieve sequencing of genomes from pasteurized milk.

General comments

• Whole genome sequencing for influenza viruses have typically been done using a universal primer approach that takes advantage of the conserved non-coding regions of influenza viruses at the end of the genomes. These contain packaging signals. A main advantage of the universal primers is that it can work for all known influenza A viruses. However, this approach also amplifies defective genomes as it only requires annealing to the ends of the genome. As a result, longer segments tend to have poor coverage in the middle.

• Potentially, the tiled approach the authors report has an advantage of having even coverage. Unfortunately, there appears to be sections where coverage drops precipitously (Figure 1). The drops in coverage appear to be 250 bp and is thus, most likely a failure of a primer set to amplify its target.

• It would be highly beneficial to compare the tiled method to universal primer set approach. A comparison would enable future users to choose the most appropriate genome amplification approach.

Specific comments

• I suggest a title change to focus on the sequencing method since the sampling was relatively limited and based on convenience (non-systematic). The major strength of the paper is the use of a novel approach to sequencing influenza viruses. However, the title suggest that systematic surveillance was conducted throughout United States whereas retail dairy products were only purchased in Wisconsin.

• Can you comment on the timings of collection/purchases? Was milk purchased weekly, daily, etc? Was each carton a distinct brand (No need to mention brand/trade names. Please anonymize)?

• Please report proportion of samples positive by qRT-PCR since this will be of interest to readers.

• From the genomes sequenced, how does it compare to other isolates from the same state?

• “Because pasteurization neutralizes infectious virus…”

o It would be more appropriate to use the term “inactivates” instead of “neutralizes” since neutralization of virus commonly refers to the preventing infectious virus to bind to host cells, whereas inactivation is a general term that refers to eliminating the ability of viruses to infect a host.

• What is the rationale behind a 20X cutoff for read depth?

• Data in Table 3 can be made more digestible with a graph

• Throughout the paper, please report log copies/mL instead of copies/mL. It is typical for viral RNA titers to be reported in the logarithmic scale because differences in orders of magnitude are more biologically relevant.

• What is the rationale for the A/bovine/Texas/24-029328-01/2024? Where is this strain located in the phylogenetic tree? While it is temporally the earliest known isolate, it is not necessarily the most ancestral/closest to the last common ancestor. In other words, the first detection of a virus is not always the first actual spillover of a virus, although it can be. It would be informative to compare your sequences to each representative subclades within the H5N1 isolates from cattle. E.g. A255V may have expanded in the lineage of A/bovine/Texas/24-029328-01/2024 and was not found in the last common ancestor within the clade of cattle H5N1 viruses.

• Please note that there is significant reassortments for clade 2.3.4.4b. Comparisons of mutations is only useful if the segments share the same lineage. Since the cattle outbreak appears to be a result of a single spillover and subsequent farm-to-farm spread, this analysis is appropriate. This may not be the case if reassortments are detected in the phylogenetic analysis.

• Figure 1. I noticed that there are areas in the genome where coverage drops precipitously. What are possible explanations of this observation? Failure of a primer set to amplify its target?

• Figure 2. Please indicate where the samples sequenced are in the phylogenetic tree

• For future reference, use of concatenated genome comparisons works for influenza given that no reassortment has occurred as is the case for the cattle H5N1 outbreak.

• Fig S1. Please use a different color scheme for 2.3.4.4 clades. It is difficult to distinguish a/b/c/d/e/f/g/h

• Table S3. There is one entry (dholab_carton_0028) that has a Ct value of 68.48. This looks like a typo.

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Dear Dr. Lail,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 13 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Ayi Vandi Kwaghe, D.V.M., M.V.Sc., P.G.D.E. Ph.D., MPH, FETP

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #5: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #5: Yes

**********

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

Reviewer #1: N/A

Reviewer #5: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #5: (No Response)

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Reviewer #1: Yes

Reviewer #5: Yes

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Reviewer #1: This added section could be more clearly written i.e. ……..Swine are often considered a mixing vessel for recombination between bird, human, and swine flu strains (add reference). This is reflected in the strict biosecurity protocols in use on pig farms. Just like dairy farms, these operations have retail outputs (such as meat ?) which may prove helpful to monitoring these viruses. Performing (asymptomatic?) genomic surveillance (on healthy/asymptomatic animals) at the population level by sampling outputs (meat?) from these locations may (generate data to) help inform risk assessments. However, it is unlikely that this approach (i.e testing milk samples to monitor H5N1 in dairy cattle ?) can be simply reconfigured to swine or another system given the need for quick turnaround and traceable supply chains (please clarify what is meant here ). Dairy ? (Milk ) is pooled between many animals, often sold within a month of production, and contains a marker of where it was produced. It's possible that extending monitoring to other agricultural settings would require orthogonal (please clarify what is meant here) approaches like wastewater or air monitoring..’

Reviewer #5: (No Response)

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Reviewer #5: No

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Amplicon sequencing of pasteurized retail dairy enables genomic surveillance of H5N1 avian influenza virus in United States cattle

PONE-D-24-59493R2

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Ayi Vandi Kwaghe, D.V.M., M.V.Sc., P.G.D.E. Ph.D., MPH, FETP

Academic Editor

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