The cow udder is a potential coinfection site for influenza A viruses

Reviewer #2 (Public review):

The authors use a library of influenza A viruses from different strains, classified in lab-adapted, human, avian, and swine according to the animal from which they were isolated. They propose that the cow mammary gland serves as a mixing vessel for influenza A viruses. As a first approach, the authors assess susceptibility to infection across different cell types, including continuous and primary cell lines, bovine mammary cells, and mammary explants. All these cells support polymerase activity. Then, they analyzed changes in the bovine virus's viral fitness relative to an avian precursor. The authors use single-gene replacement to study whether and which RNP segments improve viral transcription. As part of this section, they also test IFN-specific antagonism by NS1 to assess the input of segment 8. Quantitative glycomic analysis was performed on the continuous bovine mammary cell line to demonstrate the presence of both a2,3 and a2,6, which is consistent with their observation that these cells can be co-infected with human and avian IAVs simultaneously. The main question, however, is: what is the glycome in the explants, or directly from tissues?

Overall, the manuscript is clearly written and provides new insights into the behaviour of the cattle isolate, now compared with a representative group of model or precursor HAs of different origins.

It would be great if a consistent nomenclature for the IAV strains could be used in the study. There is a mix of origin (Texas), animal from which the virus was isolated (mallard), or abbreviations that do not follow guidelines (IAV07). Are the USSR and Udorn not lab-adapted?

The experimental setup includes bovine mammary primary and continuous cells, as well as mammary explants. Some of the most significant differences, for example, in viral fitness studies and co-infection experiments, are observed in these explants. Perhaps there could be some additional focus on this observation. The implications in comparison to the results obtained in cultured cells could be described. How will the human and other HA subtype viruses fare in the explants?

Comments on revised version.

The authors have satisfactorily addressed the reviewers' comments.

Reviewer #3 (Public review):

Summary:

This excellent manuscript by Pinto, Sharp, and colleagues examines bovine tissue tropism for influenza viruses. They find that bovine flu, as well as other strains, have strong replication in mammary tissue. They also map the genetic changes to influenza that improve replication in bovine cells. Overall, the study is well designed and executed and the results are very timely.

Strengths:

(1) The experiments are well-controlled.

(2) The figures are well-constructed and easy to follow.

(3) The Methods and legends are detailed, with sufficient information.

Comment on revised version.

The authors have strengthened the manuscript by addressing comments from the three reviewers and I have no additional concerns/suggestions.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

Here, Pinto and colleagues set out to investigate whether the cow udder is a potential mixing site for the influenza virus. The authors have demonstrated that bovine mammary epithelial cells can be infected with both avian and human influenza A viruses, supporting the idea that the cow udder may be a potential site for reassortment. Furthermore, they demonstrate that the bovine-adapted IAV replicates to similar titers in avian epithelial cells when compared to an AIV precursor virus. Thus, suggesting there is no fitness trade-off, and confirms the potential for spill-back of the cattle B3.13 into poultry, which has already been observed. Overall, I believe the authors achieved their aims. However, there are instances in which the results do not entirely support the conclusions (noted in weaknesses). Given the ongoing questions surrounding highly pathogenic avian influenza A virus in dairy cows, this work provides valuable evidence for the potential of the cow udder as a site of reassortment. These findings highlight the need for surveillance of influenza A virus incursions into livestock species, particularly cows. Some specific strengths and questions regarding weaknesses have been outlined below.

Strengths:

(1) The authors use a diverse range of cell types and influenza A virus strains, as well as a wide range of techniques to address the questions at hand.

(2) The use of cells from multiple bovine breeds for the MAC-T, bMEC and explants suggests the phenomenon is not unique to a single breed.

(3) The results suggesting there is no fitness trade-off for Cattle Texas in an avian host are interesting, and confirm the potential for spill-back of the cattle B3.13 into poultry, which has been observed.

Weaknesses:

I have listed my complete questions/concerns below. However, there are two main weaknesses of the article in its current state. Firstly, there is no apples-to-apples comparison in terms of determining a preference for IAV to infect the cow udder over other organs (Q4). The mammary gland and respiratory tract are represented by epithelial cells, but for other organs, fibroblasts were chosen. I think the fairer comparison would be to compare epithelial cells from different organs to demonstrate a preference for the mammary gland. Secondly, the main premise of the article relies on bMEC and MAC-T (primary and immortalised mammary epithelial cells), facilitating higher viral growth than the cells from other organs. Yet throughout the article, a 10x higher dose of IAV is used in the bMEC cells compared to everything else (Q6). This raises the question of how much of the results are due to a preference for the mammary epithelial cells, and how much is simply due to the increased dose.

(Q4) When we set out to test if cow mammary gland cells were particularly susceptible to IAV infection compared to other bovine cell types, we used what was available in the Roslin Institute – a mix of primary and continuous cells from various anatomical sites: three epithelial cell types (two mammary, one respiratory tract) two immune cell types and four sets of fibroblasts from various organs. Given the representation of different anatomical sites, cell types and differentiation statuses, we considered this a suitably diverse panel with which to characterise infection dynamics of a broad range of IAVs, before more focussed investigations using the bMEC and explant tissues. Both mammary epithelial cell types grew our library of influenza challenge strains significantly better than the BAT-II respiratory epithelial cells, as well as the two immune cell types and all four fibroblast populations. Of the fibroblast cells, those derived from the brain grew IAV significantly better than the skin and turbinate fibroblasts, while blood-derived macrophages grew virus significantly better than the lymphocytes and non-brain fibroblasts. So there are “apple-to-apple” comparisons as well as apple-to-pear comparisons that give significant differences. We therefore think that our conclusions (in the abstract) that mammary cells are particularly replication competent for IAV, (at the end of the introduction) that “a wide range of cow-derived cells are susceptible” and that (in the results section) that “mammary cells showed the highest susceptibility” are justifiable. However, we agree that testing a wider variety of epithelial cells would be useful and have added text to the Discussion (lines 224-228) to acknowledge this.

(Q6) We used a higher MOI for bMECs because test experiments with WT PR8 and the Cattle Texas 6:2 reassortant virus showed that MOI 0.01 infections gave more variable results than those run at MOI 0.1, perhaps because of the intrinsic variability of mixed primary cell populations. However, the end-point titres between the two conditions were not significantly different, so we therefore chose to go with the higher MOI. Accordingly, we do not think this choice is a confounding issue. This explanation (line numbers 340-345) and a new Supplementary Figure 11 showing the results of the two MOI tests have been added to the manuscript.

Reviewer #2 (Public review):

The authors use a library of influenza A viruses from different strains, classified in lab-adapted, human, avian, and swine according to the animal from which they were isolated. They propose that the cow mammary gland serves as a mixing vessel for influenza A viruses. As a first approach, the authors assess susceptibility to infection across different cell types, including continuous and primary cell lines, bovine mammary cells, and mammary explants. All these cells support polymerase activity. Then, they analyzed changes in the bovine virus's viral fitness relative to an avian precursor. The authors use single-gene replacement to study whether and which RNP segments improve viral transcription. As part of this section, they also test IFN-specific antagonism by NS1 to assess the input of segment 8. Quantitative glycomic analysis was performed on the continuous bovine mammary cell line to demonstrate the presence of both a2,3 and a2,6, which is consistent with their observation that these cells can be co-infected with human and avian IAVs simultaneously. The main question, however, is: what is the glycome in the explants, or directly from tissues?

We report quantitative glycomics for the primary bovine mammary epithelial cells as well as the continuous line the referee highlights. However, we agree with R2 that a detailed glycomic analysis of primary bovine mammary tissue would allow a better understanding of the actual glycosylation status in vivo. This has been undertaken by the authors and is available as a bioRxiv preprint. This is now cited (ref 25) in the relevant part of the results (line 184-185)

Overall, the manuscript is clearly written and provides new insights into the behaviour of the cattle isolate, now compared with a representative group of model or precursor HAs of different origins.

It would be great if a consistent nomenclature for the IAV strains could be used in the study. There is a mix of origin (Texas), animal from which the virus was isolated (mallard), or abbreviations that do not follow guidelines (IAV07). Are the USSR and Udorn not lab-adapted?

We chose the abbreviated names for a variety of reasons. Partly from common usage (e.g. PR8, Udorn), partly for consistency with other already published papers from the FluTrailMap consortia (e.g. Cattle Texas; Dholakia et al 2026), partly to make diversity obvious in certain figures (e.g. H3N1, H5N2 etc) and partly to avoid confusion between viruses that originate from the same geographic area (e.g. AIV07, AIV09, H5N8-20 etc which are all A/Ck/England/isolate numbers). Overall, we found it more confusing to use the expanded nomenclature. Re AIV07 which the referee criticises for not following naming guidelines – if this is a reference to the EURL nomenclature, AIV07 is the abbreviation for the specific virus A/Chicken/England/053052/2021, our representative virus for EURL genotype EA-2020-C, as we say in the text. This nomenclature has now been added to Table 1, to provide a fuller cross-reference for all the names.

As to whether USSR and Udorn are lab-adapted – that depends on definitions. There is a continuum of adaptive changes and/or sequence drift starting from the very first growth cycle of an isolate in the laboratory. The viruses we define here as lab adapted are ones that have been deliberately adapted to other host species or which have very long passage histories in multiple laboratory systems resulting in known functionally significant changes; for example, one lineage of PR8 was passaged 77 times in mice, 717 times in cell culture, 30 times in chick embryos, 5 times in ferrets and a further 50 times in chick embryos (https://www.medscape.com/viewarticle/812621_3?form=fpf), rendering it unarguably lab-adapted. We admit that A/USSR/77 and A/Udorn/307/1972 are probably further along this adaptive pathway than more recent isolates such as A/Norway/3433/2018, but are unaware of any specific reason that would put them into our lab-adapted category.

The experimental setup includes bovine mammary primary and continuous cells, as well as mammary explants. Some of the most significant differences, for example, in viral fitness studies and co-infection experiments, are observed in these explants. Perhaps there could be some additional focus on this observation. The implications in comparison to the results obtained in cultured cells could be described. How will the human and other HA subtype viruses fare in the explants?

We agree that this is an important and interesting question, and had already tested the strains we used for co-infections: human seasonal pdm09 H1N1 “Norway” and low pathogenic avian influenza “H3N1”, in the mammary explants. Both replicate the avian virus to 20-fold higher titres. We have added this information to the revised manuscript as new Figure panels S6E-H, called out on line 200-201 of the results.

Reviewer #3 (Public review):

Summary:

This excellent manuscript by Pinto, Sharp, and colleagues examines bovine tissue tropism for influenza viruses. They find that bovine flu, as well as other strains, has strong replication in mammary tissue. They also map the genetic changes to influenza that improve replication in bovine cells. Overall, the study is well designed and executed, and the results are very timely.

Strengths:

(1) The experiments are well-controlled.

(2) The figures are well-constructed and easy to follow.

(3) The Methods and legends are detailed, with sufficient information.

Weaknesses:

(1) A comparison to human cells would strengthen the overall impact of the results. Are human mammary cells also uniquely susceptible to influenza? Are bovine mammary cells special in some way?

This is an interesting question, but we have not tested mammary gland cells from humans (or any other species of mammal). We have however reported elsewhere (Dholakia et al., Nat Commun. 2026 Jan 16;17(1):1603. doi: 10.1038/s41467-026-68306-6.) that Cattle Texas grows well in a variety of human respiratory cells. Here, we are considering the bovine mammary organ as a potential reassortment site for IAVs because of the ongoing viral mastitis epidemic in US dairy cattle; human mammary organs seem unlikely to create a similar opportunity.

(2) For the virus infection studies with segment 8 swaps, it should at least be noted that some of the phenotypes could be driven by NEP.

We agree; as Table S1 indicates, NEP has two changes (one shared with NS1) between AIV07 and our B3.13 isolate, so we should not have conflated segment and NS1. We have changed the text to acknowledge this throughout the results (lines 127, 137 and 149) and in the discussion (line 243-244).

(3) The data demonstrating that bMEC can support co-infection are compelling and important, but would be strengthened with a comparison from a different cell type or species. Do mammary cells uniquely support higher co-infection?

We have data showing that co-infection also occurs in the continuous MAC-T udder cell line and have now included these data in a revised Figure 4D (described/called out on lines 198-206). We have not tested bovine cells from other organs for co-infection potential as they do not seem to be significant sites of infection in vivo.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) How nasal turbinate and cardiac fibroblasts are acquired/cultures is missing from the methods.

Apologies for the omissions and thank you, because rectifying this brought to light an error in cell naming. The cells originally called bovine cardiac fibroblasts were in fact a second independent preparation of skin fibroblasts. We have corrected the labelling in Figs 1, 2 and S2. The nasal turbinate cells were bought in from ATCC (code CRL-1390). This information has now been added to the methods (line 310).

(2) Please specify what cell types make up the 2D enteroids, the mammary explants and the nasal turbinates.

The composition of the 2D enteroids is described in detail in reference [60]. In precis, they are comprised predominantly of epithelial cells, including Paneth, goblet and enteroendocrine cells, as well as stem cells. This information has been added to the methods (lines 374-376) The mammary explants include duct epithelium, connective and muscle tissue, defined by H&E staining of cut sections (see new Figure S12 and text added to the methods on lines 384-386). We have also added the person who did the histology (Rebecca Ross) as an author and to the credit taxonomy (line 877). The nasal turbinate cells appear to be predominantly fibroblast morphology (Methods line 310-311).

(3) Epithelial cells are the main target for IAV, so why were fibroblasts chosen as a comparison to mammary epithelial cells? This needs justification in text. Without justification, how much can be attributed to the results being mammary-specific, rather than epithelial-specific? The brain (choroid plexus epithelium), heart (epicardium) and skin all contain epithelial cells.

We think this query is what the referee calls “Q4’ in the public part of their review. Please see our answer above.

(4) Figure 1, S1, S2 and S3 seem to suggest that mammary cells are more susceptible to IAV infection than cells from other organs. But Figure 2 demonstrates that when it comes to Cattle Texas and AIV07, most of the cell types show high viral titers. If the question is about whether the cow udder is the primary mixing site, would it not be more relevant to investigate which cell type facilitates the best growth of the potential precursor viruses, similar to Figure 4A?

Figs 1, S1, 2 and 3 all use “full” viruses whereas Fig 2 uses 6:2 reassortants between PR8 and the HPAIVs for biosafety reasons. WT PR8 replicates well in most of the bovine cells tested (Figs S1-3) so we do not see any contradiction. Figure 2 examines the contributions the internal genes make to replication in bovine cells. Re the question over the udder being a potential mixing site – this is where the virus is replicating in the real world; at least in part because of the transmission mechanism, but also because the mammary gland epithelium is highly susceptible to infection, as we show.

(5) It is misleading to compare the bMEC infection of MOI 0.1, to all other infections of MOI 0.01. This is a consistent problem throughout the article - Figures 1, S1, 2, 4. Why was the bMEC infection at a 10x greater dose? The main premise of the article relies on bMEC and MAC-T (primary and immortalised mammary epithelial cells), facilitating higher viral growth than the cells from other organs. If we compare the MOI 0.01 experiments alone, then the evidence relies on the immortalised MAC-T cells, compared to primary cell types. In this case, how much can be said about it being mammary specific, rather than immortalised vs primary? I do wonder, for example, how the epithelial nasal turbinates or type II pneumocytes would compare to the primary mammary epithelial cells if they were at the same MOI.

Please see our answer to this query earlier in the rebuttal.

(6) Why are the 2D enteroids excluded from Figure 1?

We had limited supplies of a difficult-to-grow cell model, so we only used them to test the 6:2 viruses (Fig 2).

(7) The colour scheme for Figure 3 is confusing. In Figure 3A, blue indicates European ancestry, and yellow represents North American ancestry. However, in Figure 3B, these colours now mean something different. To a reader, when there is a colour-coded schematic, it is instinctual to think that this then corresponds to the following panel(s). Since consistently throughout the article, yellow has been used for Cattle Texas, and blue has been used for AIV07, I would suggest choosing different colours to represent European and North American ancestry in Figure 3A

We’ve changed the figure as the referee suggests and modified the Fig 3 legend accordingly (line 895)

(8) I am unsure about the conclusions drawn from the results of Figure 3B. In the results, it is framed as trying to determine which segments contributed to the improved activity of Cattle Texas compared to AIV07. In lines 110-111, "... PB2 or PA from AIV07 significantly decreased Cattle Texas minireplicon activity". If PB2 is indeed significant, there is a missing yellow asterisk in Figure 3B.

Apologies, there was indeed a missing asterisk on the figure; now added.

Given the significance of PA, why was it not investigated in terms of growth kinetics similar to Figure 3C? Was it overlooked because it doesn't have a North American ancestry? The results of Figure 3B suggest that the 4 amino acid mutation in PA has significantly contributed to changes in polymerase activity.

The PA changes do indeed matter for minireplicon activity – the key change is K497R, as detailed in our related publication in Nat Comms (citation 17). However, it is less important than changes in PB2, and the PA segment swap by itself has little effect on overall virus replication.

(9) Similarly, in Figure 3C and lines 116-117, the error bars on the graph are overlapping at 48 hours, suggesting no difference in overall replication. The kinetics are slowed for AIV07 seg1-3, but not for AIV07 seg 1, indicating PB1 does not have an effect. This would then suggest that something in segment 2 or 3 contributes to the slowed kinetics in Figure 3C, which, from the Figure 3B results, is unlikely to be due to PB2. While it was not reassorted, the PA segment is potentially the driver, with its 4 amino acid mutations. I think it is worth performing growth kinetics with and without these 4 amino acid changes in PA.

We agree that visually on a log₁₀ scale, the titres of the “WT” 6:2 Cattle Texas and 5:2:1 segment 1 reassortment appear close, but the average titres are 5 and 7-fold different at 24 and 48h respectively, while a 2-way ANOVA with Dunnet’s multiple comparison post-test gives statistical significance at 48h. We have added this information to the figure and its legend (lines 905-906).

(10) In Figure 3C and E, why did you choose to perform the growth kinetics in the immortalised cell line, when you have access to primary cells? The primary cells would be a more accurate representation of what happens in situ.

The primary cells were difficult to work with and only available intermittently, so we used what was available at the time.

(11) In lines 145-147, "thus overall, the reassortment event that replaced segments 1, 2 and 8 alongside drift adaptations in segment 3 may have contributed to the ability of the B3.13 genotype virus to infect cattle". This is not clearly supported by the evidence presented. In terms of segment 1/PB2, the growth kinetics of Figure 3C have overlapping error bars at 48 hours. Where is any evidence presented for the role of segment 2/PB1? There is no change in Figure 3B.

The referee is correct, calling out seg2 here was an error; we have revised the text (line 144).

Segment 3 is overlooked in Figure 3 (as highlighted in Q9 and 10), and shows no difference in Figure S4.

Please see response to Q8; we think segment 3 contributes via PA adaptation, not via PA-X.

(12) In line 146 "... drift adaptation in segment 3". Make it clear here that you are talking about genetic drift. However, is this likely to be genetic drift? The 4 amino acid mutations are shown to have a significant impact on polymerase activity in Figure 3B, and in Figure 3C, PA potentially contributes to the reduced kinetics. When there are amino acid mutations that correspond to a beneficial phenotypic change, attributing this to drift alone rather than host adaptation is strange.

Yes, wording clarified (line 145). “Drift” was used to distinguish it from reassortment but we agree this was an incorrect term in the context.

(13) Figure 4A MAT-C cells: this is ostensibly the same experiment as Figure 3C in terms of the Cattle Texas and AIV07 viruses. If this is the case, how can you explain the difference in kinetics and overall titer? In Figure 3C, Cattle Texas reaches 10^6, and in Figure 4A it reaches almost 10^9. That's almost 3 log difference. Similarly, in Figure 3C Cattle Texas reaches 10^3, but in Figure 4A it reaches 10^6, a 3-log difference. At 24 hrs, they have roughly a 3-log difference between them in Figure 3C, but in Figure 4A this difference is much smaller. As far as I can tell, these are the same viruses, same dose and same cell model. The t0 titer is also vastly different between the two experiments.

The experiments were done at different times (several months apart, so different cell passage numbers and/or serum batches) and by different people. We have no explanation other than biological variability. However, both groups of experiments include genuine biological replicates done over the course of 2-3 weeks, so in our view represent coherent tests within themselves.

(14) In Figure 4, why weren't the a-2,6 a-2,3 proportions analysed for the explants and/or used for the co-infection experiment? It showed the greatest difference between the Cattle Texas and precursor viruses in Figure 4A.

Our data on the proportion of 2,6 and 2,3 SA in bovine udder tissue are now available in a separate preprint (now cited as [25] in our MS). We did not use the explants for co-infection experiments because it would have been technically difficult to read out the outcome by flow cytometry.

(15) Figure 4D requires a supplementary figure demonstrating the gating strategy, including one of the samples as an example.

We have compiled a figure of this and added it as new Figure S13 (called out line 556).

(16) In Figure 4D, why was an MOI of 5 chosen instead of the MOI of 0.01 used throughout the article for MAC-T cell infection? An MOI of 5 (so in a co-infection, a total of 10 virus particles per cell) is completely overloading the cells. At this dose, 10% of the cells were able to be co-infected, but how representative is this of a real co-infection scenario? While it demonstrates it is possible, it potentially remains highly unlikely, similar to the discussion around the swine respiratory tract in lines 235-240.

We had also performed the co-infections at lower MOI (1) with very similar results – this is now included in Figure 4D. Furthermore, we redid the experiments at a lower MOI of 0.05 and still see co-infection; this now replaces the MOI 5 data in Figure 4D. The text has been revised accordingly (lines 202-205)

(17) In Figure 4D, why were immortalised cells used when primary bMEC and mammary explants are available? Primary cells would provide more convincing evidence for the potential of the cow udder to be a mixing vessel. Considering that throughout the paper, a 10x higher viral dose is used in the bMEC culture, I wonder if you would need a significantly higher MOI than 5 to produce similar results in a co-infection experiment. The bMEC also has a more even a-2,3 to a-2,6 ratio compared to MAC-T in Figure 4C.

The bMECs in the original figure are primary cells. In response to other queries, we now include data from the immortalised MAC-T cell line as well.

Reviewer #2 (Recommendations for the authors):

Figure 1A, the coloured underline to discriminate continuous and primary cells is lost upon printing... perhaps another way is better?

We have changed the primary cells to italic text to make the distinction clearer.

We have made some other minor changes to wording to correct grammatical errors or improve clarity as we went through.