Genomic analysis of high pathogenicity avian influenza viruses from Antarctica reveals multiple introductions from South America

Domain: chemistry
Published: 2026-04-07
Editorial outcome: major_revision

Abstract
The spread of high pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b into Antarctica poses a major threat to polar wildlife. We report the detection of H5N1 in carcasses of eight species during the 2023-2024 and 2024-2025 austral summers in the South Shetland Islands: Antarctic shag, Antarctic tern, kelp gull, pintado petrel, Antarctic petrel, skuas, Antarctic fur seal, and southern elephant seal. Whole-genome sequencing, mutational profiling, and phylogenetic reconstruction revealed that the viruses detected in these hosts descended from distinct introduction events. One group of strains including complete and partial viral genomes from a gull, skuas, fur seals, an Antarctic tern, and a southern elephant seal clustered with H5N1 strains previously detected in marine mammals in South America and formed a polyphyletic lineage consistent with at least two independent introductions into Antarctica. A second group of strains including complete and partial viral genomes from petrels, shags, and skuas clustered with H5N1 strains previously detected in seabirds and marine mammals in South Georgia and with a previously reported HPAIV detection from Torgersen Island, Antarctic Peninsula. These findings reveal extensive epidemiological connectivity between South America and Antarctica, with South Georgia serving as a “stepping stone” for virus spread in the region.

Introduction
Global spread of the high pathogenicity avian influenza virus (HPAIV) subtype H5N1 clade 2.3.4.4b has caused unprecedented mortality among wild animals1. In late 2022, viruses of the B3.2 genotype were introduced to the Pacific coast of South America, disseminating rapidly and causing outbreaks in poultry and wildlife2–4. Phylogenetic reconstruction reveals that shortly after arrival to South America these viruses diverged into two sub-clades: an “avian” sub-clade that spread inland and was detected predominantly in poultry and terrestrial wild birds, and a “marine mammal” sub-clade that spread coastally and was detected predominantly in marine mammals and seabirds5,6. In October and November 2023, HPAIV H5N1 spread from South America onto the Subantarctic region, causing outbreaks in seabirds and marine mammals in South Georgia and Falkland (Malvinas) Islands7. Phylogenetic analysis revealed that the viruses from South Georgia were monophyletic, descending from the South American “avian” sub-clade, whereas the strains from the Falkland (Malvinas) Islands were polyphyletic, descending from the South American “marine mammal” sub-clade7.

As the virus disseminated in wildlife in South America and South Georgia, concerns were raised that it could spread further onto the Antarctic Peninsula8,9. The first reports of wildlife mortality suspected to be due to HPAIV H5N1 in the Antarctic region were made in December 202310, and several confirmed detections have been made along the Antarctic Peninsula since then10–13. During the austral summers of 2023/2024 and 2024/2025, we collected swabs and tissue samples from deceased wildlife in the South Shetland Islands, Antarctic Peninsula, to perform molecular testing and genomic characterization of HPAIV H5N1.

Results
We tested tissue and swab samples from 31 carcasses from 11 species (Supplementary Table 1), with 18 carcasses from 8 species testing positive for H5 influenza A virus: Antarctic shag (Leucocarbo bransfieldensis), Antarctic tern (Sterna vittata), kelp gull (Larus dominicanus), pintado petrel (Daption capense), Antarctic petrel (Thalassoica antarctica), skua (Stercorarius sp.), Antarctic fur seal (Arctocephalus gazella), and southern elephant seal (Mirounga leonina). In total, 18 complete genomes (from 12 individuals) and 7 partial genomes (from 6 individuals) were recovered (Table 1). A maximum likelihood tree for the HA gene segment including globally-representative Gs/Gd clade 2.3.4.4b strains during 2021–2024 revealed that all viruses detected in the Subantarctic and Antarctic region to date stem from the viruses that circulated in South America since 2022 (Supplementary Figure 1). Furthermore, all complete genomes were of the H5N1 subtype with the B3.2 genotype; partial genomes were also in agreement with this classification.

Phylogenetic reconstructions of the concatenated whole genomes indicated the viruses recovered in this study belong to two clearly distinct groups, “a” and “b” (Figure 1 and Supplementary Figure 2). Phylogenetic group “a” comprised 10 complete genomes (recovered from 3 Antarctic fur seals, 1 kelp gull, and 3 skuas) that clustered with viruses previously detected predominantly in marine mammals in South America (i.e. the South American “marine mammal” sub-clade2). Sequence identity suggests that 6 partial genomes (recovered from 1 Antarctic tern, 2 skuas, 1 Antarctic petrel, 1 southern elephant seal) were also part of this group. Phylogenetic reconstruction of concatenated whole genomes suggestRed that this group could be further divided into four subgroups, each potentially descending from a separate introduction event to Antarctica (Figure 1a). However, separate phylogenetic analysis of the longer gene segments (PB2, PB1, PA, HA, NP) only corroborated three subgroups, whereas analysis of the shorter gene segments only corroborated one (MP) or two subgroups (NA, NS) (Supplementary Figure 3).

On the other hand, phylogenetic group “b” (Figure 1b) comprised 8 complete genomes (recovered from 1 Antarctic shag, 1 pintado petrel, and 3 skuas) clustered with viruses previously detected in seabirds and marine mammals from South Georgia and with a previously reported HPAIV detection in a brown skua (Stercorarius antarcticus) from Torgersen Island in Antarctica, which in turn descended from viruses that circulated in poultry and wild birds in South America (i.e. the South American “avian” sub-clade2). Sequence identity suggests that one partial genome (recovered from Antarctic shag) was also part of this group. This group was consistently monophyletic across all phylogenetic reconstructions (Supplementary Figures 2 and 3), suggesting that these viruses and the Torgersen Island virus descended from a single introduction event to Antarctica.

A comparison of cycle threshold (Ct) values of the Real Time RT-PCR revealed that birds could be classified as either oral shedders, which had higher viral loads (i.e. lower Ct values) in the oropharyngeal swabs than in the cloacal swabs, or fecal shedders, which had higher viral loads in cloacal swabs than in oropharyngeal swabs. Birds infected with group “a” strains were more frequently oral shedders (2 skuas, 1 tern) than fecal shedders (1 skua), whereas birds infected with group “b” strains were more frequently fecal shedders (1 skua, 1 petrel) than oral shedders (1 skua) (Supplementary Figure 4).

Mutational analysis revealed amino acid substitutions that distinguished phylogenetic groups “a” and “b” across all proteins except M1 (Figure 2 and Supplementary Data 1). Most group “a” strains recovered from both birds and mammals showed two key mammalian adaptation substitutions in PB2 (PB2-Q591K and PB2-D701N); another mammalian adaptation substitution in NP (NP-N319K) was detected in a group “a” strain recovered from the brain of an Antarctic fur seal (individual O2904). All group “b” viruses had a deletion of 17 amino acids in the neuraminidase stalk (positions 58-74). Lastly, two amino acid motifs were observed in the hemagglutinin cleavage site. The original PLREKRRKRGLF motif was found in most viruses from both phylogenetic groups, comprising 14 complete genomes and 5 partial genomes. However, the PLKEKRRKRGLF motif was detected in group “a” viruses recovered from three hosts: one Antarctic fur seal (three complete genomes from the same individual), one Antarctic tern (one partial genome), and one Antarctic petrel (one partial genome) (Supplementary Table 2).

Methods
Sample collection
This study was conducted in compliance with all relevant ethical and environmental regulations. No live animals were handled or sampled, as all biological material was obtained from carcasses. Research activities were carried out under license LW-20/24 issued by the Animal Use Ethics Committee of the Vice-Presidency for Research and Biological Collections (VPPCB), Oswaldo Cruz Foundation (Fiocruz). Sampling activities in Antarctica were authorized by the Environmental Assessment Group of the Brazilian Antarctic Program (PROANTAR). Fieldwork was conducted within the framework of the FIOANTAR and VIDEANTAR projects, with logistical support provided by the Brazilian Navy. All procedures followed strict biosafety protocols to ensure sample integrity and researcher safety. Supplementary Table 1 provides detailed information on the animals sampled. Oropharyngeal, oral, nasal, and cloacal swabs and brain tissue samples were collected in tubes with 1 mL of DNA/RNA Shield (Zymo Research, Irvine, USA) and stored at –80ºC. Samples were then transported with a strict cold-chain to the Laboratory of Respiratory Viruses, Exanthematous, Enteroviruses, and Viral Emergencies at the Oswaldo Cruz Institute (IOC) for further analysis.

RNA extraction and real-time RT-PCR
RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Cat. No. 52906) following the manufacturer’s instructions. Detection and subtyping of influenza A virus were performed by real-time RT-PCR using two assays: (i) the Biomanguinhos INFA/INFB/SC2 Multiplex Kit (ANVISA Registration No. 8014217006095), and (ii) the CDC Influenza A/B Typing Panel (Flu A/B Typing Kit, Cat. No. FluIVD03-6), targeting the matrix (InfA) and hemagglutinin (H5a and H5b) genes46,47 (Supplementary Data 1). H5a was developed to detect specific groups of H5 viruses of Asian origin (A/goose/Guangdong/1996 lineage and derivatives, which include many HPAI strains). H5b is complementary, covering genetic variations of H5 that could escape detection by the H5a assay. A viral load index was calculated for each sample by subtracting the Cycle threshold (Ct) value from 40.

Whole-genome sequencing and assembly
Whole-genome sequencing of positive samples was performed using a Multisegment Reverse Transcription-PCR (M-RT-PCR) amplification protocol48,49. The sequencing library was constructed using the COVIDSeq kit (Illumina, San Diego, USA). Sequencing was conducted on the MiSeq platform (Illumina), and genome assembly was carried out using the Iterative Refinement Meta-Assembler (IRMA) pipeline50. Clade classification was performed using NextClade v3.10.1 (https://clades.nextstrain.org/).

Bayesian evolutionary analysis
To place the viruses recovered from Antarctic wildlife in a global context, we downloaded 822 globally-representative HA gene sequences from Gs/Gd clade 2.3.4.4b viruses during 2021–2024 using NextStrain (https://nextstrain.org/). Phylogenetic relationships were inferred for the HA gene using the Maximum Likelihood methods available in IQ-Tree v. 2.4.0 with a GTR model of nucleotide substitution with gamma-distributed rate variation among sites. Publicly-available sequences of influenza A viruses from South America and Antarctica were obtained through Global Initiative on Sharing All Influenza Data - GISAID (https://www.gisaid.org/) and GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/) (Supplementary Data 2). Because this initial analysis confirmed that all Antarctic/Subantarctic strains were descendent from South American strains, subsequent evolutionary analyses omitted viruses from other continents. The temporal structure of the datasets in each segment was evaluated using TempEst v1.5.351 through a root-to-tip linear regression. The significance of the correlation between collection date and genetic distance was assessed using a Pearson correlation test, with adjustments for multiple comparisons made via the Benjamini–Hochberg method for false discovery rate control. Time-scaled phylogenetic trees were estimated using the Markov Chain Monte Carlo (MCMC) approach implemented in BEAST v1.10.452, with BEAGLE v4.0.053 to improve computational efficiency. Bayesian trees were reconstructed under the nucleotide substitution model selected by jModelTest v2.1.1054, the non-parametric Bayesian Skyline coalescent demographic model55, and a relaxed molecular clock model with a continuous-time Markov chain (CTMC) rate reference prior56. The molecular clock, the time to the most recent common ancestor (TMRCA), and the spatial dynamics of each viral segment were reconstructed using a discrete phylogeographic model57 implemented in BEAST v1.10.4. A symmetric diffusion model was intentionally adopted as a conservative framework, allowing directional patterns of viral spread to emerge from the data rather than being imposed by prior assumptions, particularly given the limited and uneven geographic sampling in Antarctica.

In addition to segment-based analyses, concatenated gene datasets were also constructed and evaluated under the same Bayesian phylogeographic framework to capture genome-wide evolutionary signals. Sequences were coded according to both their sampling locations and host. For each dataset, independent MCMC runs were performed and subsequently combined to confirm that all continuous parameters achieved an effective sample size (ESS) greater than 100–200, as verified using Tracer v1.758. The maximum clade credibility (MCC) tree was summarized with TreeAnnotator v1.1052. Trees were visualized using R v4.4.159 with the packages treeio v1.30.060 and ggtree v3.14.061.

Mutational analysis
The translated sequence of all eight gene segments were compared to evaluate the presence of amino acid substitutions using NextClade v3.10.1 (https://clades.nextstrain.org/). An early B3.2 genotype strain (A/turkey/South Dakota/22-008485-001/2022; GISAID accession code EPI_ISL_11971481)62 was used as a reference.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Here the authors present PCR detection and sequencing of sequencing of H5N1 virus from 3 animals on 2 Antarctic islands.\nUnfortunately, the presented results to not provide robust support for the conclusions.\nThere are major problems with this manuscript. Only 7 carcasses were sampled. The sample numbers are so small that only\nthe most simple conclusions could be made, particularly as the phylogenetic analysis is limited. For example, the authors\nrestrict their analysis to H5 sequences from South America and Antrarctica without any justification. And overall the datasets\nare not adequately described. The authors also describe phylogeographic analysis in the methods, but these are not\npresented in the manuscript.\nDespite these major flaws, the authors are making sweeping statements about the movement of the virus throughout South\nAmerica and into Antarctica. There is no support provided for statements such as “Our phylogenetic results support the\ngrowing evidence that South Georgia has played a significant role as a “stepping stone” for the spread of HPAIV in the\nSubantarctic and Antarctic region”.\nOverall this study lacks substance and significance. Sampling size is incredibly small and inadequate and just 3 new\ngenomes were generated in this study. This manuscript does not provide any new insights into the spread of H5N1 virus into\nwild animal populations or into Antarctica.", "reviewer_2": "This manuscript presents data on the recent detection of highly pathogenic avian influenza (HPAI) in Antarctic wildlife,\nincluding viral genome sequences suggesting several independent introductions of the virus into Antarctica. The manuscript\nis overall well written and the results are clear and of timely significance. There are however some points that could be\nclarified.\nMajor comments:\nLine 231 - Please clarify which primers were used as in the cited reference they present several primers, and I did not find\nany obvious mention of the “H5a” and “H5b” labels.\nFigure 1 - Please explain the rationale for using PB2 opposed to other segments?\nCan you explain why you did not use a concatenated genome (after removing reassortants) for improved resolution as Uhart\net al., 2024, and Clessin et al., 2025 did? Also why you used a model assuming symmetric dissemination (line 257) when\nthe expectation is that the virus was at the time spreading mostly Southwards?\nI would suggest toning down the conclusion about the direct introductions from South America (e.g., line 99; header line 111,\nremove “directly”) as it is pointed out several times in the manuscript that data is sparse, and notably we have no idea of\nwhat is happening at sea, and considering how long the branches are on the phylogenetic tree.\nThe focus on biosecurity in the context of human movement (lines 49, 198 and 212) feels disconnected from the results\ngiven that animal movements are suspected to be responsible for spread (e.g., discussed in line 176).\nMinor comments:\nLine 68 - Would it be possible to include information on the number of tested samples (instead of only the ones that returned\npositive) here? E.g., a reference to Supplementary File 2. This could help build knowledge over the likelyhood to detect the\nvirus in the system.\nLines 228-232 - Please clarify product # when possible (Qiagen kit, Biomanguinhos INFA/INFB/SC2 kit, CDC Influenza A\nkit…)\nLines 230-232 - Says “detection and subtyping were performed” two times with different methods. Please clarify.\nFigure 1 - Posterior probability for kelp gull in Figure 1b is rounded up compared to Supplemental Figure 1 tree –\nrecommend consistency to avoid unnecessary confusion.\nFigure S1 - “Sequences produced in the study are highlighted in bold red” in Figure 1, but not Supplemental Figure 1 –\ncould both be highlighted for consistency.\nFigure S1 - The posterior probabilities are a bit difficult to read / match with the branch because of the overlap. I am not sure\nhow to solve this. Maybe the ones corresponding to samples from this study could be bolded? For instance, for the seal, it is\ndifficult to see if it is 0.9 or 0.3.\nTable 1 - As you use a, b, & c to refer to positive samples in the Results section, you could consider adding the a, b, & c\nlabels in Table 1.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "This manuscript presents the first genomic detections of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b\n(genotype B3.2) in Antarctica. Until very recently—apart from the sub-Antarctic outbreaks described by Banyard et al. (2024,\nNat Commun)—the Antarctic continent appeared free of HPAI H5N1. The authors now provide whole-genome sequences\nfrom three hosts (kelp gull, pintado petrel, Antarctic fur seal) collected during the 2023/24 and 2024/25 austral summers\nwithin the Antarctic Treaty area (> 60° S).\nThe study is timely, novel, and ecologically important: it documents multiple independent virus introductions into previously\nnaïve wildlife populations, including a marine mammal. The methods are rigorous, the writing is clear, and the conclusions\nare well supported. The work will interest epidemiologists, virologists, and conservation biologists alike.\nThe rapid spread of the ongoing H5N1 panzootic into the Southern Hemisphere – reaching South America in late 2022 and\nthe sub-Antarctic islands (e.g., Falklands, South Georgia) by late 2023 which has been supported by the report from CDC–\nraised serious concerns about introduction into Antarctic ecosystems. This study now provides the first confirmed whole-\ngenome sequences of H5N1 from within the Antarctic Treaty region (>60°S), collected during the 2023/24 and 2024/25\naustral summers from a kelp gull, a pintado petrel, and an Antarctic fur seal. The work is highly significant ecologically, as it\ndocuments the incursion of HPAI into previously naïve wildlife populations (including both seabirds and a marine mammal).\nThe manuscript is very well-written and the findings are presented clearly. Overall, the study’s timeliness, novelty, and\npotential conservation importance are commendable. It will be of great interest to epidemiologists, virologists, and wildlife\nhealth experts.\nThe manuscript appropriately cites Banyard et al., which first reported H5N1 incursions in South Georgia and the Falklands,\nand it makes clear that this study extends those observations to the Antarctic continent itself. In summary, the results are\nrobust, the conclusions are well-supported, and the work represents a valuable and novel contribution. I have only minor\nsuggestions detailed below:\n1- Clarify the comparison with Banyard et al. (2024): In the Introduction and Discussion, the authors should more explicitly\nposition their findings relative to the prior report by Banyard et al. I suggest the authors clearly state how their results extend\nor differ from Banyard et al.’s findings. A clearer comparison will help readers appreciate the advancement this work\nrepresents and the continuity of the epidemiological narrative. Emphasizing the distinct introduction via South Georgia vs.\ndirect South American lineages, as uncovered here, would also strengthen the discussion of routes.\n2- Mammalian adaptation markers – consider adding a summary table: Given that one of the isolates comes from a\nmammalian host (an Antarctic fur seal), the manuscript should address whether any known mammalian-adaptive mutations\nare present in these viral genomes. The authors do note in the text that no unusual adaptations were observed, which is\nreassuring. To reinforce this point, it would be useful to explicitly report the presence or absence of key mutations associated\nwith mammalian adaptation or increased virulence.\n3- Minor editing points: The manuscript is generally very well written. I did not find significant typos or errors. The flow of the\ntext is logical and concise. If not already done, a quick proofread for any minor grammatical issues or consistency (e.g., in\nnomenclature of gene segments or clades) would be worthwhile."}, "author_response": "Response to Reviewers – Manuscript NCOMMS-25-38772\nWe thank the reviewers for their careful evaluation and constructive feedback on our\nmanuscript titled \"Genomic analysis of high pathogenicity avian influenza viruses from\nAntarctica reveals multiple introductions from South America\". We appreciate the\nopportunity to revise and improve our work. Below, we provide a point-by-point response to\neach reviewer’s comments. Changes made in the manuscript are marked with the “track\nchanges” tool in the revised version.\nWe believe that these revisions substantially improve the manuscript and adequately address\nthe reviewers' comments. We thank the editorial team and all reviewers for their valuable\nfeedback and the opportunity to strengthen our work.\nSincerely,\nThe Authors\nReviewer #1:\nPoint 1) Here the authors present PCR detection and sequencing of sequencing of H5N1 virus\nfrom 3 animals on 2 Antarctic islands. Unfortunately, the presented results to not provide\nrobust support for the conclusions.\nR.: We acknowledge the reviewer’s concern regarding the limited number of genomes in the\noriginal submission. Since we submitted the original manuscript, we expanded our\ncollaboration network and were able to process additional samples from the 2024/2025\naustral summer. With these additional samples, the number of carcasses sampled increased\nfrom 7 to 30, the number of HPAIV genomes sequenced increased from 3 to 25, and the\nnumber of HPAIV-infected species increased from 3 to 7. The expanded dataset improves\nthe robustness of our analyses and supports our initial conclusion of multiple independent\nintroductions, as evidenced by the observed polyphyly. These updates are reflected in the\nrevised phylogenetic figures and supplementary materials.\nPoint 2) There are major problems with this manuscript. Only 7 carcasses were sampled. The\nsample numbers are so small that only the most simple conclusions could be made,\nparticularly as the phylogenetic analysis is limited.\nR.: In the expanded dataset, we tested 30 carcasses from 10 species, of which 17 carcasses\nfrom 7 species were positive. From these samples, we produced 18 completed genomes and\n7 partial genomes. These new genomes are now included in the revised manuscript,\nenhancing both the sample size and the phylogenetic resolution of our analyses.\nPoint 3) For example, the authors restrict their analysis to H5 sequences from South America\nand Antarctica without any justification.\nand their introductions into Antarctica. In the revised manuscript, we have expanded our\nanalysis by including additional genomes and sequencing new segments. We also performed\na concatenated genome analysis, as suggested by Reviewer #2 and following the approach\nof Signore et al. 2025 (https://doi.org/10.1126/sciadv.adu4909), thereby increasing\nphylogenetic resolution and contextualizing our findings more broadly.\nPoint 4) And overall the datasets are not adequately described. The authors also describe\nphylogeographic analysis in the methods, but these are not presented in the manuscript.\nR.: We regret the lack of details about the methods. We conducted a Bayesian\nphylogeographic analysis following the approach outlined by Lemey et al. 2009\n(https://doi.org/10.1371/journal.pcbi.1000520). The Methods section was thoroughly revised\nto ensure clarity and transparency of the analyses performed.\nPoint 5) Despite these major flaws, the authors are making sweeping statements about the\nmovement of the virus throughout South America and into Antarctica. There is no support\nprovided for statements such as “Our phylogenetic results support the growing evidence that\nSouth Georgia has played a significant role as a “stepping stone” for the spread of HPAIV in\nthe Subantarctic and Antarctic region”.\nR.: We have expanded our dataset by including additional sequenced samples and analyzing\nmultiple genome segments, which increased the robustness and resolution of our\nphylogenetic inferences. In light of these improvements, we revised the text to present\nconclusions as putative or inferred and explicitly acknowledge data limitations and\nalternative scenarios. These revisions aim to moderate the language while reinforcing the\ncredibility of our findings.\nPoint 6) Overall this study lacks substance and significance. Sampling size is incredibly small\nand inadequate and just 3 new genomes were generated in this study. This manuscript does\nnot provide any new insights into the spread of H5N1 virus into wild animal populations or\ninto Antarctica.\nR.: We respectfully disagree. This study provides valuable insight about the genomic and\nphylogenetic characteristics of HPAIV H5N1 within the Antarctic Treaty Area, and these\nfindings have ecological and conservation relevance. In response to the reviewer’s concerns,\nwe have now expanded the dataset with additional sequenced samples and genome segments,\nwhich increased the resolution and robustness of our analyses. These improvements, together\nwith a revised and more nuanced discussion, strengthen the significance and impact of our\nfindings, as also recognized by Reviewer #4 and recent studies such as Banyard et al., 2024.\nReviewer #2:\n(HPAI) in Antarctic wildlife, including viral genome sequences suggesting several\nindependent introductions of the virus into Antarctica. The manuscript is overall well written\nand the results are clear and of timely significance. There are however some points that could\nbe clarified.\nR.: We thank the reviewer for the positive assessment and constructive suggestions. Below,\nwe address each point raised and describe the changes made in the revised version of the\nmanuscript.\nPoint 1) Line 231 - Please clarify which primers were used as in the cited reference they\npresent several primers, and I did not find any obvious mention of the “H5a” and “H5b”\nlabels.\nR.: Thank you for pointing this out. We have clarified the specific primer sequences used in\nthe Methods section and indicated their correspondence to the “H5a” and “H5b” labels as\nused in our laboratory protocols. H5a was developed to detect specific groups of H5 viruses\nof Asian origin (A/goose/Guangdong/1996 lineage and derivatives, which include many\nHPAI strains). H5b is complementary, covering genetic variations of H5 that could escape\ndetection by the H5a assay. We added citations that provide the sequences of these primers\nand details of the laboratory protocols.\nPoint 2) Figure 1 - Please explain the rationale for using PB2 opposed to other segments?\nPoint 3) Can you explain why you did not use a concatenated genome (after removing\nreassortants) for improved resolution as Uhart et al., 2024, and Clessin et al., 2025 did?\nR.: We had selected PB2 due to its relatively high phylogenetic signal and lower reassortment\nrate compared to other segments. However, we agree with the reviewer that a concatenated\ngenome analysis provides better resolution. In the revised version, we now include\nphylogenetic analyses based on concatenated genomes (after reassortant removal), following\nthe approach used in Signore et al. 2025 (https://doi.org/10.1126/sciadv.adu4909). These\nresults are presented in Figure 1 and Supplemental Material 2.\nPoint 4) Also why you used a model assuming symmetric dissemination (line 257) when\nthe expectation is that the virus was at the time spreading mostly Southwards?\nR.: We appreciate the reviewer’s observation. In the revised version, we now clarify in the\nMethods section that the initial use of a symmetric diffusion model was based on default\npriors and a conservative approach given the limited number of sequences available at the\ntime. However, to address the reviewer’s concern, we re-ran the phylogeographic\nreconstruction using an asymmetric diffusion model. This updated analysis reinforces the\nsouthward movement of HPAIV from South America to South Georgia and then into\nAntarctica, as also supported by our phylogenetic reconstructions and previous studies (e.g.,\nBanyard et al., 2024; Signore et al., 2025). The revised Results section now includes a\nsummary of these updated findings.\nSouth America (e.g., line 99; header line 111, remove “directly”) as it is pointed out several\ntimes in the manuscript that data is sparse, and notably we have no idea of what is happening\nat sea, and considering how long the branches are on the phylogenetic tree.\nR.: We agree with the reviewer and have revised the text throughout the manuscript to reflect\na more cautious interpretation. The term “direct” has been removed or replaced with more\nappropriate phrasing that is better proportioned to the lack of certainty associated with the\ngaps of data.\nPoint 6) The focus on biosecurity in the context of human movement (lines 49, 198 and 212)\nfeels disconnected from the results given that animal movements are suspected to be\nresponsible for spread (e.g., discussed in line 176).\nR.: We appreciate this observation and have revised the manuscript to clarify that while our\nresults primarily support animal-mediated viral spread, the emphasis on human biosecurity\nmeasures remains relevant under the One Health framework. We now explicitly acknowledge\nthat our genomic data do not implicate human movement in the transmission events\ndescribed, but highlight that strict biosecurity remains critical to minimize potential\nanthropogenic introductions, especially in highly vulnerable and protected ecosystems like\nAntarctica.\nPoint 7) Line 68 - Would it be possible to include information on the number of tested\nsamples (instead of only the ones that returned positive) here? E.g., a reference to\nSupplementary File 2. This could help build knowledge over the likelyhood to detect the\nvirus in the system.\nR.: Thank you for the suggestion. We have now included the total number of tested samples\nand clarified this information in the main text. We also included a supplementary material\n(Supplementary File 1) that provides details on the individuals that were sampled, giving a\nbroader context for the detection rates observed in our study.\nPoint 8) Lines 228-232 - Please clarify product # when possible (Qiagen kit, Biomanguinhos\nINFA/INFB/SC2 kit, CDC Influenza A kit…)\nR.: We thank the reviewer for this suggestion. We have now updated the Methods section to\ninclude the catalog numbers of the kits used. Specifically, RNA was extracted using the\nQIAamp Viral RNA Mini Kit (Qiagen, Cat. No. 52906). Real-time RT-PCR detection was\nperformed using the Biomanguinhos INFA/INFB/SC2 Multiplex Kit (ANVISA Registration\nNo. 80142170060) and the CDC Influenza A/B Typing Panel (Flu A/B Typing Kit, Cat. No.\nFluIVD03-6). These details have been incorporated into the revised manuscript for clarity\nand reproducibility.\nPoint 9) Lines 230-232 - Says “detection and subtyping were performed” two times with\ndifferent methods. Please clarify.\nclarity. The corrected version now reads: “RNA was extracted using the QIAamp Viral RNA\nMini Kit (Qiagen, Cat. No. 52906) following the manufacturer’s instructions. Detection and\nsubtyping of influenza A virus were performed by real-time RT-PCR using two assays: (i)\nthe Biomanguinhos INFA/INFB/SC2 Multiplex Kit (ANVISA Registration No.\n8014217006095), and (ii) the CDC Influenza A/B Typing Panel (Flu A/B Typing Kit, Cat.\nNo. FluIVD03-6), targeting the matrix (InfA) and hemagglutinin (H5a and H5b) genes.”\nPoint 10) Figure 1 - Posterior probability for kelp gull in Figure 1b is rounded up compared\nto Supplemental Figure 1 tree – recommend consistency to avoid unnecessary confusion.\nR.: We apologize for the inconsistency and appreciate the reviewer’s attention to detail. Now\nthe rounding/formatting of Figure 1 is consistent with that of the trees provided in the\nsupplementary materials (specifically, Supplementary Figures 2 and 3).\nPoint 11) Figure S1 - “Sequences produced in the study are highlighted in bold red” in Figure\n1, but not Supplemental Figure 1 – could both be highlighted for consistency.\nR.: Thank you for pointing this out. We acknowledge that the sequences produced in this\nstudy were not highlighted in red in the trees provided in the supplementary materials\n(specifically, Supplementary Figures 2 and 3) as stated. We corrected the legend to these\nmaterials.\nPoint 12) Figure S1 - The posterior probabilities are a bit difficult to read / match with the\nbranch because of the overlap. I am not sure how to solve this. Maybe the ones corresponding\nto samples from this study could be bolded? For instance, for the seal, it is difficult to see if\nit is 0.9 or 0.3.\nR.: Thank you for pointing this out. We agree that the posterior probabilities in the\nsupplementary figure were difficult to interpret due to overlap. In the revised version, we will\nimprove legibility by increasing the resolution and adjusting font formatting and\ntransparency effects to ensure readability across all supplementary figures.\nPoint 13) Table 1 - As you use a, b, & c to refer to positive samples in the Results section,\nyou could consider adding the a, b, & c labels in Table 1.\nR.: With the expansion of the dataset, this part of the manuscript was thoroughly re-worked.\nThe narrative of the Results and Discussion is now centered on the phylogenetic groups “a”\nand “b”, and these labels are used consistently throughout the entire manuscript.\nReviewer #3:\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nR.: Thank you for informing us. We appreciate the initiative by Nature Communications to\nsupport the training of Early Career Researchers in peer review and are grateful for the\nthoughtful feedback provided.\nReviewer #4:\nThis manuscript presents the first genomic detections of highly pathogenic avian influenza\nA(H5N1) clade 2.3.4.4b (genotype B3.2) in Antarctica. Until very recently—apart from the\nsub-Antarctic outbreaks described by Banyard et al. (2024, Nat Commun)—the Antarctic\ncontinent appeared free of HPAI H5N1. The authors now provide whole-genome sequences\nfrom three hosts (kelp gull, pintado petrel, Antarctic fur seal) collected during the 2023/24\nand 2024/25 austral summers within the Antarctic Treaty area (> 60° S).\nThe study is timely, novel, and ecologically important: it documents multiple independent\nvirus introductions into previously naïve wildlife populations, including a marine mammal.\nThe methods are rigorous, the writing is clear, and the conclusions are well supported. The\nwork will interest epidemiologists, virologists, and conservation biologists alike.\nThe rapid spread of the ongoing H5N1 panzootic into the Southern Hemisphere – reaching\nSouth America in late 2022 and the sub-Antarctic islands (e.g., Falklands, South Georgia) by\nlate 2023 which has been supported by the report from CDC– raised serious concerns about\nintroduction into Antarctic ecosystems. This study now provides the first confirmed whole-\ngenome sequences of H5N1 from within the Antarctic Treaty region (>60°S), collected\nduring the 2023/24 and 2024/25 austral summers from a kelp gull, a pintado petrel, and an\nAntarctic fur seal. The work is highly significant ecologically, as it documents the incursion\nof HPAI into previously naïve wildlife populations (including both seabirds and a marine\nmammal). The manuscript is very well-written and the findings are presented clearly.\nOverall, the study’s timeliness, novelty, and potential conservation importance are\ncommendable. It will be of great interest to epidemiologists, virologists, and wildlife health\nexperts.\nThe manuscript appropriately cites Banyard et al., which first reported H5N1 incursions in\nSouth Georgia and the Falklands, and it makes clear that this study extends those observations\nto the Antarctic continent itself. In summary, the results are robust, the conclusions are well-\nsupported, and the work represents a valuable and novel contribution. I have only minor\nsuggestions detailed below:\nR.: Thank you for the thoughtful and encouraging comments. We are pleased that the novelty,\necological importance, and clarity of our study were recognized. We have carefully\nconsidered the minor suggestions provided below and implemented revisions accordingly to\nfurther strengthen the manuscript.\nDiscussion, the authors should more explicitly position their findings relative to the prior\nreport by Banyard et al. I suggest the authors clearly state how their results extend or differ\nfrom Banyard et al.’s findings. A clearer comparison will help readers appreciate the\nadvancement this work represents and the continuity of the epidemiological narrative.\nEmphasizing the distinct introduction via South Georgia vs. direct South American lineages,\nas uncovered here, would also strengthen the discussion of routes.\nR.: Thank you for this insightful suggestion. We expanded the Introduction to provide more\ncontext on the epidemiology of HPAIV in South America leading up to the spread of these\nviruses to South Georgia (as documented by Banyard et al.) and then onto the Antarctic\nPeninsula. We further clarify that our phylogenetic analyses support several independent\nvirus introductions into the Antarctic Peninsula region, one deriving from the South Georgia\nstrains described by Banyard et al. and another three introductions deriving from the South\nAmerican “marine mammal clade”. This distinction highlights the complexity of viral\nincursion routes and reinforces the need for ongoing surveillance across the Southern Ocean.\nPoint 2) Mammalian adaptation markers – consider adding a summary table: Given that one\nof the isolates comes from a mammalian host (an Antarctic fur seal), the manuscript should\naddress whether any known mammalian-adaptive mutations are present in these viral\ngenomes. The authors do note in the text that no unusual adaptations were observed, which\nis reassuring. To reinforce this point, it would be useful to explicitly report the presence or\nabsence of key mutations associated with mammalian adaptation or increased virulence.\nR.: We added a mutational analysis to the manuscript, including a new figure (Figure 2) and\nsupplementary materials (Supplementary File 4) that provide an inventory of amino acid\nsubstitutions. A new “Genomic features” section was added to the Discussion to highlight\nsome of the most significant mutations and discuss how these findings relate to prior\nexperimental data. These analyses reveal the presence of several mammalian-adaptive\nmutations in the strains identified in this study, which may enhance virulence for Antarctic\nmarine mammals and potentially contribute to an increased zoonotic risk.\nPoint 3) Minor editing points: The manuscript is generally very well written. I did not find\nsignificant typos or errors. The flow of the text is logical and concise. If not already done, a\nquick proofread for any minor grammatical issues or consistency (e.g., in nomenclature of\ngene segments or clades) would be worthwhile.\nR.: We performed a thorough proofreading of the revised manuscript to ensure consistency\nin gene segment and clade nomenclature, grammar, and figure annotations. Minor issues have\nbeen corrected, and terminology has been standardized throughout the text.\n7"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The inclusion of additional sampling and sequencing has somewhat improved this paper. But there are still major flaws with the genetic analysis.\nIn the phylogenetic trees there are long branches leading into the sequences from Antarctica that represent over 1 year of evolution. My previous suggestion to expand the geographic origin of the viruses included in the phylogeny was partly from concern that the analysis might be missing links to viruses from other locations apart from South America (although this is the most logical source). I would still prefer to see a phylogeny (perhaps just limited to the concatenated genome) that includes a globally representative dataset. This could be easily done with Nextstrain and would most likely address my concern and support the restriction of the genetic analysis to South American strains.\nThe authors say that they have conducted a phylogeographic analysis, but there is no actual presentation of the phylogeography results in the manuscript. It is inexplicable, particularly as transmission dynamics would be informative for this study. There are no statistical supports provided in Figure 3, there are lines drawn on a map that represent phylogenetic relationships from the trees. Unfortunately these results would also be subject to sampling biases in the datasets, a well known problem with phylogeography, none of which is discussed.\nThe structure of the paper could be improved. There is a very small results section and then 5 pages of discussion that includes a rehashing of the results. In my opinion the manuscript requires reorganization. The \"Conservation and management implications\" could form the bulk of the discussion with everything prior to that under results.\nOverall this study lacks significance due to flawed genetic analyses.", "reviewer_2": "The manuscript has been considerably improved through revision, including notably larger sample sizes, and more appropriate and better explained methods. I really appreciate the paragraph on oral vs fecal shedders, which will help guide future work on HPAI in wildlife. The conclusions are now well supported by the manuscript.\nLine 128 - I am confused about whether this motif was found in 1 (\"an\") or 3 (\"3 complete genomes [from the same individual?]\") fur seals.\nLine 162 - It is not obvious to me why multiple introductions would be more conservative than diversification.\nLine 184 (or somewhere else in the discussion) - I would add a mention about unsampled species here, which could also have played a role in the dissemination of the virus, such a fulmars and giant petrels, which have been observed to carry the virus and suspected to play a role in its dissemination (Banyard et al. 2024; Clessin et al. 2025), but can be harder to access, or less likely to die, than the sampled species. I do not mean listing all potential species, but only acknowledging that we only observe part of the process, notably because some species are more difficult to access.\nLine 265 - I would suggest spelling out SSAFS each time as the abbreviation is used only a few times.\nLine 338 - In the response, the authors talk about an asymmetric model but I only see mentions of the symmetric model in the manuscript."}, "author_response": "R2- NCOMMS-25-38772A\nWe are once again very grateful for the reviewers’ constructive feedback. Below,\nwe provide our point-by-point responses to all comments and suggestions.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nComment: The inclusion of additional sampling and sequencing has somewhat\nimproved this paper. But there are still major flaws with the genetic analysis.\nIn the phylogenetic trees there are long branches leading into the sequences from\nAntarctica that represent over 1 year of evolution. My previous suggestion to\nexpand the geographic origin of the viruses included in the phylogeny was partly\nfrom concern that the analysis might be missing links to viruses from other\nlocations apart from South America (although this is the most logical source). I\nwould still prefer to see a phylogeny (perhaps just limited to the concatenated\ngenome) that includes a globally representative dataset. This could be easily\ndone with Nextstrain and would most likely address my concern and support the\nrestriction of the genetic analysis to South American strains.\nResponse: We thank the reviewer for this important observation. With regards to\nthe long branches of the phylogenetic trees, this is unfortunately due to the gaps\nin sampling and also reflects the inherent limitations of the Antarctic region.\nAntarctica is an unusual ecosystem with extreme seasonality, being almost\ncompletely abandoned by wildlife (with rare exceptions that breed in winter such\nas emperor penguins, which are absent from our study area) and virtually\ninaccessible to researchers during winter (hence even environmental sampling\nwould be hardly feasible). It is only feasible to obtain samples in spring/summer\nand there is an unavoidable data gap in autumn/winter. Because our phylogenetic\ntrees are time-calibrated, this dynamic visually translates into unusually long\nbranches. Hence, while in other epidemiological systems the existence of long\nbranches in the phylogenetic trees could be interpreted as a red flag that indicates\nfailures in surveillance/sequencing effort, in the Antarctic context these long\nbranches are not unexpected and are virtually impossible to avoid.\nWith regards to the restriction of the genetic analysis to South American,\nSubantarctic and Antarctic strains, there is some background that is worth\nconsidering. Previous studies have established that, with the exception of a few\ninitial introductions to Colombia and Venezuela that were unsuccessful, all strains\nthat disseminated throughout South America originated from a single introduction\nof clade 2.3.4.4b genotype B3.2 from North America to Peru in October 2022\n(Leguía et al. 2023; Ospina-Jimenez et al. 2024; Uhart et al. 2024; Bruno et al.\n2025; Pardo-Roa et al. 2025; Uhart & Vanstreels 2025). And, more importantly,\nnone of the strains detected to date in the Subantarctic/Antarctic region have\nshown association with strains from continents other than South America.\nFurthermore, all virus sequenced in South America in 2023 and 2024 show no\nevidence of genomic reassortment, and all were directly descendent from this\ninitial October 2022 introduction, retaining the genotype B3.2 (Rimondi et al.\n2025; Vanstreels et al. 2025). Genomic analyses corroborate that all viruses in\nthat region descended from South American strains and retained the B3.2\ngenotype (Banyard et al. 2024; Clessin et al. 2025). Our data and that of McCulley\net al. 2025 show that the same is true for the viruses that spread onto Antarctica,\ni.e. all Subantarctic/Antarctic HPAI viruses sequenced thus far were descendants\nof the introduction to South America dating back to October 2022. Specifically,\nthis is demonstrated by our phylogenetic trees for each gene segment (trees in\nSupplementary File 4), which show how all Subantarctic/Antarctic virus\nsequences are placed well within the clade of South American viruses, with high\nstatistical support and highly consistent tree structures across all eight gene\nsegments as well as in the concatenated genome (Supplementary File 3).\nNevertheless, in response to the reviewer’s concern, we have added a new\nmaximum likelihood tree for the HA gene of >800 sequences of globally\nrepresentative strains of the H5 clade 2.3.4.4b. This analysis has been included\nin the supplementary materials (new Supplementary File 2), and offers a further\ndemonstration that all Subantarctic and Antarctic viruses descend from South\nAmerican viruses.\nLiterature cited in the response:\nBanyard et al. 2024 https://doi.org/10.1038/s41467-024-51490-8\nBruno et al. 2025 https://doi.org/10.1016/j.virol.2025.110602\nClessin et al. 2025 https://doi.org/10.1038/s41467-025-64297-y\nLeguía et al. 2023 https://doi.org/10.1038/s41467-023-41182-0\nMcCulley et al. 2025 https://doi.org/10.1128/mra.00041-25\nOspina-Jimenez et al. 2024 https://doi.org/10.3389/fvets.2024.1347509\nPardo-Roa et al. 2025 https://doi.org/10.1038/s41467-025-57338-z\nRimondi et al. 2025 https://doi.org/10.21203/rs.3.rs-7960151/v1\nVanstreels et al. 2025 https://doi.org/10.1101/2025.05.23.655175\nUhart et al. 2024 https://doi.org/10.1038/s41467-024-53766-5\nUhart & Vanstreels 2025 https://doi.org/10.1139/cjm-2025-0189\nComment: The authors say that they have conducted a phylogeographic\nanalysis, but there is no actual presentation of the phylogeography results in the\nmanuscript. It is inexplicable, particularly as transmission dynamics would be\ninformative for this study. There are no statistical supports provided in Figure 3,\nthere are lines drawn on a map that represent phylogenetic relationships from the\ntrees. Unfortunately these results would also be subject to sampling biases in the\ndatasets, a well known problem with phylogeography, none of which is discussed.\nResponse: Fair enough, our previous description may have incorrectly implied\nthat a formal phylogeographic analysis had been performed when this is not the\ncase. In reality, what we did was a phylogenetic analysis that incorporates a\ndiscrete phylogeographic model, which is not the same as a phylogeographic\nanalysis sensu stricto. We edited the sub-heading of the Methods were this was\nimplied to correct this.\nWith regards to Figure 3, we should clarify that our intention was not to present a\nstatistical phylogeographic reconstruction, but rather to provide a conceptual\nvisualization of the putative routes of HPAI H5N1 spread in the Subantarctic and\nAntarctic regions. The arrows in this figure are drawn from formal\nphylogeographic reconstructions performed by other studies (Banyard et al.\n2024; Uhart et al. 2024; Clessin et al. 2025) and from inference based from\nphylogenetic analyses in this and previous studies (McCulley et al. 2025;\nSteinfurth et al. 2025). By integrating our findings with those of earlier studies we\nbelieve this figure will help readers better understand the geographic context\n(distances and positions of the various Subantarctic islands and study sites) and\npotential pathways of spread that are being discussed in text. It is true that this\nfigure is subject to limitations related to geographic sampling biases, however this\nis the case of everything else in this and other studies, it is a recurring challenge\nin all research of HPAI viruses in the Subantarctic and Antarctic region, and it is\nsomething that we already mention in the Discussion.\nIn response to the reviewer’s concerns, we edited the caption of Figure 3 to clarify\nthat the map does not represent a phylogeographic analysis per se but instead is\na conceptual representation integrating the works of others and our own\nphylogenetic analysis. The caption now reads as follows:\n“Figure 3. Conceptual representation of the hypothesized pathways of\nspread of HPAIV H5N1 in the Subantarctic and Antarctic region, based on\nthe findings of this study and prior studies that performed phylogeographic\nand phylogenetic reconstruction5,7,13,27,28. Arrows represent the potential\nroutes of spatial dissemination of the HPAIV H5N1 South American\n“marine mammal” (blue) and “avian” (red) sub-clades (sensu Uhart and\ncolleagues5).”\nLiterature cited in the response:\nBanyard et al. 2024 https://doi.org/10.1038/s41467-024-51490-8\nClessin et al. 2025 https://doi.org/10.1038/s41467-025-64297-y\nMcCulley et al. 2025 https://doi.org/10.1128/mra.00041-25\nSteinfurth et al. 2025 https://doi.org/10.1101/2025.09.06.674618\nUhart et al. 2024 https://doi.org/10.1038/s41467-024-53766-5\nComment: The structure of the paper could be improved. There is a very small\nresults section and then 5 pages of discussion that includes a rehashing of the\nresults. In my opinion the manuscript requires reorganization. The \"Conservation\nand management implications\" could form the bulk of the discussion with\neverything prior to that under results.\nResponse: We appreciate the constructive comment regarding the manuscript\nstructure. To our understanding, the Results section presents the findings of a\nstudy in a strictly objective approach (i.e. the results of the experiment in a\nvaccuum), whereas the Discussion section interprets these findings in light of the\nliterature and discussing hypotheses that may explain them (i.e. the experiment\nunder the light of human knowledge). This is of course a subjective and\nsomewhat nuanced distinction that also reflects stylistic preferences and\nacademic traditions across different fields of research and cultures. Nevertheless,\nwe feel this approach is well suited to the scope of this study and, in our\nexperience, it has been the most adopted in the biological literature.\nIn this sense, we would prefer to keep the text that is under the sub-headings\n\"Dissemination of “marine mammal” viruses from South America to Antarctica\"\nand \"Virus dissemination through South Georgia as a ‘stepping stone’\"\nand \"Genomic features\" in the Discussion rather than moving it to the Results. This is\nbecause in those sections we are taking information that was objectively\npresented in the Results and interpreting it in light of other studies and collective\nhuman knowledge, proposing hypothesis to explain them and drawing\nimplications to our broader understanding of the ecology and evolution of HPAI\nviruses. Nevertheless, because we understand this is a stylistic choice, if the\neditors disagree and would like us to restructure our Results and Discussion\nsections as suggested by the reviewer or in another way that the editors feel\nwould be more suitable to the journal, please let us know and we can do so.\nComment: Overall this study lacks significance due to flawed genetic analyses.\nResponse: We respectfully disagree. Of course the study has flaws, such as the\nlimited sample size when compared to studies on poultry or on wildlife in less\nremote regions of the world, or the limited availability of regional sequencing data\nto strengthen the phylogenetic inferrence that can be drawn from our sequences.\nHowever, we feel that given the practical challenges of collecting samples in\nAntarctica, our study provides a robust contribution to the literature and to our\ncurrent understanding of HPAI viruses in Antarctic wildlife.\nReviewer #2 (Remarks to the Author):\nComment: The manuscript has been considerably improved through revision,\nincluding notably larger sample sizes, and more appropriate and better explained\nmethods. I really appreciate the paragraph on oral vs fecal shedders, which will\nhelp guide future work on HPAI in wildlife. The conclusions are now well\nsupported by the manuscript.\nResponse: We thank the reviewer for the positive assessment of our revised\nmanuscript.\nComment: Line 128 - I am confused about whether this motif was found in 1 (\"an\")\nor 3 (\"3 complete genomes [from the same individual?]\") fur seals.\nResponse: We thank the reviewer for pointing out the ambiguity. This motif was\ndetected in only one Antarctic fur seal. We recovered three complete genomes\nfrom this same individual, obtained from the nasal, oral, and brain samples, and\nall of them exhibited the PLKEKRRKRGLF motif.\nWe revised the text as follows, which should convey this more clearly:\n“The original PLREKRRKRGLF motif was found in most viruses from both\nphylogenetic groups, comprising 14 complete genomes and 5 partial\ngenomes. However, the PLKEKRRKRGLF motif was detected in group “a”\nviruses recovered from three hosts: one Antarctic fur seal (three complete\ngenomes from the same individual), one Antarctic tern (one partial\ngenome), and one Antarctic petrel (one partial genome) (Supplementary\nFile 6).”\nComment: Line 162 - It is not obvious to me why multiple introductions would be\nmore conservative than diversification.\nResponse: Thank you for pointing this out, the text was incorrect. We agree with\nthe reviewer that it is more conservative to assume less, not more, introduction\nevents, but when writing this paragraph we failed to convey this. Furthermore,\nduring drafting we had gone back and forth about the best approach to interpret\nthe discrepancies among the different phylogenetic analyses, and this caused\nsome parts of the text to become incongruent.\nWhen it comes to the strains of the group “a”, some of our phylogenetic analyses\npointed to there having been 4 introduction events (concatenated genome),\nothers pointed to 3 introduction events (PB2, PB1, PA, HA, and NP) and others\npointed to 2 introduction events (NA and NS). In this sense, we consider it more\n“conservative” to consider that there were less introduction events, i.e. the\nhypothesis of local diversification is more conservative than the hypothesis of\nadditional introductions. And therefore our more “conservative” interpretation is\nthat there were only 2 introduction events in group “a”. Adding to the other\nintroduction event that led to group “b”, we feel that our study provides robust\nevidence for 3 introduction events in total.\nOn this basis and in consideration of the reviewer’s comments, we made minor\nedits to Figure 3 and to the Abstract to reflect this interpretation. Furthermore, the\nparagraph of the Discussion was revised and now reads as follows:\n“Our analyses suggest that group “a” strains recovered from Antarctic\nwildlife were polyphyletic; however, different analytical methods disagreed\non the number of introduction events that might have originated them. All\nanalyses agreed that the virus from the kelp gull sampled at Livingston\nIsland in January 2024 (individual O2601) was clearly distinct from the\nremaining group “a” strains, representing an isolated introduction event.\nHowever, the analyses disagreed on whether the remaining group “a”\nstrains resulted from multiple introduction events or not. Hence, until\nfurther data is available to corroborate additional introduction events, we\nconservatively consider that the group “a” strains detected in this study\ndescended from only two separate introduction events. Future genomic\ndata from South American, Antarctic and Subantarctic HPAIV H5N1\nviruses may help clarifying the number of introduction events and whether\nthey occurred directly from South America or indirectly via Subantarctic\nislands. In particular, it is worth noting that one of the strains recovered\nfrom Antarctic fur seal (individual O2913) at King George Island in\nDecember 2024 was closely related to a strain from a southern fulmar\n(Fulmarus glacialoides) sampled in the Falkland (Malvinas) Islands in\nOctober 2023, hinting at the possibility that the archipelago may have\nserved as a “stepping stone”.”\nComment: Line 184 (or somewhere else in the discussion) - I would add a\nmention about unsampled species here, which could also have played a role in\nthe dissemination of the virus, such a fulmars and giant petrels, which have been\nobserved to carry the virus and suspected to play a role in its dissemination\n(Banyard et al. 2024; Clessin et al. 2025), but can be harder to access, or less\nlikely to die, than the sampled species. I do not mean listing all potential species,\nbut only acknowledging that we only observe part of the process, notably because\nsome species are more difficult to access.\nResponse: Thank you for this suggestion. We agree that the role of unsampled\nspecies in the dissemination of HPAIV H5N1 is an important point that should be\nacknowledged. In response to this comment, we have added the following\nsentence to the Discussion acknowledging that our observations represent only\na subset of the ecological process, as some key species are difficult to sample or\ndetect:\n“It is also important to acknowledge the potential role of other birds species\nas fulmars and giant petrels—both of which have been reported as\ninfected or suspected to participate in HPAIV H5N1 spread7,27—were not\nsampled in this study, either because they are locally less abundant,\nexperienced less mortality, or are less likely to be detected as carcasses.”\nComment: Line 265 - I would suggest spelling out SSAFS each time as the\nabbreviation is used only a few times.\nResponse: Done.\nComment: Line 338 - In the response, the authors talk about an asymmetric\nmodel but I only see mentions of the symmetric model in the manuscript.\nResponse: Thank you for drawing attention to this inconsistency. We confirm that\nthe phylogeographic analyses presented in the manuscript were conducted using\na symmetric discrete CTMC diffusion model, as correctly described in the\nMethods. The mention to an asymmetric diffusion model in our response to a\ncomment in the first review round resulted from miscommunication between the\ncoauthors, leading to an imprecise wording in the response letter.\nWe should clarify that the use of a symmetric discrete phylogeographic model\ndoes not imply an assumption of bidirectional or non-directional viral spread, but\nrather reflects a conservative prior structure in which transition rates between\nlocations are assumed to be equal a priori. Although epidemiological and\necological evidence suggests that HPAIV was predominantly spreading\nsouthwards during the study period, the availability of sequence data from the\nSubantarctic and Antarctic region remains limited and unevenly distributed\nacross locations. Under such conditions, the use of a symmetric model is\ncommonly recommended to avoid overparameterization and identifiability issues\nthat may arise when fitting asymmetric diffusion models with sparse data.\nImportantly, despite this conservative modeling choice, the inferred spatial\ndynamics consistently support a southward dissemination of HPAIV from South\nAmerica to Antarctica or to South Georgia and subsequently into Antarctica. This\ndirectionality emerges from the posterior reconstruction of ancestral locations and\nTMRCA estimates, rather than being imposed by directional priors.\nWe have revised the Methods section to clarify the rationale for adopting a\nsymmetric diffusion model. We apologize for any confusion this may have caused\nand appreciate the reviewer’s careful reading."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns and clarified the issues around the phylogeography."}, "author_response": ""}]

Decision letter