Detection and spread of high pathogenicity avian influenza virus H5N1 in the Antarctic Region

Domain: earth_science
Published: 2024-09-03
Editorial outcome: major_revision

Abstract
Until recent events, the Antarctic was the only major geographical region in which high pathogenicity avian influenza virus (HPAIV) had never previously been detected. Here we report on the detection of clade 2.3.4.4b H5N1 HPAIV in the Antarctic and sub-Antarctic regions of South Georgia and the Falkland Islands, respectively. We initially detected H5N1 HPAIV in samples collected from brown skuas at Bird Island, South Georgia on 8th October 2023. Since this detection, mortalities were observed in several avian and mammalian species at multiple sites across South Georgia. Subsequent testing confirmed H5N1 HPAIV across several sampling locations in multiple avian species and two seal species. Simultaneously, we also confirmed H5N1 HPAIV in southern fulmar and black-browed albatross in the Falkland Islands. Genetic assessment of the virus indicates spread from South America, likely through movement of migratory birds. Critically, genetic assessment of sequences from mammalian species demonstrates no increased risk to human populations above that observed in other instances of mammalian infections globally. Here we describe the detection, species impact and genetic composition of the virus and propose both introductory routes and potential long-term impact on avian and mammalian species across the Antarctic region. We also speculate on the threat to specific populations following recent reports in the area.

Introduction
Following the emergence and global expansion of A/goose/Guangdong/1/96 (GsGd)-lineage H5 high pathogenicity avian influenza viruses (HPAIV) there have been repeat epizootics in wild birds and poultry populations globally. This lineage spread globally, evolving into diverse clades classified according to the H5 haemagglutinin (HA) gene phylogeny1. In the autumn of 2021, the situation escalated considerably with the detection of a clade 2.3.4.4b HPAIV subtype H5N1 in Europe2. Subsequently, two unprecedented epizootic waves with this lineage in 2021/22 and 2022/23 were associated with mass mortality events in wild birds together with unprecedented numbers of incursions into poultry premises2–6. High levels of viral adaptation to wild bird species7, and increased fitness advantage through continued genetic reassortment8 likely underpin the broad impact infection has had across many avian species3. This wide host range has facilitated the transmission of the lineage across a large geographic area, including from Europe to North America9,10, where it has since rapidly expanded its range southward into South America via migratory avian species.

Incursion into South American countries, starting in November of 2022, represented the first recorded instances of GsGd-lineage H5 HPAIV in the region11–13. Mass mortality events in the region have been particularly severe and reported across several different bird species in addition to marine mammals3,11,14, highlighting the extensive ecological impact of HPAIV and the ongoing threat it presents to naïve hosts.

The Antarctic region includes the ice shelves, waters, and all the island territories in the Southern Ocean situated inside of the ‘Antarctic Convergence’ or ‘Antarctic Polar Front’, a marine belt encircling Antarctica where Antarctic waters meet those of the warmer sub-Antarctic15. Several islands are located inside the Antarctic region, including South Georgia, while the Falkland Islands among others are located outside the Antarctic Convergence in the sub-Antarctic zone (Fig. 1). There have been no previous reports of HPAIV inside the Antarctic region16,17. Antarctica and the sub-Antarctic islands possess unique ecosystems which support the population strongholds of several avian and marine mammal species. The relative isolation of these islands from human populations has provided species across the Antarctic with only limited protection from anthropogenic environmental change18. Indeed, wildlife populations in the Antarctic face a broad range of challenges from introduced species19, to longline fisheries20,21, and rapid climate change22–24. Several native bird species including wandering albatross (Diomedea exulans), macaroni penguins (Eudyptes chrysolophus), grey-headed albatross (Thalassarche chrysostoma), and white-chinned petrel (Procellaria aequinoctialis), are listed as either vulnerable or endangered25. Iconic long-lived species with late maturity, such as albatross, exhibit low resilience to rapid increases in population mortality26. High mortality disease outbreaks therefore represent a substantial threat to already vulnerable seabird populations27,28.

While geographically isolated, several Antarctic seabird species routinely range between the South Atlantic and Southern Ocean, visiting the South American coast to either forage or overwinter29. Brown skuas (Stercorarius antarcticus), kelp gulls (Larus dominicanus), southern giant petrel (Macronectes giganteus), and snowy sheathbills (Chionis albus) have previously been identified as potential vectors of infectious pathogens into this vulnerable ecosystem due to their migratory traits, scavenging behaviour, and previously identified roles as carriers of low pathogenicity avian influenza viruses (LPAIV)30–37.

Evidence of LPAIV in the Antarctic region was first identified from serological studies in the 1980s38–40. A range of subtypes have since been reported (H140, H331, H435, H541, H632, H739, H931, and H1133) including genetic analysis of the influenza virus subtypes H4N735, H5N533,41, H6N832 and H11N233,42,43. In contrast to the more prevalent H11N2 influenza viruses, which likely circulate silently through local populations, H4-H6 subtypes were found to share high sequence similarity with viruses from South America, indicating more recent introduction events32,33,35. Evidence of LPAIV transmission to the continent from the Americas demonstrates the elevated risk of clade 2.3.4.4b H5 HPAIV being introduced to the Antarctic, encouraging researchers in 2022 to employ additional biosecurity measures while maintaining surveillance activities30,44. During the austral summer of 2022/23, sampling and surveillance was conducted at several sites in the Antarctic region, and as of March 2023, HPAIV had not been detected16.

Here we describe the observation of morbidity and mortality events across different species as well as the positive detection of H5N1 HPAIV in a variety of species in South Georgia, inside the Antarctic region and the sub-Antarctic Falkland Islands. We detail the suspicion, diagnostic evaluation, and clinical presentations of HPAIV in the region. Genetic analysis is used to characterise potential introduction routes and the consequences of HPAIV circulation in this region are considered.

Results
Case description and virus detection
Provision for diagnostic investigation of avian influenza is limited in the Antarctic and sub-Antarctic regions. For some samples in the description below, local RT-PCR testing at the KEMH Pathology and Food, Water & Environmental Laboratory on the Falkland Islands was used to screen for the presence of avian influenza virus H5N1 viral RNA (vRNA). This was initially undertaken for a Southern Fulmar as described below and this detection, alongside the increase clinical disease and mortalities observed on South Georgia triggered shipment of samples to the World Organisation for Animal Health (WOAH) and Food and Agriculture Organisation (FAO) International Reference Laboratory for avian influenza, swine influenza and Newcastle disease virus at the Animal and Plant Health Agency (APHA), Weybridge, UK for confirmatory and further diagnostic evaluation. An overview of both observations and testing is detailed below.

On the 17th September, researchers of the British Antarctic Survey (BAS) on Bird Island, South Georgia (an island approximately 0.5 km Northwest of the main island of South Georgia) (Fig. 2A) discovered a single southern giant petrel showing behaviours indicative of loss of coordination, neurological twitching, and lethargy. This individual was observed being preyed and scavenged upon by brown skuas and other southern giant petrels (Macronectes giganteus). On 8th October, brown skuas were observed in the same locality showing lethargy, neck spasms, twitching, and an inability to fly, and by 10th October, bird mortality was seen on Bird Island, with the highest number of mortalities occurring at the roosting site of non-breeding birds. Swab samples were collected from the three brown skua (Stercorarius antarcticus) on 8th October 2023 and a further brown skua on the 11th October which were all later found dead on Bird Island. These initial sampling events all yielded PCR positivity for HPAIV H5N1. The series of sampling events and species testing positive from this point onwards are detailed in Table 1 and Supplementary Table S1.

Briefly, both brown skuas and kelp gulls then tested positive at two different locations on South Georgia (Hound Bay and St. Andrews Bay) on the 30th October with further gulls and skuas (Moltke Harbour) swabbed a day later (31st October) also testing positive. Further positive samples were taken on the 3rd (Harpon Bay) and 8th (Penguin River) November from both brown skuas and kelp gulls. Further escalation in mortality occurred by 17th November 2023, when 57 skuas were observed to have died at Bird Island although samples could not be retrieved. On the 27th of November a South Georgia shag tested positive from King Edward Cove alongside an Antarctic tern sampled dead on the 6th of December.

Alongside avian species, in early December clinical disease consistent with mammalian infection with HPAIV was observed in colonies of southern elephant seal (Mirounga leonine) and Antarctic fur seal (Arctocephalus gazella) at Jason Harbour on South Georgia (Supplementary Table S2). Clinical presentation included difficulty breathing, with coughing and short sharp breath intake. Individuals also showed accumulation of viscous fluid around the nasal passage. Five southern elephant seals and 1 Antarctic fur seal from Jason Harbour collected on the 9th December tested positive for the virus (Supplementary Table S2).

Overall, between the initial detection of HPAIV on the 8th of October and 9th December, from South Georgia a total of 33 avian carcasses and 17 mammalian carcasses were sampled including representatives of five different avian species and two mammalian species across eight different locations. Of these, 66% (n = 28/33 avian carcasses; n = 6/17 mammalian carcasses) tested positive for HPAIV H5N1.

Concurrent to the events occurring on South Georgia, on 30th October, a southern fulmar (Fulmarus glacialoides) was reported dead on Stanley, Falkland Islands and tested positive for the virus (Fig. 2B). Numerous other swabs were taken from different avian species across the Falkland Islands (Supplementary Table 1) but only three black-browed albatross (Thalassarche melanophris) tested positive from Saunders Island (n = 1) and Steeple Jason (n = 2) being collected on the 19th and the 26th of November, respectively. In total between the 30th October and 10th December samples were taken and tested from a total of 13 carcasses from nine different bird species with 31% testing positive for HPAIV H5N1 (Table 1 and Supplementary Table S1).

Virus isolation
Wherever samples tested positive for HPAIV H5N1, attempts to isolate viable virus were undertaken. From the samples collected from South Georgia, virus isolation was successful from two brown skuas and a kelp gull taken from Hound Bay on the 30th October, a brown skua and kelp gull collected from Moltke harbour on the 31st October, a kelp gull from Harpon Bay on the 3rd November and both a South Georgia shag and an Antarctic tern from King Edward Cove on the 27th November and the 6th December, respectively.

Virus isolation was also successful from swabs taken from carcasses found dead on the Falklands Islands, including from samples from two southern fulmar collected on the 30th October and the 13rd November from the Falklands Islands, and from black-browed albatross from Saunders Island and Steeple Jason on the 19th November and the 26th November, respectively.

From a mammalian perspective, no viable virus could be recovered from any of the samples testing positive by the generic AIV, HPAIV H5-specific assay and N1-specific rRT-PCR assays45–48 (Supplementary Table 2).

Genomic and phylogenetic analysis
Whilst successful virus isolation gave material for full virus genomic sequencing and analysis, wherever possible, samples for which only rRT-PCR detection was possible were also assessed for full genome generation and sequence analysis. A total of 20 full genomes were generated from avian samples and a further three full genomes were generated from samples collected from seals. These included ten full virus genomes from brown skuas (from Bird Island, Hound Bay, Moltke Harbour, and Penguin River); six full virus genomes from kelp gulls (from Hound Bay, Moltke Harbour, Harpon Bay, and Penguin River); and both a South Georgia shag and an Antarctic tern (both from King Edward Cove). A further avian full virus genome was generated from two Southern Fulmars, both from Stanley Island on the Falklands Islands (Supplementary Table S1).

From a mammalian perspective we generated two full virus genomes from southern elephant seals (both from Jason Harbour); and one full virus genome from an Antarctic fur seal (from Jason Harbour) (Supplementary Table S2).

Comparison of the full dataset generated from samples described revealed that they shared 99.86–100% nucleotide identity across all eight influenza gene segments (Supplementary Table S3). The sequences generated were then analysed with representative global H5N1 clade 2.3.4.4b full-genome sequences to assess genetic ancestry by inferring maximum-likelihood phylogenetic trees (Supplementary Fig 1). The sequences from South Georgia and the Falkland Islands clustered with H5N1 viral sequences obtained from South America between October 2022 and October 2023, in particular Argentina, Uruguay, Peru, and Chile across all gene segments. All sequences from South Georgia and the Falkland Islands were genotyped according to the United States of America H5N1 schema, given the spread of these viruses from North to South America in early 202211, and found to be part of the B3.2 genotype49.

To further investigate the introduction of H5N1 HPAIV into South Georgia and the Falkland Islands, representative H5N1 clade 2.3.4.4b HA sequences from North and South America were used to perform time-resolved phylogenetic analysis (Fig. 3A and Supplementary Fig 2). This analysis provided evidence for distinct, separate introductions of H5N1 into South Georgia and the Falkland Islands, with both sets of sequences sharing a common ancestor with sequences from South America dating back to July 2023 (Falkland Islands, range: December 2022 to November 2023) and August 2023 (South Georgia, range: February November 2023). However, both sets of sequences produced long branch lengths compared to South American sequences, potentially due to unsampled ancestry. The sequences from South Georgia formed a single monophyletic group suggestive of a single introduction, whilst those from the Falkland Islands formed a paraphyletic group, with sequences from Argentina and Brazil. To investigate the source of these introductions, a Bayesian stochastic search variable selection (BSSVS) analysis was performed using the country or location from which the sequences originated as a discrete trait and then the support for these transmissions was quantified using Bayes factors (BFs). This analysis demonstrated that Argentina was the likely source of H5N1 HPAIV for both South Georgia (moderate support with a BF of 3–10) and the Falkland Islands (very strong support with a BF of 30–100) (Fig. 3B). The spread of H5N1 HPAIV within South Georgia was then also investigated using the same approach (Fig. 3C). This analysis further supported that the initial introduction into South Georgia occurred on Bird Island, before onward spread to Penguin River (definitive support with a BF > 100), Hound Bay (strong support with a BF of 10–30) and Moltke Harbour (moderate support with a BF of 3–10). Interestingly, both Harpon Bay and Jason Harbour did not demonstrate a statistically relevant connection to Bird Island based on the BSSVS analysis, but also there was no connection between these sampling locations and any outside of South Georgia either. Nevertheless, this demonstrates that after introduction into South Georgia, H5N1 HPAIV was then spread onward to multiple locations across the islands, whereby it infected multiple avian and mammalian species.

Alongside interrogation of phylogenetic ancestry, the sequences derived from all species were assessed for the presence or absence of adaptive mutations that may indicate adaptation to replication in mammals. From the mammalian samples, only a single amino acid change of interest (PB2 E627K in one southern elephant seal sequence from Jason Harbour) was observed. None of the sequences contained any of the other PB2 mutations of particular interest (i.e., T271A, K526R, Q591K or D701N) or any other mutations associated with adaptation to replication in mammals. Interestingly, a kelp gull sequence from Harpon Bay displayed a single amino acid change of interest, PB2 D701N, but no others. Both southern fulmar sequences obtained from the Falkland Islands contained Q591K and D701N mutations. There were also no mutations in either mammalian or avian species that would affect the susceptibility of this virus to antivirals (Supplementary Table S4). WGS read coverage for all sequences generated can be found in Supplementary Table S5.

Methods
Sample collection
All samples were collected from carcasses found dead on the islands with the permissions of the Falkland Islands government and the government of South Georgia through collaboration with the British Antarctic survey. Sampling was only conducted where safe to do so and to minimize potential pathogen spread by fomite transfer to healthy animals. Data was collected by staff on the ground to define location, species and approximate age.

Virological detection
On the Falkland Islands, initial diagnostic assessment of samples was undertaken at the KEMH Pathology and Food, Water & Environmental Laboratory utilising the QIAamp Viral RNA Mini Kit (Qiagen) and the Oasig OneStep RT-qPCR kit for H5N1 (Genesig). A preliminary diagnosis was made of avian influenza H5N1 infection. Following reports of increasing mortalities and the observation of disease consistent with HPAIV infection in avian and mammalian species in South Georgia, oropharyngeal (OP) and cloacal (C) swabs collected from birds were submitted to the Animal and Plant Health Agency (APHA)-Weybridge for laboratory virological investigation. Total nucleic acid was extracted from all samples45 for testing by a suite of three AIV real-time reverse transcription polymerase chain reaction (rRT-PCR) assays consisting of the Matrix (M)-gene assay for generic influenza A virus detection47,48, an assay for specific detection of HPAIV H5 clade 2.3.4.4b45, and an N1-specific rRT-PCR to confirm the neuraminidase type46. All primers and probes used in these assays can be found in Supplementary Table 6 A positive result was denoted in each case by a Cq value ≤ 36.0. The samples were also screened for avian paramyxovirus type 1 (APMV-1) by an rRT–PCR assay targeting the large polymerase (L) gene74 where a positive result was denoted by a Cq value ≤ 37.0. All amplifications were carried out in an AriaMx qPCR System (Agilent, United Kingdom). The OP swabs, C swabs or brain material were used for virus isolation in 9- to 11-day-old specific pathogen-free (SPF) embryonated fowls’ eggs (EFEs) according to the internationally recognised methods75.

Whole-genome sequencing and phylogenetic analysis
Wherever possible, sequence generation was undertaken on original clinical material. However, material derived following successful virus isolation in eggs was also utilised for sequence generation. For whole-genome sequence analysis, the extracted vRNA was converted to double-stranded cDNA and amplified using a one-step RT-PCR using SuperScript III One-Step RT-PCR kit (Thermo Fisher Scientific) as previously described76,77. The primers used can be found in Supplementary Table 6. PCR products were purified with Agencourt AMPure XP beads (Beckman Coultrer) prior to sequencing library preparation using the Native Barcoding Kit (Oxford Nanopore Technologies) and sequenced using a GridION Mk1 (Oxford Nanopore Technologies) according to manufacturer’s instructions. Assembly of the influenza A viral genomes was performed using a custom in-house pipeline as described previously8 but adapted for nanopore sequence reads. All influenza sequences generated and used in this study are available through the GISAID EpiFlu Database (https://www.gisaid.org). All H5N1 HPAIV clade 2.3.4.4b sequences available in the EpiFlu database between 1st September 2020 and 22nd January 2024 were downloaded to create a sequence dataset. As North America and Europe were over-represented in this dataset, these were sub-sampled to maintain representative sequences using PARNAS78. The remaining dataset was separated by segment and aligned using Mafft v7.52079, and manually trimmed to the open-reading frame using Aliview version 1.2680 The trimmed alignments were then used to a infer maximum-likelihood phylogenetic tree using IQ-Tree version 2.2.381 along with ModelFinder8 and 1000 ultrafast bootstraps82. For the time-resolved phylogenetic analysis, all HA sequences available from South America, and representatives from North America were combined with these sequences from South Georgia and the Falkland Islands and used to infer phylogeny using BEAST version 1.10.483 with the BEAGLE library84. The Shapiro-Rambaut-Drummond-2006 (SRD06)85 nucleotide substitution model was implemented with a four-category gamma distribution model of site-specific rate variation and separate partitions for 83 with the BEAGLE library83,84. The Shapiro-Rambaut-Drummond-2006 (SRD06)85 nucleotide substitution model was implemented with a four-category gamma distribution model of site-specific rate variation and separate partitions for codon positions 1 plus 2 versus position 3 with the Hasegawa-Kishino-Yano (HKY) HKY substitution models one each with a strict clock and a coalescent GMRF Bayesian skyride tree prior. Three independent Markov Chain Monte Carlo (MCMC) runs were performed and combined using the Log Combiner tool in the BEAST package. Each chain consisted of 200,000,000 steps and was sampled every 20,000 steps, and the first 10% of samples were discarded as the burn-in. Discrete geographical transition events were reconstructed using asymmetric continuous-time Markov Chain model with an incorporated Bayesian stochastic search variable selection (BSSVS) to determine which transition rates sufficiently summarize connectivity86. SpreaD3 was used to determine the rates of transmission using a Bayes factor (BF) test. The BF represents the ratio of two competing statistical models, represented by their marginal likelihood, and, in this case, was used to determine the likelihood of transmission between geographic locations. The support of the BF for transmission was interpreted as described previously87). BF and representative transitions related to South Georgia and the Falkland Islands were visualised on maps in R (v4.3.2) using the packages GGPlot288, rnaturalearth89, rnaturalearthdata and sf90. Nucleotide identity between sequences was determined as described previously82. Sequences were genotyped according to the USDA schema, using the GenoFLU tool (https://github.com/USDA-VS/GenoFLU)49.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary:\nThe manuscript by Bennison et al. entitled \"Detection and spread of high pathogenicity avian\ninfluenza virus H5N1 in the Antarctic Region\" describes several avian species infected with HPAI\nH5N1 that were primarily sampled in South Georgia Island (within the Antarctic Polar Front), and\nsecondarily in the Falkland Islands (adjacently external to the Antarctic Polar Front) between\nOctober and November 2023. In total, 49 samples, from 24 individuals representing 5 species of\nbirds, and 6 samples, from 3 individuals representing 1 species of mammal, were sampled. All\nmammalian samples were negative for HPAI H5N1. Most avian samples (~3/4) were positive for\nHPAI H5N1, but virus isolation was successful in only 4 cases, and genomes were reported to\npublic repositories also in only 4 cases. However, the genomes reported to public repositories are\nnot derived from the same samples where virus isolation was successful. Based on these data, and\nusing epidemiological meta-data as support, the authors describe emergence of HPAI H5N1 within\nthe Antarctic region, and then speculate on possible spread forward.\nMajor comments:\nThe identification of HPAI H5N1 within the Antarctic region is, in and of itself, compelling enough to\nwarrant publication in Nature Communications, provided that the data are presented clearly and\ncompletely in order to communicate the importance and relevance of the findings. Several of the\nauthors in this manuscript have extensive experience in the influenza field and knowledge of how\nto conduct these types of analyses properly. It is because of this experience that this reviewer was\nsurprised to find that the manuscript was not particularly well written, and that many key data\nwere not included, or partially described, or insufficiently explained. Often, shortcomings were not\ndiscussed/addressed. A basic example: the summary list I provide above, which includes how\nmany samples, how many species, how many positives, etc., were tested is something I have had\nto count myself from the Supplementary Data provided. These details are super important, yet the\nauthors never clearly state them in the main text. Additionally, key information, like the CT values\nfor PCR-positive samples, or the coverage length and depth for the NGS data deposited in public\nrepositories is missing. The methods are often quite thin, and not enough for a reader to be able\nto fully replicate their work. There is no photographic record of the animals (much less mention of\npathology reports), or explanations for why virus isolation was only possible in 4 cases, or for why\nonly 4 genomes were generated. In fact, there is no explanation for why the 4 samples where\nvirus isolation was possible, were also not sequenced (that’s something that should be done, and\nthe pros and cons of sequencing a primary sample vs. an isolate should be addressed). Because of\nthese reasons, this reviewer considers that the paper in its current form is not suitable for\npublication in Nature Communications unless it undergoes major revisions, that all the missing\ndata are included, and that the viral isolates that were successful are sequenced and subsequently\nincluded in the phylogenetic analysis (which even the authors admit reveals massive under\nsampling. So, if you already know your data is under sampled, why would you not take advantage\nof all the samples you have on hand and try to reduce your under sampling?\nMinor Comments:\nLine 28: comma missing after “Falkland Islands”\nLine 30: check and be consistent with your plurals… I think you mean “fur seals”\nLine 32: your wording is repetitive. You just mentioned “South Georgia” in the sentence above…\nrewrite for consistency and flow.\nLine 42: reference if missing for “Europe”\nLine 52: careful with reference #13. That was not primary literature and it cited a BioRx paper that\nsubsequently retracted a lot of the data they shared in their original submission to BioRx.\nLine 56: “several islands are located within the Antarctic region…” a map early on would be helpful\n(a clear one, that includes which islands are within the Antarctic Polar Front (consider defining this\narea too) and which are not. The map you include later on is hard to see, so not very informative\nor helpful.\nLine 96: First mention of Fig 1A, which is a map, but not a great one. As mentioned above,\nconsider providing a complete map of Antarctica, including the Antarctic Polar Front, as well as\nthe location of both S Georgia and Falklands relative to the polar front.\nLine 100: Figure 1B doesn't match the statement \"these individuals had died\" or the symptoms\ndescribed. Please provide photographic record of dead animals or change the statement that goes\nwith Fig 1B.\nLine 107 onwards: “…swab samples were collected from six found dead kelp gull and four found\ndead brown skua from Hound Bay, South Georgia in addition to four found dead brown skua from\nSt Andrews, South Georgia (Figure 1B)” is a strange sentence. Repetitive and difficult to read. The\nantithesis of clarity. Check grammar and try to help your reader understand you easily, without\nhaving to re-read your sentences/paragraphs. \"Six found dead kelp gull\" sounds super strange.\nAnd then you repeat the same phrase structure twice more. Perhaps try to convey all of this\nrepetitive info in a more useful and informative way? A timeline? In figure form, or in a table? Also,\ntry to provide info in order. Right now in this paragraph you are all over the place: You first talk\nabout sample types collected, then species, then location, then symptoms, then back to sample\ntypes, back to species, and back to locations. It makes for super inconvenient reading. And the\nentire time you refer to either Fig 1A or B, both of which are not helpful and not relevant to either\nsample types of species (only locations, which you can barely distinguish on your maps). Consider\none figure, where A is a map (a clear, useful one that includes the polar front) and B is perhaps\npics of the animals sampled.\nLine 125: “local molecular testing”??? Can you be more specific? Can you explicitly say “RT-PCR”,\nor whatever it is, so that the reader doesn’t have to go to the materials and methods to dig up the\ninfo?\nLine 133: first mention of RT-PCR, but references are not provided and reader sent to the\nsupplementary materials to continue digging for important information. That is not acceptable,\nespecially not for a manuscript submitted to a journal of this caliber.\nLine 133: Supplementary Table 1 doesn’t include CT values for positive samples (please include\nthose), or metrics on the virus isolation (how long till you saw CPE? Or did you test in a different\nway? How did you confirm isolation?), or metrics on NGS (at the very least, discussion of\ncoverage length and depth, with minimum coverage depth for all complete genome segments. These\ndata are also important because they may help explain some of your other observations, like \"infectious\nvirus could not be isolated\" (and yet your Supplementary Table lists 4 positive isolations, so\nwhere’s the mistake?).\nLine 134: You say \"infectious virus could not be isolated\" but Sup Table 1 shows 4 virus isolations\nin embryonated eggs. Not only that, but one of these is from a brain sample, which usually has\nmuch higher viral loads, so it makes total sense that you would be able to isolate from such a\nsample. So where’s the discrepancy? There isn’t one, only that you present your data in\nchronological order, which is not very helpful and in fact leads the reader to confusion. Careful that\npoor presentation of the data doesn’t lead to misinterpretations, like did you do the experiments or\nnot? \"virus could not be isolated\" is a very poor/limited description of what you did or what\nhappened. Clearly state if you tried and failed? Or did you not try? Explain things clearly to your\nreader, so that they don’t have to go fishing in the data to find support for your statements. Also\nconsider that separate from virus isolation, you have other options in the lab, including partial\nsequencing of your PCR products, or better yet, full genome sequencing of the entire genome.\nYou don’t explicitly state any of this in the Results section. You should, as much as possible, use\nconcise language.\nLine 140: “were negative in each assay” is super vague. Which assays are you referring to?\nLine 142: “tested positive” is also super vague… explicitly name the assay.\nLine 143: “in each assay” is more vagueness. What are you talking about? PCR and sequencing?\nPCR and isolation?\nLine 146: “Three full genome sequences…” again, you are reporting your findings in chronological\norder, which is not helpful at all. You have a total of 4 genomes. You should be discussing these as\na whole. Where did they come from, when, do you have full coverage, do you have partial\ncoverage, what’s the CT value of the samples, etc.\nLine 148: “shared 99.86-100% nucleotide identity…” Can you provide some evidence? A figure?\nCan you back-up your statements with data? Or is the reader supposed to go to GenBank,\ndownload the sequence and do the comparison themselves? Same for Line 149 “a single sequence\nwas also generated”… All of the important details/metrics are missing from the narration.\nLine 151: “greater than 98%” this statement is also missing evidence.\nLine 152: “combined” combined how? Provide details on which sequences were used… this is\nimportant to assess the quality of your analysis\nLine 153: “assess genetic ancestry” Again, few details are provided on how the phylogenetic trees\nwere constructed. Yes, they explain this in the materials and methods, but the results section\nneeds enough info to stand on its own. The authors seem to rely/build heavily on the analysis of\nreference #10, but don't explain if what they did is a completely separate analysis that reaches the\nsame conclusions, or a build-on. Authors talk about 131/140 sequences (Line 163), but the\npresent study only generated 4 genomes, so its very easy for the reader to get lost. All of this\ncould be clarified with a thorough re-write.\nLine 170: “produced long branch lengths compared to South American sequences…” Meaning?\nProvide and interpretation please\nLine 171: “discrete trait analysis based upon the country of origin was performed…” What does\nthis mean? Also, how is Supplementary Fig 2 different from Fig 2 (other than they are colored\ndifferently?). What is this figure contributing to the analysis? Define “discrete trait analysis”\nLine 187: “had” needs to be checked for grammar\nLine 181: “being considered” avoid the passive voice\nLine 192: “It is therefore, not unreasonable to suspect that birds on South Georgia may show high\nconnectivity, which may aid the spread of disease, as has been documented previously33, but also\nmay be evidenced by the rapid collection of samples from different areas within South Georgia.”\nThis sentence makes little sense. Please reword.\nLine 199: first definition of “connectivity.” This This explanation/definition needs to be mentioned\nearlier, when the first bring up the topic of \"connectivity\"\nLine 210: “despite the consistency of clinical presentation seen in elephant seals with that reported\nelsewhere.” Can you think of any other explanations?\nLine 214: “invasive sampling of avian and mammalian species remains challenging to undertake in\nareas where appropriate facilities are lacking” Verbose and repetitive. Consider editing down.\nLine 226: “from rapid spread” Again, this is poorly phrased. You mean that the virus is spreading,\nbut the sentence reads as if you are talking about a species spreading.\nLine 249: “detected” check for grammar\nLine 254: “during summer 2023” grammar\nLine 256: “precludes a conclusive assessment of potential incursion routes substantially more\ndifficult” makes no sense\nLine 276: “separately pooled” what do you mean? Define pooled separately\nLine 290: “all influenza sequences generated” there is no clarity on how many samples, how many\nindividuals, how many species, how many genomes you have. One needs to go diving into sup\nmaterials to find out. You need to provide complete details, including NGS specs, coverage,\nCT values, etc.\nLine 298: “bootstraps” full stop missing\nLine 303: “mugration model” define or provide a reference\nLine 358: “Discrete trait analysis” What does this mean? Figures 2 and Sup Figure 2: How are\nthese different?", "reviewer_2": "NCOMMS-23-54471\nDetection and spread of high pathogenicity avian influenza H5N1 in the Antarctic Region\nBennison ad colleagues describe the relevant (and depressing) first observations of HPAI H5N1 in\nthe Antarctic and sub-Antarctic regions of South Georgia and the Falkland Islands. The paper\ndescribes a highly relevant observation and is well written. Relevant data is provided for the\ndescribed observations and conclusions.\nI have only minor remarks to consider:\n- Consider including Suppl table to the main manuscript. It is relevant for the reader to have a\nclear overview of samples collected and outcome of analyses on these samples.\n- Line 41 and further: Consider shortly introducing the different genetic clades that have arisen\nsince 1996 before jumping to mentioning clade 2.3.4.4b\n- Line 42: it now seems clade 2.3.4.4b was first observed in 2021 in Europe. I do not think that is\ncorrect.\n- Line 44: ‘thousands’ of outbreaks at poultry farms seems off.\n- Line 100: not sure to what the reference to the figure actually refers to in this sentence.\n- Line 170: consider explaining in a bit more detail what the long branches actually indicate/mean.\n- Unfortunately there is no uniform classification system yet for reassortments/genotypes. IS there\na labelling by EFSA for genotype B.3.2 as well? Genotype Herring_Gull/France like, is labeled BB\nby EFSA, but labeled differently by You et al who described the definition of B3.2. Due to lack of\na uniform genotype labelling system, a schematic of the gene segment reassortment and their\norigin could be insightful\n- Figure 1A: consider zooming out a bit more to make clearer which region it is", "reviewer_3": "I appreciate the opportunity to read and review the manuscript, ‘Detection and spread of high\npathogenicity avian influenza virus H5N1 in the Antarctic Region’. In this manuscript, the authors\ndescribe observations of sick and dead birds on South Georgia and the Falkland Island during\nOctober–December 2023 as well as diagnostic findings confirming the occurrence of highly\npathogenic H5N1 avian influenza (HPAI) among a relatively small number of the diagnostic\nsamples collected. This report signifies the first confirmed detection of HPAI in the Antarctic region\nand documents further geographic spread of the ongoing panzootic caused by infection with\ngoose/Guangdong lineage viruses. The authors discuss the first detection of HPAI in the Antarctic\nregion in the context of potential ecologic consequences.\nIn general, I found this manuscript to be well-written, to be based on apparently robust data, and\nto contain reasonable inference given the data summarized. I did not identify any obvious\ndeficiencies pertaining to sampling design or diagnostic methods employed. My critiques are\nrelatively minor and generally pertain to a perceived lack of clarity or precision which could\nintroduce reader confusion. To this end, I’ve appended numerous, mostly editorial, suggestions\nbelow which I hope the authors may find helpful in the revision of their product.\nI thank the authors for submitting their best work to Nature Communications.\nSpecific comments…\nLines 27, 34, and throughout: The authors might consider whether this manuscript describe the\n‘emergence’ of HPAI in the Antarctic region or (geographic) spread thereto. Though an argument\ncan certainly be made for the former, the latter seems irrefutable to me given the evidence.\nLines 64-67: My question here may be naïve, but are these ‘resident’ species, or rather, migratory\nspecies endemic to the Southern Ocean?\nLine 81: What is meant by ‘continental America’?\nLine 82: The modifiers ‘likely’ and ‘high’ seem unnecessary to me. Also, ‘dispersal’ or ‘spread’\nmight be more precise than ‘transmission’ in this sentence when referencing spatial dissemination.\nFinally, this statement might be improved if it were made past tense.\nLines 100-102: I presume Bird Island is considered to be part of South Georgia or a broader South\nGeorgia island group. It would be helpful to your reader to clarify (e.g., Bird Island and South\nGeorgia appear, at times, to be used interchangeably throughout the manuscript). Also, was this\nmortality on Bird Island also among brown skuas or other species? It is somewhat unclear as\nwritten.\nLine 118: Perhaps events might be described as ‘occurring’ rather than ‘emerging’.\nLine 121: The article ‘a’ should be added before ‘Falkland steamer duck’.\nLines 95-143: You might consider referencing Supplementary Table 1 periodically throughout this\ntext to help out your reader(s).\nLines 158-163: This same information is conveyed almost verbatim in the Discussion (lines 239-\n246). Please include only once to avoid repetition. Also, please consider using ‘dispersal’ or\n‘spread’ rather than ‘transmission’ to refer to spatial dissemination.\nLine 166: Perhaps ‘provided evidence for’ rather than ‘demonstrated’.\nLines 170-173: I personally found this analysis to be weak given probable sampling/reporting\nbiases (e.g., the dearth of sequences from Argentina). I note that the authors do not reference this\nanalysis in the Discussion. Given inherent uncertainty in this analysis and the lack of contribution\nto the overall narrative, I suggest that the authors consider omitting from their product.\nLine 178: The authors might clarify that they are referencing the Antarctic mainland here.\nLine 187: Consider using ‘susceptible bird populations’ rather than ‘the populations of sensitive\nbird species’.\n194: The authors might consider ending their statement after reference 33, perhaps adding\nreference 49. I’m dubious that sample collection alone supports their claim (e.g., in the absence of\ngenomic information or animal movement data) and the description of reference 49 doesn’t add\nsignificant value to the narrative in my opinion.\nLine 198-204: I don’t follow the logic of this argument. I feel that the Subantarctic and Antarctic\necosystems are quite diverse and that pelagic birds may or may not be particularly likely to\nfacilitate rapid HPAI spread. The authors should please ‘take or leave’ this viewpoint, but I’d\nencourage them to omit this particularly subjective text. Alternatively, they might revise/re-frame\nas I acknowledge that I may be misunderstanding the intent of messages.\nLine 249: There is a grammatical issue here.\nFigure 2: Given that HPAI detections in the Antarctic region and those in South America are\nconsistently of genotype B3.2 viruses, phylogenetic analysis of complete concatenated viral\ngenomes would potentially provide higher resolution inference than a tree generated using only\ndata for the H5 HA gene segment. Also, the authors might consider using ‘dispersal’ or ‘spread’\nrather than ‘transmission’ in the legend."}, "author_response": "Response to Reviewers – NCOMMS-23-54471 – Detection and spread of high pathogenicity avian\ninfluenza H5N1 in the Antarctic Region\nWe thank the reviewers for their fulsome assessment of the work we submitted. Alongside amending\nthe manuscript to satisfy the reviewer’s comments we have also added a considerable amount of data\nto further define the situation in the area. As such, the manuscript has been thoroughly amended with\nhighlighting throughout to demonstrate where changes have been made. Below we offer a point-by-\npoint rebuttal to comments on the original article with our responses being italicised and in bold font\nfor clarity.\n\n[... Author responses to Reviewer 1, 2, and 3 points ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I guess many of my comments have been addressed (especially the minor ones), though in all\nhonesty it has been hard to assess this, as the responses are mostly “the comments have been\naddressed” or “we have amended the text,” but when one goes to the text it is very hard/impossible\nto find the change since the new manuscript is completely different from the old one and there is no\nexplicit mention in the response to reviewers how/where it is that they have “addressed the\ncomments” or “amended the text.” Also, the new version of the manuscript does not include track\nchanges, which makes the work of a conscientious reviewer who provided honest feedback in good\nfaith harder than it should be. I will say that this version of the manuscript is MUCH improved over\nthe previous one (congrats and thank you!), and I was hoping that I could say yes, go ahead and\npublish as is, as the discovery of HPAI H5N1 in Antartica is super compelling on its own. However, there\nare still some remaining points that I think are important and where I emphatically disagree\nwith the authors.\nLine 37: They say “Critically, genetic assessment of sequences from mammalian species demonstrates\nno increased risk to human populations.” And again later in Line 329: “Critically, assessment of\nsequences derived from mammalian species did not indicate any adaptive mutations or increased risk\nto human populations. A single sequence from a single Southern elephant Seal sequence from Jason\nHarbour) contained the PB2 E627K mutation that is associated with adaptation to replication in\nmammalian species...” My issue is: how can you claim no increased risk to human populations when\nyou find one of *THE* most important markers of mammalian host adaptation/pathogenicity, which is\nPB2 E627K present in a mammal?! Is it because you only find it in one sample? Isn’t it also true that\nyou recognize that you are vastly under-sampled? Your trees show it! Not to mention issues with your\nsample collection/testing. It doesn’t escape me that working in Antarctica is incredibly difficult, and\nthe logistics perhaps sometimes insurmountable. But the explanations provided to rebut the good\ncritical feedback that was given during the first round of reviews are disappointing. For example, Line\n258: “Information to date suggests that HPAIV infection in seals often leads to a neurological\npresentation with infrequent detection of viral material being detected through standard swab\nsampling activities. This may explain the initial lack of influenza vRNA detection in elephant seal swab\nsamples taken in this study.” Claiming “infrequent detection” in swabs is misleading. What is true is\nthat the brains of infected animals have higher viral loads than other tissues, especially when they\nhave encephalitis, but that does not mean you cannot pull this from swabs (or other tissues), so a\nmore thoughtful and honest discussion of why you didn’t or couldn’t would be appreciated.\nAnother point: I asked you to include minimum coverage depth for your sequences, but you claim that\nthis has “little relevance” since you are reporting consensus genomes. I completely disagree that\ncoverage depth is of little relevance, even for consensus genomes. Yes, we often translate NGS data\ninto consensus genomes, particularly in a situation like this one, where one is not looking at intra-host\nviral diversity, but a consensus genome with a coverage of 1x is completely different from a\nconsensus genome with coverage of 10,000x. Coverage depth is a measure of certainty, of probability (in a\nsense) that the sequence you are reporting is correct, so for you to dismiss this as “not relevant” is\nnot appropriate. Asking for coverage depth is the least I could ask you for. I could be asking you to\ndeposit your raw data into public repositories for others to scrutinize, and yet, because of the gravity\nof the situation I am asking for simple information that you already have. Why do you not want to\nshow it? Is your data not solid? Or is it that you don’t trust it? Or do you not find it compelling\nenough? I would suspect that it is because there are parts of the genomes where your coverage depth\nis zero –if so, this should be discussed, not hidden. We all generate data where some parts of the\ngenome have low or zero coverage depth. I went to GISAID to look for the sequences and they were\nhard to find (you also don’t provide accession numbers), so along with the fact that you claim “no\nincreased risk to humans” despite having E627K and being under-sampled worries me tremendously.\nFinally, line 340: “should the situation change…” I would argue the situation is already changing, only\nthat you are catching it early and therefore you don’t have all the evidence clearly displayed in front\nof you (just a few previews). And rather than seeing the situation for what it is (you have E627K!!!!! how\nmuch more compelling can it get?!?!?!), you don’t grasp its potential severity, and the potentially\ncritical finding of E627K in a mammal. Yes, it is true you only find it only in one sample thus far, but if\nit is in one sample already, it is a matter of time before it is found in others.", "reviewer_3": "I appreciate the opportunity to review the revised manuscript, ‘Detection and spread of high\npathogenicity avian influenza virus H5N1 in the Antarctic Region’ submitted to Nature\nCommunications. The authors have substantially reworked their original submission in response to\nthree reviewer critiques. Additionally, the authors have expanded the scope of the manuscript to\ninclude numerous additional detections of highly pathogenic avian influenza (HPAI) viruses in the\nAntarctic Region. They have also bolstered analyses through the inclusion of additional genetic\ninformation on HPAI viruses. The manuscript is much improved and also substantially altered as\ncompared to the original submission.\nFrom my perspective, the revised manuscript is conceptually sound and lacking any fatal flaws in\nreporting of information or interpretation. My assessment is that modest revisions would help to\nshore up several minor deficiencies within the narrative. I’ve appended (below) numerous comments\ntowards this end. I thank the authors for their important contribution to Nature Communications and\nthe broader scientific literature.\nLines 45-46: It is a gross over-simplification to state that GsGd HPAIV ‘spread to other Asian\ncountries’. Such viruses also spread through Europe, much of Africa, and into North America prior to\n2021. Please revise this statement to make more congruent with the preceding and following\nstatements.\nLine 87: It is not explained how H11N2 viruses contrast with those of H4-H6 HA subtypes.\nLine 89: Perhaps spread of low pathogenic avian influenza viruses to the Antarctic Region from both\nthe Americas and elsewhere provides evidence on HPAI virus introduction risk? See prior comment.\nLine 115: I believe ‘preyed’, rather than predated, may be the more appropriate term here.\nLine 142: 6/17 mammalian carcasses are reported as HPAI virus positive in tables 1 and S2.\nLine 178-180: There is a grammatical issue in this statement.\nLine 188-189; I understand the ‘full data set’ to include only sequences generated in this study and\nnot the representative sequences included in the maximum likelihood phylogenetic analyses as\ndescribed throughout the preceding statements in the paragraph. As such, the statement explaining\ncomparisons of nucleotide identity should probably be presented first with explanation of maximum\nlikelihood phylogenetic analyses following thereafter.\nLine 192: The full tree (Figure 2A) with complete tip labels including strain names and/or accession\nnumbers should be provided to readers for transparency and so the analysis can be replicated. Such\na figure could be provided as a supplemental file.\nLine 226: There is a tense issue with the verb ‘has not’ (e.g., should be ‘had’ not given the report of\nHPAI in the Antarctic Region in this product).\nLine 262-264: I’m not understanding the logic that invasive sampling demonstrated that swab\nsamples were positive. Are the authors trying to convey that testing of tissue samples collected\nthrough necropsy was often consistent with diagnostic results for swab samples generated from the\nsame individuals?\nLines 266-268, 289-295: I encourage the authors to focus discussion on information presented in their\nreport, not unreported/unpublished/unreviewed observations that cannot be verified by readers.\nLine 304: The phrasing ‘the original H5N1’ is not appropriate nor correct. Please rephrase along the\nlines of, ‘genotype 3.2 emerged following co-infection (of a presumably avian host) with a viral\ndescendant of the first GsGd HPAI virus detected in North America during 2021 (purportedly\nintroduced from Europe) with a North American lineage virus’."}, "author_response": "Bennison et al., 2024- Rebuttal to revision\nAgain we thank the reviewer’s for their insightful comments on our manuscript. A rebuttal to each\npoint is listed below and a line number added for ease of checking against the amended\nmanuscript.\n\n[... Author responses to Reviewer 1 and 3 points ...]"}]

Decision letter