# Mass mortality events in the sub-Antarctic Indian Ocean caused by long-distance circumpolar spread of highly pathogenic avian influenza H5N1 clade 2.3.4.4b

Augustin Clessin<fnref n="1" />, François-Xavier Briand<fnref n="2" />, Jérémy Tornos<fnref n="1" />, Mathilde Lejeune<fnref n="1" />, Camille De Pasquale<fnref n="1" />, Romain Fischer<fnref n="1" />, Florent Souchaud<fnref n="2" />, Edouard Hirchaud<fnref n="2" />, Tristan Bralet<fnref n="1" />,<fnref n="3" />, Christophe Guinet<fnref n="4" />, Clive R. McMahon<fnref n="5" />, Béatrice Grasland<fnref n="2" />, Guy Baele<fnref n="6" />, Thierry Boulinier<fnref n="1" />

Author contributions: TBo, AC, GB and BG conceived the study; TBo, JT and ML led the field work; CDP, RF, TBr, CG, and CRM contributed to field sampling; FXB and FS performed the molecular analyses; EH performed the sequencing data curation; GB, FXB, AC and BG performed the sequencing analyses; ML performed the serological analyses; AC, TBo and GB wrote the first version of the manuscript and all authors provided inputs.

<footnote n="1">¹ Centre d'Ecologie Fonctionnelle et Evolutive (CEFE), CNRS, Université Montpellier, EPHE, IRD, Montpellier, France</footnote>

<footnote n="2">² ANSES, Laboratoire National de Référence, Ploufragan, France</footnote>

<footnote n="3">³ ANSES, UZB, Maisons-Alfort, France</footnote>

<footnote n="4">⁴ Centre d'Etudes Biologiques de Chizé (CEBC), UMR 7372, CNRS-La Rochelle Université, 79360 Villiers-en-Bois, France</footnote>

<footnote n="5">⁵ IMOS Animal Tagging, Sydney Institute of Marine Science, Mosman, 2088, New South Wales, Australia</footnote>

<footnote n="6">⁶ Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium</footnote>
Abstract

Since 2020, the outbreak of highly pathogenic avian influenza (HPAI) virus clade 2.3.4.4b
has turned into the largest documented panzootic to date, reaching the sub-Antarctic region
and Antarctica via the tip of South America in 2023. Here, we describe its recent arrival into
the Indian Ocean sub-Antarctic archipelagos of Crozet and Kerguelen, where we first
detected the virus in October 2024 in dead southern elephant seals, king penguins, gentoo
penguins, brown skuas and kelp gulls. While the panzootic is ongoing, it has already caused
unprecedented and alarming mortalities of southern elephant seals. We collected brain
swabs from various seal and bird carcasses, subsequently isolated the virus and obtained
25 novel HPAI H5N1 clade 2.3.4.4b sequences. Our phylogenetic and phylogeographic
analyses show that there have been independent introductions of the virus to Crozet and
Kerguelen, from the distant South Georgia Islands in the Southern Atlantic, and not from the
more nearby coasts of South Africa. Our results point to a year-long gap in genomic
surveillance in the south polar region, obscuring how HPAI H5N1 clade 2.3.4.4b is spreading
in the sub-Antarctic and illustrating the difficulties in tracking pathogen dispersal in the
region. Locally, our phylogenetic analyses show that the virus is transmitted between
different species. Moreover, our serological analyses show that some southern elephant seal
pups had mounted an anti-H5 antibody response. With the spread to Crozet and Kerguelen,
HPAI H5N1 2.3.4.4b is moving ever closer to Australia and New Zealand, which currently
remain free from infections with this strain, and represents a major threat to the sub-Antarctic
wildlife. Our results provide key elements to enable stakeholders to anticipate the arrival and
spread of the virus in remote areas of critical wildlife conservation concerns.
Introduction

The ongoing outbreak of clade 2.3.4.4b of highly pathogenic avian influenza (HPAI) H5N1
virus has been unique not only in the large number of host species affected, with a wide
range of both birds and mammals reported dead ¹⁻³, but also in the scale of the panzootic, in
that all continents except Oceania have been affected ⁴. Originating in Asia in 1996 ⁵,⁶, it
spread across the world in different waves. In the large outbreak of 2016-2017, it reached
South Africa ⁷. In 2021 and 2022 it started to severely impact wild bird species and poultry
across Europe ⁸⁻¹⁰, in southern Africa ⁷,¹¹, across North America ¹², and South America where
it resulted in die-offs of tens of thousands of seabirds ¹³ and marine mammals ¹⁴. At the time
of writing, HPAI H5N1 clade 2.3.4.4b has not been detected in any birds or other animals in
Australia and the southern Pacific region ¹⁵. While there were no incursions of H5N1 into the
Antarctic region during the summer season 2022/2023 ¹⁶, HPAI H5N1 clade 2.3.4.4b was
detected for the first time in the Antarctic continent and the surrounding sub-Antarctic islands
in October, 2023 ⁴, when it spread from the tip of South America to the Falkland and South
Georgia islands. On route down the coast of South America, the virus had a devastating
effect on seal and bird populations ¹³,¹⁷, hence the scientific community was extremely
concerned about the impact the virus could have on the dense seabird and pinniped
communities of the sub-Antarctic and Antarctic regions. The situation on South Georgia
Island turned quickly into a mass mortality event of pinnipeds and seabirds. The mortality
levels were especially worrying for two species, the wandering albatross (Diomedea
exulans) ¹⁸ and the southern elephant seals (Mirounga leonina) ⁴. Moreover, scavenging
species, like the brown skua (Stercorarius antarcticus) and the giant petrels (Macronectes
spp), have been suspected to play a key role in virus spread ¹⁶.

The sub-Antarctic vertebrate communities are characterised by pinnipeds and
seabirds that forage at sea and breed in dense and often mixed-species colonies on the
sparse islands and archipelagos that span the region ¹⁹. This results in an annual pulse of
high numbers of concentrated animal congregations to breed and moult. For instance, the
French Austral Lands and Seas (Crozet, Kerguelen, Saint-Paul and Amsterdam Islands) are home to 50 million seabirds of up to 47 species, many of which aggregate in large numbers for breeding on Crozet and Kerguelen each year, along with hundreds of thousands of marine mammals²⁰. In addition to being at risk of massive die-offs because they are densely packed during breeding, several of the seabird populations are especially threatened because they are only present at a few locations and their populations are already under pressure from global change²¹. Seabirds and seals make extensive movements at sea within and outside the breeding season, and it is critical to track the potential spread of the virus over vast areas of oceanic water and identify its mechanisms of transmission within and among wild host communities²².

The Crozet and Kerguelen archipelagos are both located in the Southern Indian ocean and are among the most isolated islands of the world, being respectively 2300 and 3800 kilometres from the nearest continents. While much has been learnt about the at-sea movements of seabirds and seals from and to the archipelagoes ²³, it is unclear how these movements may facilitate the spread of disease agents like H5N1 given the limited information on infectious period length, mortality rates and pathogeny in key potential hosts. Consequently, and despite growing concerns of the scientific community given the extent of the HPAI epizootic in the Atlantic sub-Antarctic region, the eventual arrival of the virus to the sub-Antarctic Indian region remained feared and uncertain during the entire 2023-2024 austral summer ¹⁶. In October 2024, we recorded abnormally high numbers of deaths in southern elephant seal colonies on Possession Island, in the Crozet archipelago, Southern Indian Ocean (Figure 1). Later, we recorded deaths of several seabird species, in lower numbers. We detected the first abnormal mortality of southern elephant seals almost a month later on Kerguelen island. We here describe the first confirmed HPAI outbreaks on the Crozet and Kerguelen archipelagos and report the results of phylogeographic analyses of the virus sequences we obtained. This provides us with key insights into the origin and number of introductions, on the similarities of viral isolates between mammals and seabirds,
as well as evidence of mammal-to-mammal transmissions. Initial serological investigations
suggest that a number of southern elephant seals mount an antibody response.

<figure><img src="image_1.png" /><figcaption>Figure 1. Carcasses of southern elephant seals and king penguins on Petite Manchotière, Possession Island, Crozet. A) Brown skua scavenging on a penguin carcass (yellow arrow); B) Aggregation of dead elephant seal pups; C) Brown skua scavenging on a penguin carcass (yellow arrow) next to a dead adult elephant seal; D) Dead elephant seals and king penguins in very close proximity to high densities of king penguins; E) Carcasses of king penguins at the river mouth at the edge of a large breeding colony. We refer to Supplementary Figure S1 for more detailed pictures of A) and C), to Supplementary Figure S2 for a map that shows the location of Crozet and other sub-Antarctic islands, and to Supplementary Figure S3 for detailed maps of our sampling sites on Crozet and Kerguelen. Picture credit: Jérémy Tornos/Mathilde Lejeune, CNRS/IPEV.</figcaption></figure>

**Methods**

*Study areas*

The Crozet (46°24' S, 51°45' E) and Kerguelen (49°15' S, 69°10' E) archipelagos are part of the French Southern and Antarctic Lands (TAAF), and together with the Prince Edwards Islands (Marion and Prince Edwards, South Africa) and Heard and McDonald Islands
(Australia) they constitute the main Southern Indian Ocean Islands, where millions of seabirds and marine mammals aggregate each year to breed (Supplementary Figure S2). Situated near the oceanic polar front, these islands are exposed to windy and cold oceanic climates. They have no permanently settled inhabitants nor domestic animals. Approximately 60 (in the winter) to 150 (in the summer) people are usually present on Crozet and Kerguelen at any time, mainly made up of military personnel, officials, scientific researchers and support staff. The islands are serviced four times a year by the research and supply ship RV Marion Dufresne, from Réunion Island.

Sample collection

Breeding colonies of seabirds and marine mammals on Possession Island, Crozet archipelago, and Kerguelen islands are regularly monitored as part of long-term research projects supported by the French Polar Institute (IPEV) and management activities of the French Southern Territories National Nature Reserve. As part of disease ecology research and surveillance, field personnel working with wildlife had been trained to contribute to the detection and reporting of abnormal mortalities that could be due to infectious diseases. After the first abnormal mortalities were detected and led to suspicion of HPAI emergence on Possession Island, Crozet, specific sampling was organized at the site of the first detections as well at other locations with aggregations of seabirds and pinnipeds (Supplementary Figure S3). Special biosecurity measures were taken to prevent risks of contributing to the spread of infectious agents and exposure of personnel, according to international guidelines adapted to specific field conditions ²⁴. We reached the sampling sites on foot. Sites close to refuges enabled a longer presence and therefore a more complete sampling effort while applying appropriate biosecurity measures. The Baie Américaine refuge was closed for sanitary reasons (as it was very close to an elephant seal colony), and this site was particularly difficult to access because of the distance to the station (8 km) and the
biosecurity constraints that needed to be respected. On Kerguelen, we accessed our
sampling site on the north eastern side of the Courbet Peninsula by helicopter.

At each site, we focused sampling on the freshest accessible carcasses, with care to
limit disturbance to the local wildlife. We focused our sampling efforts on collecting swab
samples from the brain - using contendant tools and sterile swabs - given prior reports of a
tropism of the HPAI virus for the central nervous system in marine mammals¹⁴, and from the
oral cavity for birds. Nasal and rectal samples are difficult to collect due to scavenging. We
stored the first series of swab samples, collected between October 20th and 27th, in the field
in dry sterile microcentrifuge tubes, before freezing and storing them at -80°C within 6 hours.
Samples were then brought back to Réunion Island and transported to the French National
Reference Laboratory for avian influenza, at ANSES (Ploufragan). We collected a second
series of swabs over the following four weeks. These samples were stored and transported
in the same way, except samples collected in the most remote part of the island where dry
swabs were maintained at ambient temperature (circa 6°C) for several days before being
frozen at -80°C. On Kerguelen, we carried out the sampling of carcasses out on December
1st and 2nd, 2024. In addition, we had previously collected a series of blood samples from
adult southern elephant seals in Kerguelen in 2018 (n=41), 2022 (n=46) and 2023 (n=57).
We spinned the blood samples and separated the plasma within 24 hours after collection
with heparinized syringes. In 2024, in parallel to having sampled carcasses, we sampled
blood from 26 asymptomatic elephant seal pups. Due to the timing of collection of those
samples, concomitant with the ongoing mortality, the seroprevalence could not be used to
infer the proportion of pups that had been infected among those that eventually survived the
event. After centrifugation in the field, we stored the plasma samples at -20°C until serology
analyses.

Avian influenza antigenic testing
We used qualitative rapid immunochromatographic strip tests (BIO K 106, Dpifit AIV Ag, BioX) in the field to detect the potential presence of avian influenza virus in swab samples. The test detects the presence of NP protein of avian influenza viruses. The detection level is expected to be 10⁴.⁸ DIE50 / mL. Given the expected high viremic load in the brains of dead animals, we expected brain samples to be excellent in-field indicators of HPAI induced mortality. We reported a test as positive when the control and T bands came out visible, but note that all samples were subject to further RT-qPCR testing, validation and confirmation at the French National Reference Laboratory for avian influenza.

## Detection and sequencing of HPAI H5N1 clade 2.3.4.4b

We eluted each dry swab in 1 mL of minimal essential medium (MEM). We extracted RNA from 100µl of the elution medium with the addition of a mixture of RLT buffer and β-mercaptoethanol for the lysis step. The following steps were carried out inline with the NucleoMag Vet (Macherey) extraction procedures using the KingFisher automated system. We performed detection of avian influenza H5 HP clade 2.3.4.4b by real-time qRT-PCR (rt RT-PCR) according to the protocol recommended by the European Union Reference Laboratory for avian influenza virus (personal communication; Calogero Terregino). For full genome sequencing, we amplified the eight viral segments by RT-PCR ²⁵, followed by library preparation and deep sequencing on a NextSeq2000 sequencer (Illumina).

## Phylogenetic analysis

On December 26th, 2024, we downloaded all available H5N1 HA sequences as well as full genomes from GenBank ²⁶ and GISAID ²⁷,²⁸. We first removed duplicate entries and complemented the resulting data set with our 25 newly generated H5N1 2.3.4.4b sequences from the Crozet and Kerguelen archipelagos (Table 1). We aligned the resulting HA data set using MAFFT v7.490 ²⁹, followed by manual / visual inspection. This yielded an initial data set containing 15072 sequences. We then performed maximum-likelihood (ML) phylogenetic
inference using IQ-TREE v2.2.2.6<sup>30</sup> with automated model selection using ModelFinder<sup>31</sup>, which selected the GTR+F+I+R6 model according to the Bayesian Information Criterion. We performed 1000 bootstrap replicates using the ultrafast bootstrap approximation approach (UFBoot)<sup>32,33</sup>. From this large ML phylogeny, we extracted a large, well-supported (bootstrap support 94%) cluster that contained our 25 novel HPAI H5N1 2.3.4.4b sequences from the Crozet and Kerguelen archipelagos, to be used in subsequent analyses. Within this cluster, our 25 sequences were included within an 83-sequences clade that had 100% bootstrap support and contained sequences from South Georgia and the South Sandwich Islands, as well as Antarctica.

We proceeded to construct a full-genome data set for this cluster to improve phylogenetic resolution in a subsequent Bayesian phylogenetic analysis. We followed the approach in Uhart et al.<sup>14</sup> in that such a workflow limits the time for H5N1 to accrue mutations and diversify, thereby limiting genetic diversity, but did not restrict ourselves to only complete genomes in order to reduce both geographic and genetic biases<sup>34</sup>. We removed reassortant sequences while constructing ML trees to assess temporal signal in this complete genome data set and remove outliers using TempEst<sup>35</sup>. This resulted in a final data set consisting of 1221 genomic sequences which exhibited a clear temporal signal.

We continued to perform a Bayesian time-calibrated phylogenetic analysis on this data set using Markov chain Monte Carlo as implemented in BEAST v1.10.5<sup>36</sup>, employing the BEAGLE high-performance computational library for computational efficiency to run on a powerful graphics processing unit<sup>37</sup>. We employed a non-parametric coalescent model<sup>38,39</sup> as the tree prior, a general time-reversible substitution model<sup>40</sup> with a discretized gamma distribution to accommodate among-site rate heterogeneity<sup>41</sup>, and an uncorrelated relaxed clock with an underlying lognormal distribution<sup>42</sup>. We used the default priors in BEAST v1.10.5<sup>36</sup>, including a conditional reference prior on the mean evolutionary rate<sup>43</sup>. We ran this analysis until all relevant effective sample sizes reached at least 200, as assessed in Tracer v1.7.3<sup>44</sup>, and checked if independent replicate analyses converged to the same
posterior distribution. Based on the sampled posterior phylogenetic trees from this analysis,
we performed a Bayesian discrete phylogeographic analysis using an asymmetric
continuous-time Markov chain model with Bayesian stochastic search variable selection <sup>45</sup>.

The following locations were present in our 1221-taxa data set and used as discrete states:
Antarctica, Argentina, Bolivia, Brasil, Canada, Chile, Colombia, Costa Rica, Crozet Islands,
Ecuador, Falkland Islands, Guatemala, Honduras, Kerguelen Islands, Panama, Peru, South
Georgia Islands, United States, and Uruguay. We used the highest independent posterior
subtree reconstruction (HIPSTR) approach in TreeAnnotator X <sup>46</sup> to construct a
time-calibrated consensus phylogeny, after removing 10% of the chain as burn-in.

Identification of molecular markers with zoonotic potential or mammal adaptation

The identification of the main molecular markers already identified in the literature was
integrated just after the workflow for determining the consensus sequences and
automatically applied to each sample using the FluMut application <sup>47</sup>.

Serological analyses

We collected blood samples from southern elephant seals using heparinized syringes and
separated the plasma in the field by centrifugation, which we then stored at -20°C until
further analysis. We placed plasma samples at 56°C for 30 minutes before performing
analyses in order to ensure inactivation of the H5 HP viruses contained in plasma samples
and decomplementation of the plasma. We used commercially-available enzyme-linked
immunosorbent assay (ELISA) kits to detect antibodies against avian influenza viruses in
plasma samples. To detect and quantify specific antibodies against H5-AIV protein in plasma
samples, we used a competitive ELISA (ID Screen® Influenza H5 Antibody Competition,
reference no. FLUACH5-5P, Innovative Diagnostics SARL, Grabels, France). We conducted
the assay according to the kit instructions. We obtained plasma antibody titre by reading
absorbance at 450 nm on a microplate reader (Tecan Infinite® 200 Pro; Tecan Group Ltd.,
Mannendorf, Switzerland) and corrected raw absorbance values using optical densities of the negative control provided by the ELISA kit (OD<sub>NC</sub>). Anti-H5 AIV antibody levels in plasma samples are expressed through an inhibition value calculated as: Inhibition<sub>sample</sub> = OD<sub>sample</sub>/OD<sub>NC</sub>. We measured the between and within-assay repeatability using inhibition values calculated from a repeated sub-samples run on each plate-run (between-assay coefficient of variation: 4.4 ± 3.7%, n=8 samples; within-assay coefficient of variation: 1.7 ± 1.1%, n=6 samples).

# Results

## Descriptive epidemiology

The abnormal die-offs on Possession Island, Crozet, which one of us (CDP) detected in the last part of October 2024, consisted mostly of southern elephant seal pups, but also included several adult elephant seals, king penguins, brown skuas, wandering albatrosses and a Cape petrel (Daption capense) (Figure 1). The sites where we detected the initial massive die-off are Petite Manchotière and Baie Américaine beach, where on October 17th we counted more than 90 dead southern elephant seal pups over a total of approximately 800 (Supplementary Figure S3). Some of the alive elephant seals showed symptoms, mainly convulsion, conjunctivitis, uveitis and nasal discharge. We saw one adult vomiting. On Baie du Marin, near the base, we detected more than 50 dead elephant seal pups and 2 adults on October 28th, as well as dozens of dead king penguins. On the two days when we conducted sampling at Jardin Japonais (October 27th and November 7th, 2024), we respectively counted 17 dead pups out of a harem of 21 in total, and only 8 live pups out of a total of more than 270 that we had counted a few weeks earlier. Three weeks later, the death rate had risen considerably at all sites, but the exact proportion is difficult to assess, as the carcasses are rapidly washed away by seawater, buried partly in the sand or eaten by scavengers. Nevertheless, we estimate that more than half of the pups died at most of the
sites we visited by mid-November, and mortality was still high at that time. We also found
significant numbers of king penguins (over 150 detected on the edges of the colony) dead or
showing clinical symptomatic signs at Petite manchotière and Jardin Japonais - lethargy,
shivering, eye conjunctivitis and nasal discharge. The number of affected penguins was
nevertheless small compared to the total number of alive penguins present - tens of
thousands at those three large colonies. Note that mostly moulting adult king penguins were
affected. We detected mortalities at many southern elephant seal breeding sites and several
seabird breeding sites on Possession island, including 2 nestlings of wandering albatross
(positive with antigenic test) on November 6th at Petite manchotière. Further intense
mortalities of southern elephant seals carried on after those early reports.

On mainland Kerguelen, on November 11th at the Ratmanoff king penguin and southern elephant seal colony, a first dead pup was detected and turned up as avian influenza positive with an antigenic test. On the following days, dozens of dead brown skuas, kelp gulls (Larus dominicanus) and southern elephant seal pups were detected on the close perimeter of the large penguin colony. At all sites, scavengers - brown skuas, southern giant petrels (Macronectes giganteus), northern giant petrels (Macronectes halli) and lesser sheathbills (Chionis minor) - were observed foraging on dead carcasses (Figure 1). We also observed scavenging at sea on floating carcasses, by *procellariiformes* and brown skuas.
On Crozet, we conducted a full necropsy of a king penguin in the field and observed macrological signs of pericardial hemorrhage and broad internal tissue inflammation (Supplementary Figure S4). Histological examination was not available at the time of writing.
The on-site avian influenza antigenic tests we conducted on brain swabs from dead animals were positive for most sampled individuals or pools of samples, and the presence of HPAI H5N1 virus was confirmed by rtRT-PCR conducted at the French National Reference Laboratory. The virus was detected in 65 samples and sequencing could be conducted on 25 samples from southern elephant seals and seabird species (Table 1 and Supplementary Table 1). Among the 46 samples of southern elephant seals tested by RT-PCR, 42 came out
positive for H5 clade 2.3.4.4b highly pathogenic avian influenza (Supplementary Table 1). 11
king penguins, 7 brown skuas, 3 kelp gulls and one Cape petrel were also found to be
RT-PCR-positive for HPAI H5N1. Among the 21 avian influenza antigenic tests conducted,
all that came out positive (16) corresponded to carcasses for which at least a positive H5
RT-PCR sample was found (Supplementary Table 1). Of the four that came out negative and
for which a RT-PCR could be conducted, two came out negative by RT-PCR, but two - one
king and one gentoo penguin (*Pygoscelis papua*) - came out positive.
<table><caption>Table 1. Samples from which we obtained genomic sequences, with species name, location, type of sample, and sampling date. We provide accession numbers for GISAID. We refer to Supplementary Table 1 for an overview of all PCR-tested samples.</caption><thead><tr><th>Host</th><th>Site</th><th>Location</th><th>Sample</th><th>Age</th><th>Collection date</th><th>GISAID accession no.</th></tr></thead><tbody><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>EPI_ISL_19747197</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>EPI_ISL_19747198</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>EPI_ISL_19747199</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>EPI_ISL_19747200</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>EPI_ISL_19747211</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>EPI_ISL_19747210</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Adult</td><td>20 Oct. 2024</td><td>EPI_ISL_19747201</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Adult</td><td>20 Oct. 2024</td><td>EPI_ISL_19747202</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Pup</td><td>27 Oct. 2024</td><td>EPI_ISL_19747204</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Pup</td><td>27 Oct. 2024</td><td>EPI_ISL_19747205</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Mare aux éléphants</td><td>Brain</td><td>Pup</td><td>06 Nov. 2024</td><td>EPI_ISL_19747207</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Pup</td><td>07 Nov. 2024</td><td>EPI_ISL_19747206</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>07 Nov. 2024</td><td>EPI_ISL_19747213</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>08 Nov. 2024</td><td>EPI_ISL_19747212</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Petite Manchotière</td><td>Brain</td><td>Adult</td><td>11 Nov. 2024</td><td>EPI_ISL_19747214</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td>EPI_ISL_19747208</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td>EPI_ISL_19747209</td></tr><tr><td>Stercorarius antarcticus</td><td>Crozet</td><td>Baie Américaine</td><td>Brain</td><td>Adult</td><td>14 Nov. 2024</td><td>EPI_ISL_19747215</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>16 Nov. 2024</td><td>EPI_ISL_19747203</td></tr><tr><td>Daption capense</td><td>Crozet</td><td>Sphinx</td><td>Brain</td><td>Adult</td><td>22 Nov. 2024</td><td>EPI_ISL_19747216</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>EPI_ISL_19747217</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>EPI_ISL_19747218</td></tr><tr><td>Larus dominicanus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Immature</td><td>01 Dec. 2024</td><td>EPI_ISL_19747219</td></tr><tr><td>Stercorarius antarcticus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Adult</td><td>01 Dec. 2024</td><td>EPI_ISL_19747220</td></tr><tr><td>Stercorarius antarcticus</td><td>Kerguelen</td><td>Port aux Français</td><td>Brain</td><td>Adult</td><td>02 Dec. 2024</td><td>EPI_ISL_19747221</td></tr></tbody></table>
## Phylogenetic analyses

The detected virus in our samples belongs to the genotype B3.2 (according to the American nomenclature), which is the highly pathogenic genotype mainly detected in South America and the one that had been detected in south polar areas¹⁴. Bayesian time-calibrated phylogenetic analysis yielded a mean evolutionary rate of 4.9 x 10⁻³ (4.6-5.2 x 10⁻³ 95% highest posterior density interval; HPD) substitutions per site per year, in line with the estimate of Uhart et al. ¹⁴, who reported a mean avian rate of 5.4 x 10⁻³ substitutions per site per year (4.9-5.9 x 10⁻³ 95% HPD) using a host-specific local clock model ⁴⁸. Figure 2 shows the consensus phylogeny as obtained using the highest independent posterior subtree reconstruction (HIPSTR) approach in TreeAnnotator X ⁴⁶. Immediately apparent are the very long branches relating to when HPAI H5N1 clade 2.3.4.4b arrived in the sub-Antarctic region - specifically on South Georgia Island - which is estimated to have taken nearly six months, and which we estimate to have come in from Argentina (but see also Figure 3 and Supplementary Figure S5). We estimate subsequent spread eastward in the sub-Antarctic and southward in the Antarctic region - to Antarctica, the Crozet Islands and the Kerguelen Islands - to have occurred with South Georgia Island as the source. However, the subsequent dispersal to the (estimated most recent common ancestor in the) Crozet Islands and the Kerguelen Islands is estimated to have taken approximately one year from their estimated most recent common ancestor, based on the currently available data. While these islands are geographically very distant, it is remarkable that such long-distance pathogen dispersal took place without there currently being any samples / genomes from (other) locations that could break up the very long branches in Figure 2. These two long-term dispersal events could hence be due to gaps in current sampling efforts in the sub-Antarctic and Antarctic regions or to genomic data not being yet available at the time of our data analysis (but see the Discussion).

Finally, Figure 2 shows that in the two clusters of sequences from the Southern Indian Ocean islands - one from the Crozet Islands and one from the Kerguelen Islands -
mixtures of species can be observed, with southern elephant seals in each, but also king penguins, a brown skua, a kelp gull and a Cape petrel. This is in contrast with the other parts of the phylogeny, where each cluster is estimated to be host-specific, based on the currently available data. However, it is important to note that those host-specific clusters might result from a sampling bias rather than from host-specific transmission. In the South Georgia Islands, differences between personal protective equipment (PPE) and staff training requirements for seal and bird carcass sampling led to much fewer seal carcasses being sampled than bird carcasses ¹⁸. We attempted to avoid this sampling bias on Crozet and Kerguelen by having a more extensive sampling strategy, covering all species that were found dead at each sampling occasion, and with several individuals per species sampled at each site, when possible. This enabled us to detect multi-species clusters in our phylogeny, which was not detected previously (Figure 2). Importantly, this points to very frequent cross-species transmission, specifically from seals to scavengers (Figure 1).

The presence of multi-species clusters may also be attributed to southern elephant seals spending time in close vicinity with high aggregation of seabirds on mixed-species colonies, even though they do not feed on seabirds or marine mammals (Figure 1). This may have been critical for the inferred chain of transmission events (Figure 2). Further, southern elephant seals undertake very long trips at sea over their life cycle ⁴⁹, although their movements are much slower than those of birds, which may limit their ability to vector the virus, depending on how long an individual may stay infected. In their annual migration, they can move from South Georgia, Crozet or Kerguelen to the Antarctica coastal waters, several thousands of kilometers away, in several weeks ⁴⁹. Many of the movements are made at a limited range of latitudes, with possible overlap at the sole of the roaming areas of widely-spread seabird populations such as those of South Georgia, Crozet and Kerguelen.
<figure><img src="image_2.png" /><figcaption>Figure 2. The main cluster of interest in our consensus tree, containing our novel sequences from the Crozet and Kerguelen Islands, in October and November of 2024. These sequences cluster among a larger number of sequences from the South Georgia Islands. This most likely implies two separate introductions - one into the Crozet Islands and another into the Kerguelen Islands - from the South Georgia Islands, based on the currently available genetic data. Importantly, the very long branches of approximately 1 year, on top of a branch of nearly six months for the first introduction into the South Georgia Islands, illustrate significant gaps in (animal / pathogen) surveillance in the Antarctic and sub-Antarctic regions. Horizontal blue bars represent the 95% HPD intervals for the ancestral node date estimates. Color scheme according to ColorBrewer 2.0⁵⁰.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 3. Phylogeographic analysis focused on our newly obtained sequences elucidates the spread of H5N1 around Antarctica, based on the currently available data. After having been imported into the South Georgia Islands from Argentina, the latter serves as the source for further dispersal of H5N1 across the sub-Antarctic region (Possession Island is one of the Crozet Islands; all available sequences from South Georgia and the Sandwich Islands are actually from the South Georgia Islands). Note that the South Georgia Islands are approximately 2100 kilometres east of Argentina, 5800 kilometres west from the Crozet Islands and 6600 kilometres west from the Kerguelen Islands. We refer to Supplementary Figure S5 to the location-annotated phylogeny. Map modified from Mortimer et al. ⁵¹, with the same color scheme as in Figure 2 (arrows colored according to destination).</figcaption></figure>

## Molecular markers

The viral sequences obtained display certain molecular markers on the HA protein (S133A, S154N, T156A) which have been shown to increase binding to human-type receptors in vitro. These markers have also been found in the majority of highly pathogenic H5 viruses
since 2020. One of our sequences from a southern elephant seal analysed also features an E627K mutation in the PB2 protein. This mutation is considered to be one of the most important markers of adaptation to mammals, and has been identified in one other southern elephant seal sequence from the South Georgia Islands ⁴. However, our phylogenetic analysis (Figure 2 and Supplementary Figure S5) does not show a monophyletic clustering of our (southern elephant seal) sequence from Crozet and Kerguelen with the southern elephant seal from the South Georgia Islands, raising questions regarding the emergence and spread of this mutation. We will focus on unraveling the evolutionary relationship of this mutation in a future study, when more sequences from different sub-Antarctic islands have become available. We refer to the Supplementary File for additional details / output from the FluMut software package ⁴⁷.

## Serological analyses of southern elephant seal samples

To explore whether the populations of southern elephant seals of Kerguelen had been exposed to avian influenza virus prior to the outbreak, we screened plasma samples of adult elephant seals collected in 2018, 2022 and 2023 using ELISA competitive assays. We found no evidence for the presence of antibodies against protein H5 in the series of samples prior to 2024 (Figure 4A). A few adults (n=4) sampled in 2023 showed elevated H5-antibody levels, however we cannot confidently interpret these values as H5-seropositive individuals. In 2024, we collected samples from 26 asymptomatic elephant seal pups after the outbreak had started, at a time when there were still many individuals dying. Among those sampled pups, Figure 4B shows that two pups exhibited high levels of antibodies against H5 protein, which suggests that they had been exposed and mounted a humoral immune response. The sampling was cross-sectional and it was not possible to determine whether those pups survived.
<figure><img src="image_4.png" /><figcaption>Figure 4. Quantification of antibodies against the AIV-H5 protein from adult southern elephant seals sampled in Kerguelen in 2018, 2022 and 2023 (A), and from pups sampled in 2024 during the outbreak (B). The grey area displays a H5-seropositivity threshold: two H5-seropositive pups are highlighted with red dots, while grey dots represent H5-seronegative individuals.</figcaption></figure>

Discussion

In addition to the pressing threats it poses to the local biodiversity, our detection of the HPAI H5N1 clade 2.3.4.4.b on the isolated Crozet and Kerguelen archipelagos is of global concern as it shows the virus's ability to spread over exceptionally long distances and across isolated oceanic basins. This implies that the hosts can remain infectious for sufficiently long periods to cover thousands of kilometers. This is especially relevant for the sub-Antarctic region, where several archipelagos are to date thought to be free of HPAI but potentially at risk of exposure. Further dispersal across the Southern Ocean should hence be considered at high risk and would threaten many other wildlife populations, most notably in the Australia and New Zealand sub-Antarctic islands.

The Indian Ocean sub-Antarctic islands are remote, hence the chances of between-distant-island transmission events could be thought of as being low, but our results show that this is not the case, likely because of significant connections via the movements of
the widely roaming vertebrates they are hosting. In the meantime, the virus is also suspected
to have arrived on Marion Island, of the Prince Edwards Islands (South Africa)⁵², awaiting
testing in February 2025⁵³, but it is not known whether it has been responsible for die-offs
on other significant regional biodiversity hotspots, such as Est and Cochon Island
(Supplementary Figure S2), in the Crozet archipelago, the more remote parts of Kerguelen,
or Heard and McDonald Islands (Australia). Several sub-Antarctic islands are classified as
UNESCO World Heritage Sites ²⁰ because of their environmental significance
(Supplementary Figure S2), unique pristineness and abundant but sensitive fauna, and it is
especially worrying that highly pathogenic avian influenza virus H5N1 has reached those
areas already at least twice independently from the South Atlantic subantarctic.

Evidence of between-mammal transmission has already been provided in colonies of
elephant seals in South America⁵⁴ and other species in North America⁵⁴,⁵⁵. Vertical
transmission has recently been shown as a mode of transmission of HPAI between
pinnipeds⁵⁵. We recurrently observed terrestrial scavengers like giant petrels and brown
skuas feeding on carcasses (Figure 1 and Supplementary Figure S1) and they may hence
be part of the transmission chain between individuals, groups, colonies and across broader
spatial scales such as island and between islands. Scavenging by species on floating
carcasses as we observed (Supplementary Figure S6) may also be important. Differentiating
exposure from susceptibility will be important to further understand the eco-epizoological
dynamics, which should be possible via serology analyses coupled with demographic
studies.

Southern elephant seals are known to occasionally travel over thousands of
kilometres⁵⁴, but this takes several weeks. Conversely, circumpolar movements of
procellariiformes are well-documented⁵⁶ (Supplementary Figure S7), notably eastward,
following wind direction, and species like giant petrels can cover thousands of kilometers in a
week⁵⁷. Giant petrels, as scavengers and widely roaming species at high southern latitudes,
are an obvious candidate species to have played a role in the spread of the virus between
the South Georgia Islands area and the Southern Indian Ocean islands. These species have already been identified as a potential spreader of avian influenza virus <sup>58</sup>. Brown skuas and kelp gulls, as other well-known scavengers on marine mammals and seabird colonies, could also have been involved, but perhaps less likely for the large-scale eastward spread due to their more restricted movements and their winter migration that involves move north-south movements than eastward or westward movements at high latitude <sup>59</sup>. Migratory movements are the obvious type of movement that may have been involved in the spread of HPAI H5N1, but large-scale foraging movements of breeding scavengers and predators may also have played a role, as well as less directed movements of individuals not currently involved in breeding, such as pre-breeders <sup>60</sup>. The long-distance carriage of the virus from the southern Atlantic to the southern Indian could thus have happened through scavenging seabirds and not seals. This would imply that bird-to-seal spillovers initiated both the Crozet and the Kerguelen massive mortality of southern elephant seals, and that the introduction on the islands could have happened earlier through scavenger seabirds. Bird-to-seal transmission events have happened several times in South America but are uncommon <sup>4,13,14</sup>. The timing of mortalities on Kerguelen, delayed more than two months after the return of the southern elephant seals at their colony, is supportive of that introduction scenario.

## Data availability

We have deposited the 25 sequences generated in this study into the publicly accessible GISAID <sup>27,28</sup> database with accession numbers EPI_ISL_19747197 to EPI_ISL_19747221. We provide an Excel table that maps the GISAID accession numbers to the individual gene sequence accession numbers as supplementary material.

## Acknowledgements
We acknowledge support from the French Polar Institute (IPEV ECOPATH-1151), ANR AAPG ECOPATHS project (ANR-21-CE35-0016), REMOVE_DISEASE project (ANR-21-BIRE-0006; Biodiversa+ and Water JPI joint call for projects under the BiodivRestore ERA-NET Cofund GA N°101003777) and CNRS Ecologie & Environnement SEE-Life ECOPATH. We also acknowledge specific support from Ceva Wildlife Research Fund since the beginning of the recent HPAI panzootic affecting wild species. We thank Martha I. Nelson for helpful discussions regarding the detection of reassortant sequences. G.B. acknowledges support from the Research Foundation - Flanders ("Fonds voor Wetenschappelijk Onderzoek - Vlaanderen," G0E1420N, G098321N), from the European Union Horizon 2023 RIA project LEAPS (grant agreement no. 101094685), and from the DURABLE EU4Health project 02/2023-01/2027 which is co-funded by the European Union (call EU4H-2021-PJ4) under Grant Agreement No. 101102733. We also acknowledge support from Zone Atelier Terres Australes et Antarctiques (ZATA) and Direction de l'Environnement of TAAF. We conducted serological analyses at the GEMEX (CEFE) platform. We thank the French Polar Institute (IPEV) and TAAF for long-term and reactive support. We thank Amandine Gamble, Nicolas Keck, Gregory Jouvion, Rozenn Le Net, Karin Lemberger, Célia Lesage, Maxime Amy and SAGIR/Office Français de la Biodiversité (OFB) network for having contributed to organize the training of field personnel for wildlife disease surveillance, which helped responding to HPAI emergence on Crozet and Kerguelen. We also thank Antoine Stier, Lucia Llorente, Nathan Thenon, Blaise Raymond, Marina Oger, Sara Boucheron, Loïs Angelvin, Francesco Bonadonna, Céline Lebohec, Christophe Barbraud, Aude Noiret and Bastien Bauger for their help at various stages of the study. The "NextSeq™ 2000" sequencing system and the "ThinkSystem SR650 V3 servers" used to obtain and analyse the data, were funded by a European Union grant.

## Ethical issues and permits
Permits to handle animals were obtained from the French Ministry of Research and the Animal experimental research authority (APAFIS #31773-2019112519421390 v4). Sampling was conducted under permits from Préfecture des TAAF (A-2023-82, A-2023-159) after evaluation by the Conseil de l'Environnement Polaire and Conseil National de la Protection de la Nature. After the suspicion of HPAI, permits were granted by the Préfecture des TAAF to pursue eco-epidemiological fieldwork (A-2024-92, A-2024-95, A-2024-144).
Supplementary Materials

<table><thead><tr><th>Host</th><th>Site</th><th>Location</th><th>Sample</th><th>Age</th><th>Collection date</th><th>Influenza A antigenic test</th><th>qRT-PCR H5 HP clade 2.3.4.4b</th><th>GISAID accession no.</th></tr></thead><tbody><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747197</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747198</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747199</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747200</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Adult</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747201</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Adult</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Adult</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747202</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747210</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747211</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>20 Oct. 2024</td><td>n.t.</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>27 Oct. 2024</td><td>-</td><td>Inconclusive</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Pup</td><td>27 Oct. 2024</td><td>+</td><td>Detected</td><td>EPI_ISL_19747204</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Pup</td><td>27 Oct. 2024</td><td>+</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Pup</td><td>27 Oct. 2024</td><td>+</td><td>Detected</td><td>EPI_ISL_19747205</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Mare aux éléphants</td><td>Brain</td><td>Adult</td><td>06 Nov. 2024</td><td>+</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Mare aux éléphants</td><td>Brain</td><td>Pup</td><td>06 Nov. 2024</td><td></td><td>Not detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Mare aux éléphants</td><td>Brain</td><td>Pup</td><td>06 Nov. 2024</td><td>+</td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Mare aux éléphants</td><td>Brain</td><td>Pup</td><td>06 Nov. 2024</td><td></td><td>Detected</td><td></td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Mare aux éléphants</td><td>Brain</td><td>Pup</td><td>06 Nov. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747207</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Pup</td><td>07 Nov. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747206</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>07 Nov. 2024</td><td>+</td><td>Detected</td><td>EPI_ISL_19747213</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>07 Nov. 2024</td><td></td><td>Detected</td><td></td></tr></tbody></table>
<table><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>08 Nov. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>08 Nov. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747212</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Jardin Japonais</td><td>Brain</td><td>Adult</td><td>08 Nov. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Pygoscelis papua</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Adult</td><td>10 Nov. 2024</td><td>-</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Petite Manchotière</td><td>Brain</td><td>Adult</td><td>11 Nov. 2024</td><td>n.t.</td><td>Not detected</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Petite Manchotière</td><td>Brain</td><td>Adult</td><td>11 Nov. 2024</td><td>n.t.</td><td>Detected</td><td>EPI_ISL_19747214</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td>+</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td></td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td></td><td>Detected</td><td>EPI_ISL_19747208</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td>+</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td></td><td>Detected</td><td>EPI_ISL_19747209</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Pup</td><td>12 Nov. 2024</td><td></td><td>Detected</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Chaloupe</td><td>Brain</td><td>Adult</td><td>12 Nov. 2024</td><td>-</td><td>Detected</td></tr><tr><td>Stercorarius antarcticus</td><td>Crozet</td><td>Baie Américaine</td><td>Brain</td><td>Adult</td><td>14 Nov. 2024</td><td>+</td><td>Detected</td></tr><tr><td>Stercorarius antarcticus</td><td>Crozet</td><td>Baie Américaine</td><td>Brain</td><td>Adult</td><td>14 Nov. 2024</td><td></td><td>Detected</td><td>EPI_ISL_19747215</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>Baie Américaine</td><td>Brain</td><td>Adult</td><td>14 Nov. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>16 Nov. 2024</td><td>+</td><td>Detected</td><td>EPI_ISL_19747203</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>16 Nov. 2024</td><td></td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>16 Nov. 2024</td><td></td><td>Detected</td></tr><tr><td>Pygoscelis papua</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Chick</td><td>16 Nov. 2024</td><td>-</td><td>Not detected</td></tr><tr><td>Aptenodytes patagonicus</td><td>Crozet</td><td>La Pérouse</td><td>Brain</td><td>Adult</td><td>19 Nov. 2024</td><td>-</td><td>Not detected</td></tr><tr><td>Daption capense</td><td>Crozet</td><td>Sphinx</td><td>Brain</td><td>Adult</td><td>22 Nov. 2024</td><td>+</td><td>Detected</td><td>EPI_ISL_19747216</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>24 Nov. 2024</td><td>+</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Crozet</td><td>Baie du Marin</td><td>Brain</td><td>Pup</td><td>24 Nov. 2024</td><td></td><td>Not detected</td></tr><tr><td>Stercorarius antarcticus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Adult</td><td>01 Dec. 2024</td><td>+</td><td>Detected</td></tr><tr><td>Stercorarius antarcticus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Adult</td><td>01 Dec. 2024</td><td></td><td>Detected</td></tr><tr><td>Stercorarius antarcticus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Adult</td><td>01 Dec. 2024</td><td></td><td>Detected</td><td>EPI_ISL_19747220</td></tr><tr><td>Stercorarius antarcticus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Adult</td><td>01 Dec. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>+</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td></td><td>Detected</td><td>EPI_ISL_19747217</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td></td><td>Detected</td><td>EPI_ISL_19747218</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>n.t.</td><td>Detected</td></tr></table>
<table><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Mirounga leonina</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Pup</td><td>01 Dec. 2024</td><td>n.t.</td><td>Detected</td></tr><tr><td>Larus dominicanus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Immature</td><td>01 Dec. 2024</td><td>+</td><td>Detected</td></tr><tr><td>Larus dominicanus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Immature</td><td>01 Dec. 2024</td><td></td><td>Detected</td></tr><tr><td>Larus dominicanus</td><td>Kerguelen</td><td>Ratmanoff</td><td>Brain</td><td>Immature</td><td>01 Dec. 2024</td><td></td><td>Detected</td></tr><tr><td>Stercorarius antarcticus</td><td>Kerguelen</td><td>Port aux Français</td><td>Brain</td><td>Adult</td><td>02 Dec. 2024</td><td>+</td><td>Detected</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>EPI_ISL_19747219</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>EPI_ISL_19747221</td></tr></table>

Supplementary Table 1. List of the samples we collected as part of our study. The table indicates the species name, the location, the organ sampled, the age of the individual sampled, the sampling date, the influenza A antigenic test and the qRT-PCR result. Influenza A antigenic tests were conducted directly on the field (“+” and “-” indicates respectively a positive and a negative antigenic test; “n.t.” indicates that the sample has not been tested). Pooled antigenic tests are indicated when a result spans multiple rows. qRT-PCR H5 HP clade 2.3.4.4b were conducted on all collected samples and results are presented as “Detected”, “Not detected” or “Inconclusive” for each sample. We provide accession numbers for GISAID for samples that have been sequenced.
<figure><img src="image_5.png" /><figcaption>Supplementary Figure S1. Detailed pictures of brown skuas scavenging on king penguins on Petite Manchotière, Possession Island, Crozet. Picture credit: Jérémy Tornos/Mathilde Lejeune, CNRS/IPEV.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Supplementary Figure S2. Map showing sub-Antarctic islands of high relevance for southern marine mammal and seabird populations, with islands and areas of UNESCO World Heritage Sites in pink (modified from Earthstar Geographics, UNESCO World Heritage Sites, https://whc.unesco.org/en/list/).</figcaption></figure>
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Supplementary Figure S3. The Crozet (Possession Island) and Kerguelen (Courbet peninsula) Islands, with details of our sampling sites (S: number of samples sequenced; P: number of PCR-positive samples; T: total number of samples analysed) and species.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Supplementary Figure S4. Picture of a necropsied king penguin, positive by PCR for HPAI H5N1 clade 2.3.4.4b virus, showing hemorrhage into the pericardial space (haemopericardium) as a macroscopic lesion. Picture credit: Jérémy Tornos/Mathilde Lejeune, CNRS/IPEV.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Supplementary Figure S5. Discrete phylogeographic reconstruction reveals South Georgia Island as the source for the (independent) introductions of H5N1 into the Crozet Islands, the Kerguelen Islands, and Antarctica. All inferred ancestral locations in this phylogeny - the most relevant ones indicated by semi-transparent colored circles - have 100% posterior probability.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Supplementary Figure S6. A northern giant petrel scavenging on a southern elephant seal pup carcass. Picture credit: Augustin Clessin.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Supplementary Figure S7. Captured on the 17th of January, 2025, in South Georgia by one of the authors of this study (AC), a northern giant petrel was found to have been ringed on Crozet as a chick, at Pointe Basse, on the 14th of January, 2024. Picture credit: Augustin Clessin.</figcaption></figure>
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