On October 31, 2022, a 12-year-old Tibetan black bear (Ursus thibetanus) was found in Sigean Wildlife-park (southern France) in a lateral decubitus position associated with polypnea and hyperthermia (39.9°C). This bear (bear1) was anesthetized for examination, and blood samples were taken. The bear was found dead the next day, and the necropsy revealed extremely congested lungs. On November 07, a second 6-year-old Tibetan black bear (bear2) was found exhausted with respiratory distress. The animal showed signs of fatigue, anorexia, and coughing for about two weeks before fully recovering from the infection. A few days later, two other bears from the same enclosure as the two previous bears presented moderate polypnea associated with anorexia, also suggesting a similar infection. Almost concomitantly with the clinical signs observed in bears, some mortalities in wild and captive birds were observed in the wildlife park. The first avian deaths were observed on November 04, 2022, in captive pink-backed pelicans, and on November 15, 2022, others in captive pink-backed pelicans wild western jackdaws and black-headed gulls. All birds were found near the bear enclosure in the wildlife park. For bear1 and bear2, viral RNA was extracted from tracheal and sinonasal swabs using the NucleoSpin VET kit (Macherey-Nagel, Düren, Germany) and tested for HPAI (H5N1, clade 2.3.4.4b) for SARS-CoV-2 and adenoviruses (Nyaruaba et al. 2022) by digital PCR (dPCR) using the QIAcuity One-Step Viral RT-PCR kit (Qiagen, Hilden, Germany). In birds, viral RNA was extracted from cloacal and tracheal swabs using the Adiamag kit (BioX Diagnostics, Rochefort, Belgique) and tested for the M and H5 genes by rRT-PCR using the Adiavet AIV M and H5-H7. Subsequently, the RNA samples from bears and birds were analyzed by real-time RT-PCR specific for the H5 HA gene of clade 2.3.4.4b and neuraminidase (N1) genes (Hoffmann et al., 2016; Naguib et al., 2017) using Quantitect virus kit (Qiagen, Hilden, Germany).

One sample from each animal was found positive for H5 HA of clade 2.3.4.4b, and the full genome sequencing of detected HPAI H5N1 was performed after amplification of the eight viral genomic segments by conventional RT-PCR (Zhou et al., 2009). These viral sequences were aligned using the MAFFT software with the closest sequences of HPAI H5N1 viruses circulating in France and Europe during the winter of 2022-2023, and the sequences were subjected to phylogenetic analysis. The maximum likelihood method based on the Kimura 2-parameter model was performed to obtain phylogenetic trees with 1000 bootstrap replicates using the MEGA7 software (Kumar et al. 2008).

Samples obtained from bear1 showed a dark red or necrotic tracheal mucosa. There was disseminated intravascular coagulation, probably of pulmonary origin, sepsis with generalized congestion. Histopathological analysis showed severe multifocal necro suppurative hepatitis, multifocal fibro-necrotic splenitis, marked pulmonary congestion, and edema. Biochemical analyses revealed severe neutropenia and thrombocytopenia, a slight increase in uremia, severe hypercreatininemia, and an increase in alanine aminotransferase. However, the biochemical results of bear2 showed a very slight neutrophilia without leukocytosis.

Virus testing of the bears was positive for HPAI (H5N1, clade 2.3.4.4b) by digital PCR (dPCR) and negative for SARS-CoV-2 and adenoviruses. Results from bear2 suggested a positivity for H5N1, clade 2.3.4.4b, but with a much lower viral load. With the reference methods for AIV detection, results showed the presence of the HPAI H5N1 clade 2.3.4.4b virus in samples from bear1 and birds but not from the bear2 sample (Table 1), and others from Europe (Table 2).

Four complete viral genomes were obtained: one from the bear1, one from wild black-headed gulls, and two from captive pink-backed pelicans. Sequences are available in the GISAID database (https://www.gisaid.org) under accession nos. EPI_ISL_17233426 (bear), EPI_ISL_18722087, and EPI_ISL_18722088 (pelican) and black-headed gull (EPI_ISL_18722086). Phylogenetic analyses of HPAI H5N1 of the eight segments revealed that all viruses belonged to the A/duck/Saratov/29–02/2021–like genotype, which was the predominant virus genotype circulating in France and Europe during this epizootic period (EFSA et al. 2023b). In addition, the viral sequence from bear1 differed by 12 to 14 nucleotides (out of 13005 total nucleotides) from the sequences identified in birds from the Sigean Wildlife Park. The phylogenetic analysis of the HA segment showed that the sequences from the Wildlife-park (bear and birds) were closely related among themselves and were closely related to viruses detected from September 2022 to November 2022 in the central French departments in captive and wild birds (Figure 1).

The phylogenetic trees based on the seven remaining avian influenza segments showed the same topology. These results suggest likely contamination of the bear and captive birds of the wildlife park directly from the infected wild birds. Among the distinctive amino acid mutations observed between wild/captive birds and bear1, the E627K mutation on the PB2 protein was only identified in the H5N1 virus isolated from the bear. This mutation has previously been described as a major marker of influenza virus adaptation to mammalian hosts (Gabriel et al., 2013). The absence of this mutation in the closest viral sequences found in wild and captive birds from the Sigean Wildlife-park suggests a rapid selection of viruses with this mutation after crossing the bird/mammal species barrier as already observed (Bordes et al. 2023, Briand et al. 2023).