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. 2024 Mar 5;12(3):e0373623.
doi: 10.1128/spectrum.03736-23. Epub 2024 Feb 2.

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022

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High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022

Pierre Bessière et al. Microbiol Spectr. .

Abstract

High pathogenicity avian influenza viruses (HPAIVs) H5Nx of clade 2.3.4.4b have been circulating increasingly in both wild and domestic birds in recent years. In turn, this has led to an increase in the number of spillover events affecting mammals. In November 2022, an HPAIV H5N1 caused an outbreak in a zoological park in the south of France, resulting in the death of a Tibetan black bear (Ursus thibetanus) and several captive and wild bird species. We detected the virus in various tissues of the bear and a wild black-headed gull (Chroicocephalus ridibundus) found dead in its enclosure using histopathology, two different in situ detection techniques, and next-generation sequencing, all performed on formalin-fixed paraffin-embedded tissues. Phylogenetic analysis performed on the hemagglutinin gene segment showed that bear and gull strains shared 99.998% genetic identity, making the bird strain the closest related strain. We detected the PB2 E627K mutation in minute quantities in the gull, whereas it predominated in the bear, which suggests that this mammalian adaptation marker was selected during the bear infection. Our results provide the first molecular and histopathological characterization of an H5N1 virus infection in this bear species.

Importance: Avian influenza viruses are able to cross the species barrier between birds and mammals because of their high genetic diversity and mutation rate. Using formalin-fixed paraffin-embedded tissues, we were able to investigate a Tibetan black bear's infection by a high pathogenicity H5N1 avian influenza virus at the molecular, phylogenetic, and histological levels. Our results highlight the importance of virological surveillance programs in mammals and the importance of raising awareness among veterinarians and zookeepers of the clinical presentations associated with H5Nx virus infection in mammals.

Keywords: epidemiology; influenza; zoonotic infections.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Histopathology, viral antigen, and RNA detection in tissues obtained from infected bear. (a) Visceral lymph node: necrotizing vasculitis (arrowhead) with thrombosis and hemorrhages. H&E stain. (b) Visceral lymph node: viral antigen is observed associated with vasculitis and extending to the surrounding lympho-nodal parenchyma. Anti-nucleoprotein influenza A IHC (anti-NP IHC). (c) Visceral lymph node: viral RNA is intralesionally detected within areas of vasculitis. M gene RNAscope ISH. (d) Trachea: thrombosis (arrowhead) and perivascular leukocytic infiltration are observed within the mucosa and submucosa. The overlying epithelium is sloughed (arrowhead) (H&E stain). (e) Trachea: no viral antigen detection is observed (anti-NP IHC). (f) Trachea: positive viral RNA detection is observed in the interstitium of mucosa and submucosa (RNAscope ISH). (g) Lung: diffuse congestion and edema (H&E stain). (h) Lung: IHC shows moderate non-specific background staining with no significant detection of viral antigen at low magnification (anti-NP IHC). (i) Lung: viral RNA is widely distributed within the lobular and interlobular interstitium (RNAscope ISH). Intestine: autolytic changes are present in the mucosa, including cell sloughing (arrowhead). The submucosa, tunica muscularis, and serosa appear within normal limits. The myenteric plexus (insert and asterisk) is readily identifiable and also normal (H&E stain). (j) Intestine: no viral antigen detection is observed (anti-NP IHC). (l) Intestine: viral RNA is focally associated with the myenteric plexus nerve trunk and connective tissue (insert) (RNAscope ISH). Scale bars: 50 (d–f), 100 (a–c and g–i), and 200 µm (j–l).
Fig 2
Fig 2
HA maximum likelihood phylogenetic tree. Bear- and gull-derived sequences are labeled in red and blue, respectively. Bootstrap support values > 90 are shown at branches. Scale bar: number of nucleotide substitution per site.

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