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. 2023 Jan 20;12(2):168.
doi: 10.3390/pathogens12020168.

Zoonotic Mutation of Highly Pathogenic Avian Influenza H5N1 Virus Identified in the Brain of Multiple Wild Carnivore Species

Sandra Vreman  1 , Marja Kik  2   3 , Evelien Germeraad  1 , Rene Heutink  1 , Frank Harders  1 , Marcel Spierenburg  4 , Marc Engelsma  1 , Jolianne Rijks  2 , Judith van den Brand  2   3 , Nancy Beerens  1
Affiliations

Zoonotic Mutation of Highly Pathogenic Avian Influenza H5N1 Virus Identified in the Brain of Multiple Wild Carnivore Species

Sandra Vreman et al. Pathogens. .

Abstract

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021-2022 outbreak in the Netherlands included red fox (Vulpes vulpes), polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals were submitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal. Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1 virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness.

Keywords: HPAI H5N1 influenza; brain; carnivores; full genome sequences; immunohistochemistry; pathology; phylogenetic analysis; virology; wildlife; zoonotic mutation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Map of the locations were HPAI H5N1 infected carnivores were found during the 2021–2022 epizootic in the Netherlands. The infected carnivores analyzed in this study are marked in orange, and numbers are corresponding to Table 1. The infected mammals analyzed in previous studies [8] are depicted in grey. The map was generated using the R software package tmap [24].
Figure 2
Figure 2
Phylogenetic tree of the HA segment obtained with the maximum likelihood method showing the viruses detected in the carnivores from the current study (orange). The previous carnivore H5N1 virus sequences from the period 2021–2022 (dark grey), related sequences from wild birds (green), and relevant H5N1 sequences from the previous 2020–2021 avian influenza season (light grey) are also shown. Bootstrap values above 50% are shown at the nodes.
Figure 3
Figure 3
Brain histopathology and virus expression in the cerebrum of an otter infected with HPAI H5N1. (A) Severe changes in the white matter with loss of neurons, neuronal degeneration (asterisk), cellular debris (arrow), gliosis, and hemorrhages (arrowhead). Hematoxylin and eosin stain (HE), objective 40×. (B) Virus expression in neurons (arrowhead), immunohistochemistry (IHC) influenza A protein, objective 40×. (C) Perivascular cuffing (arrowhead) and meningitis (arrow). HE, objective 40×. (D) Only minimal virus expression in a few degenerated neurons (arrow), IHC, objective 20×. (E) Cerebrum with mild to moderate gliosis in neuropil and surrounding neurons (arrowhead), HE, objective 40×. (F) Clear virus expression in neurons (arrowhead), IHC, objective 40×.
See this image and copyright information in PMC

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