Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations

Abstract

During the 2020 to 2022 epizootic of highly pathogenic avian influenza virus (HPAI), several infections of mammalian species were reported in Europe. In the Netherlands, HPAI H5N1 virus infections were detected in three wild red foxes (Vulpes vulpes) that were submitted with neurological symptoms between December of 2021 and February of 2022. A histopathological analysis demonstrated that the virus was mainly present in the brain, with limited or no detection in the respiratory tract or other organs. Limited or no virus shedding was observed in throat and rectal swabs. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to HPAI H5N1 clade 2.3.4.4b viruses that are found in wild birds. This suggests that the virus was not transmitted between the foxes. A genetic analysis demonstrated the presence of the mammalian adaptation E627K in the polymerase basic two (PB2) protein of the two fox viruses. In both foxes, the avian (PB2-627E) and the mammalian (PB2-627K) variants were present as a mixture in the virus population, which suggests that the mutation emerged in these specific animals. The two variant viruses were isolated, and virus replication and passaging experiments were performed. These experiments showed that the mutation PB2-627K increases the replication of the virus in mammalian cell lines, compared to the chicken cell line, and at the lower temperatures of the mammalian upper respiratory tract. This study showed that the HPAI H5N1 virus is capable of adaptation to mammals; however, more adaptive mutations are required to allow for efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses caused high mortality among wild birds from 2021 to 2022 in the Netherlands. Recently, three wild foxes were found to be infected with HPAI H5N1 viruses, likely due to the foxes feeding on infected birds. Although HPAI is a respiratory virus, in these foxes, the viruses were mostly detected in the brain. Two viruses isolated from the foxes contained a mutation that is associated with adaptation to mammals. We show that the mutant virus replicates better in mammalian cells than in avian cells and at the lower body temperature of mammals. More mutations are required before viruses can transmit between mammals or can be transmitted to humans. However, infections in mammalian species should be closely monitored to swiftly detect mutations that may increase the zoonotic potential of HPAI H5N1 viruses, as these may threaten public health.

Keywords: HPAI H5N1; fox; neurotropism; virology; zoonotic infections.

FIG 1

Locations of the three infected foxes and wild birds from which closely related viruses were isolated during the 2021 to 2022 HPAI H5N1 epizootic in the Netherlands. The map was generated using the R software package tmap (35).

FIG 2

Histopathology and virus protein expression in tissues of foxes. Immunohistochemistry (IHC) was performed on tissue sections against influenza A NP. The insets show the magnification of IHC stained. (A) Fox-Dorst moderate purulent broncho-interstitial pneumonia (less affected area), no virus protein expression; (B) Fox-Oosterbeek severe purulent broncho-interstitial pneumonia with intralesional larvae (Angiostrongylus vasorum), no virus protein expression; (C) Fox-Heemskerk mild to moderate purulent rhinitis with positive staining of olfactory epithelial cells; (D) Fox-Dorst severe necropurulent tracheitis with intralesional parasite eggs (Capillaria spp), no virus protein expression; (E) Fox-Dorst cerebrum moderate nonsuppurative encephalitis with virus protein expression in neurons and microglia cells in the neuropil; (F) Fox-Heemskerk cerebellum moderate nonsuppurative encephalitis with virus protein expression in neurons and microglia cells in the neuropil; (G) Fox-Oosterbeek bulbus olfactorius, no significant histopathology or virus protein expression; (H) Fox-Heemskerk moderate lymphoplasmacytic myocarditis with mild myocardial degeneration and necrosis with positive virus protein staining of cardiomyocytes; (I) Fox-Oosterbeek colon, no significant histopathology or virus protein expression. Panels A, B, C, D, H, and I, magnification ×20; panel F, magnification ×10; panels E–G, original magnification 2.5×; insets, magnification ×40.

FIG 3

Phylogenetic tree for the HA segment obtained via the maximum likelihood method that shows the viruses detected in the samples of the three foxes (orange), closely related HA sequences from other viruses detected in the Netherlands (green) and in Europe (blue), and relevant sequences from the 2020 to 2021 epizootic (blue).

FIG 4

Virus replication curves of the PB2-627K (blue) and PB2-627E (red) H5N1 HPAI viruses on A549 (human), MDCK (dog), and DF-1 (chicken) cells cultured at 37°C, which is the temperature of the avian upper respiratory tract, and at 33°C, which is the temperature of the mammalian upper respiratory tract. The differences in infectious virus titers between viruses are statistically significant from 48 h p.i. onwards (P < 0.05). No significant differences were found between the infectious virus titers of the two viruses on DF-1 cells at 37°C (P > 0.05). The virus titers of cells cultured at 33°C instead of 37°C were significantly lower at 24 h p.i. (P < 0.0001) and 48 h p.i. (P < 0.05), but they were significantly higher at 72 h p.i. (P < 0.05).