Highly pathogenic avian influenza H5N1 virus infections in wild red foxes (Vulpes vulpes) show neurotropism and adaptive virus mutations

Abstract

During the 2020-2022 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 2021 and February 2022. Histopathological analysis demonstrated the virus was mainly present in the brain, with limited or no detection in the respiratory tract and other organs. Phylogenetic analysis showed the three fox viruses were not closely related, but were related to HPAI H5N1 clade 2.3.4.4b viruses found in wild birds. In addition, limited virus shedding was detected suggesting the virus was not transmitted between the foxes. Genetic analysis demonstrated the presence of 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) variant were present as a mixture in the virus population, which suggests the mutation emerged in these specific animals. The two variant viruses were isolated and virus replication and passaging experiments were performed. These experiments showed mutation PB2-627K increases 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 the HPAI H5N1 virus is capable of adaptation to mammals, however more adaptive mutations are required to allow efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness.

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

“…The virus was detected in nine red foxes, three polecats, one otter and one badger. This study confirmed the initial findings in three of the red foxes [ 8 ] by greater numbers of animals and across four species. Higher viral RNA loads were consistently detected in the brains of the animals studied compared to those detected in throat or anal swabs.…”

“…In addition, virus pathogenesis was studied to identify the potential entry route of the virus, the sites of virus replication, and the associated lesions. Collectively, we showed that: (1) the carnivore viruses were less related to each other than to wild birds; (2) across species, the PB2-E627K mutation associated with mammalian adaptation was found as detected in foxes in the previous study [ 8 ]; (3) the brain of most mammals tested positive for viral RNA whereas limited viral RNA or protein was observed in other organs. We conclude that brain samples are important for reliable detection of the HPAI H5N1 virus in mammals, and therefore must be routinely included besides throat and anal swabs in the surveillance program, and HPAI infections in mammals must be made notifiable to the Veterinary Authority.…”

“…Of these, 14 animals tested positive for influenza A virus using the M-PCR. Three red foxes ( Vulpes vulpes ) were analyzed in a previous study [ 8 ]. The current study describes the analysis of the 11 other carnivores: red fox ( n = 6), polecat ( Mustela putorius ) ( n = 3), badger ( Meles meles ) ( n = 1), and otter (Lutra lutra) ( n = 1) ( Table 1 ).…”

“…An overview of sampled material per animal is provided in Table S1 for virology and Table S2 for pathology. Swabs and tissue samples for virological examination were processed as described previously [ 8 ]. Tissue samples for histology were processed and embedded in paraffin and stained with hematoxylin and eosin (HE).…”

“…Genetic analysis identified mutation E627K in PB2 in two of the HPAI H5N1 viruses isolated from these animals. The PB2-627K mutation increased virus replication in mammalian cell lines [ 8 ]. This mutation is an adaptation of the virus polymerase protein, that likely stimulates virus replication in the lower body temperature environment in mammals compared to birds [ 9 , 10 ].…”