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

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.

Incursion of Novel Highly Pathogenic Avian Influenza A(H5N8) Virus, the Netherlands, October 2020.

Highly pathogenic avian influenza A(H5N8) virus was detected in mute swans in the Netherlands during October 2020. The virus shares a common ancestor with clade 2.3.4.4b viruses detected in Egypt during 2018-2019 and has similar genetic composition. The virus is not directly related to H5N8 viruses from Europe detected in the first half of 2020.

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 viruses.

Strategies to control spread of highly pathogenic avian influenza (HPAI) viruses by wild birds appear limited, hence timely characterization of novel viruses is important to mitigate the risk for the poultry sector and human health. In this study we characterize three recent H5-clade 2.3.4.4 viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the two H5N8 viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, which showed that chickens are efficiently infected by (fecal) contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector.

Circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands, 2006-2016.

In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.

Modifications of the 3'-UTR stem-loop of infectious bursal disease virus are allowed without influencing replication or virulence.

Many questions regarding the initiation of replication and translation of the segmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) remain to be solved. Computer analysis shows that the non-polyadenylated extreme 3'-untranslated regions (UTRs) of the coding strand of both genomic segments are able to fold into a single stem-loop structure. To assess the determinants for a functional 3'-UTR, we mutagenized the 3'-UTR stem-loop structure of the B-segment. Rescue of infectious virus from mutagenized cDNA plasmids was impaired in all cases. However, after one passage, the replication kinetics of these viruses were restored. Sequence analysis revealed that additional mutations had been acquired in most of the stem-loop structures, which compensated the introduced ones. A rescued virus with a modified stem-loop structure containing four nucleotide substitutions, but preserving its overall secondary structure, was phenotypically indistinguishable from wild-type virus, both in vitro (cell culture) and in vivo (chickens, natural host). Sequence analysis showed that the modified stem-loop structure of this virus was fully preserved after four serial passages. Apparently, it is the stem-loop structure and not the primary sequence that is the functional determinant in the 3'-UTRs of IBDV.