Spread of highly pathogenic avian influenza (HPAI) virus H5N1 clade 2.3.4.4b of the goose/Guangdong (gs/GD) lineage, has exacerbated since early 2022 into a panzootic (1). Regional enzootic status in wild bird populations in Europe and North America, with lethal courses of HPAI virus infection in some species, produced large numbers of wild bird carcasses, easy prey for raptors and scavengers. Exposure of terrestrial carnivores and marine mammals resulted in sporadic infections, some of those terminating with fatal encephalitis (2). Frequent spill-over events, rather than consistent mammal-to-mammal transmission, were at the basis of these cases (Figure, panel A). However, recently reported HPAI outbreaks among sea lions along the Pacific coast of South America and an outbreak in a mink farm in Spain (3) may constitute first examples of avian-independent transmission chains and increase public health concerns about zoonotic transmissions of this virus. Still, the total of 11 human cases globally reported for the currently dominating H5N1 2.3.4.4b lineage did not point toward increased zoonotic propensity (4).

Possible adaption of avian influenza virus (AIV) to mammalian livestock hosts and subsequent human exposure is of particular concern. In this respect, the role of pigs as a “mixing vessel” for HPAI viruses is largely unresolved. AIV can potentially be transmitted to pigs, and further reassortment with swine influenza A viruses (swIAVs) may contribute to the emergence of pandemic strains. Rare and subclinical infections of pigs with gs/GD HPAI virus have been confirmed serologically in Vietnam, Thailand, and France (5) and virologically in Indonesia (clades 2.1.1 and 2.1.3), Nigeria (clade 2.3.2.1c), China (clade 2.3.4), and Italy (clade 2.3.4.4.b) (6–8).

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Figure. Phylogeny and experimental design for study of susceptibility of pigs against experimental infection with highly pathogenic avian influenza (HPAI) (H5N1) virus clade 2.3.4.4b. A) Maximum-likelihood phylogenetic tree (RAxML, https://cme.h-its.org/exelixis/web/software/raxml...

For our study, we purchased 4-month-old pigs (6 male, 4 female) from a conventional pig holding in Germany and exposed them nasally or by the alimentary route to high doses of the recent avian-derived HPAI virus H5N1 2.3.4.4b isolate A/chicken/Germany/AI04286/2022 (genotype Ger-10.21-N1.5). The egg-grown isolate was closely related to a mammal case but lacked any mammalian-adaptive mutations (Figure, panel A). We inoculated 2 groups of 4 pigs each intranasally or by feeding 1 infected embryonated chicken egg per animal. One sentinel pig per group was associated at day 1 postinoculation to assess the transmission by direct contact to those inoculated (Figure, panel B).

After exposure, HPAI virus H5N1 detection by real-time RT-PCR was limited to day 2 (intranasal group) and day 4 (oral group) postinoculation, with a range of 10–150 genome copy equivalents per 0.1 mL (Table). An avian-derived, swine-adapted H1avN1 strain intranasally inoculated into naïve pigs at the same dose and by the same device produced 3–4 log₁₀ levels greater nasal excretion in comparison (Table) (9). It cannot be excluded that HPAI virus H5N1 detected in nasal swabs at day 2 postinoculation still represents residual inoculum. Correspondingly, except for 1 alimentary inoculated that showed tracheal viral loads at day 4 postinoculation close to the detection limit, samples from the remaining 3 pigs euthanized at day 4 postinoculation gave no indication of viral replication in respiratory or gut tissues, regardless of the inoculation route. Only in 1 intranasally inoculated animal, euthanized at day 14 postinoculation, was viral RNA detected at low levels in organ samples. In addition to samples from the respiratory and alimentary tracts, minute amounts of RNA were found also in a brain sample of that pig. The virus could not be isolated using chicken hepatoma cells and MDCK-II cells, and histologic and immunohistochemical investigations gave no evidence for inflammatory reactions or presence of viral antigen. Low viral loads in this pig impeded sequence analysis of eventual mutants. Nonetheless, this pig was the only animal that seroconverted at 14 dpi. In agreement with the virologic findings, none of the pigs showed any clinical signs or fever within 14 days of observation.

In conclusion, only 1 of 8 pigs inoculated intranasally with HPAI virus H5N1 underwent transient, low-level infection that resulted in the presence of viral RNA in several tissue specimens and seroconversion at 14 dpi. In naturally infected wild mammals, this virus was prominently detected in the brain (2). Given the detection of viral RNA in the brain of 1 intranasally inoculated pig, it cannot be excluded that longer observation might have revealed continuing viral replication in the brain of this animal. Sialic acid α2,3-gal receptors dominate on porcine brain cells, which might have fostered replication of α2,3-adapted viruses, such as HPAI virus H5N1 (10).

Overall, we conclude that pigs are unlikely vehicles in transmitting this genotype of HPAI virus H5N1 clade 2.3.4.4b among pigs and across interfaces. However, considering the ongoing massive panzootic of this virus, a plethora of new genotypes of the circulating strain is emerging, with possibly higher permissiveness for pigs. Therefore, swine populations need to be part of HPAI virus surveillance programs, and periodic reassessment of prepandemic propensity of circulating HPAI virus H5N1 genotypes in the swine model is required.

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