Avian influenza is one of the largest known threats to domestic poultry. Influenza outbreaks on poultry farms typically lead to the complete slaughter of the entire domestic bird population, causing severe economic losses worldwide. Moreover, there are highly pathogenic avian influenza (HPAI) strains that are able to infect the swine or human population in addition to their primary avian host and, as such, have the potential of being a global zoonotic and pandemic threat. Migratory birds, especially waterfowl, are a natural reservoir of the avian influenza virus; they carry and exchange different virus strains along their migration routes, leading to antigenic drift and antigenic shift, which results in the emergence of novel HPAI viruses. This requires monitoring over time and in different locations to allow for the upkeep of relevant knowledge on avian influenza virus evolution and the prevention of novel epizootic and epidemic outbreaks. In this review, we assess the role of migratory birds in the spread and introduction of influenza strains on a global level, based on recent data. Our analysis sheds light on the details of viral dissemination linked to avian migration, the viral exchange between migratory waterfowl and domestic poultry, virus ecology in general, and viral evolution as a process tightly linked to bird migration. We also provide insight into methods used to detect and quantify avian influenza in the wild. This review may be beneficial for the influenza research community and may pave the way to novel strategies of avian influenza and HPAI zoonosis outbreak monitoring and prevention.

Rapid diagnosis is essential for the control and prevention of H5 highly pathogenic avian influenza viruses (HPAIVs). However, highly sensitive and rapid diagnostic systems have shown limited performance due to specific antibody scarcity. In this study, two novel specific monoclonal antibodies (mAbs) for clade 2.3.4.4 H5Nx viruses were developed by using an immunogen from a reversed genetic influenza virus (RGV). These mAbs were combined with fluorescence europium nanoparticles and an optimized lysis buffer, which were further used for developing a fluorescent immunochromatographic rapid strip test (FICT) for early detection of H5Nx influenza viruses on chicken stool samples. The result indicates that the limit of detection (LoD) of the developed FICT was 40 HAU/mL for detection of HPAIV H5 clade 2.3.4.4b in spiked chicken stool samples, which corresponded to 4.78 × 10⁴ RNA copies as obtained from real-time polymerase chain reaction (RT-PCR). An experimental challenge of chicken with H5N6 HPAIV is lethal for chicken three days post-infection (DPI). Interestingly, our FICT could detect H5N6 in stool samples at 2 DPI earlier, with 100% relative sensitivity in comparison with RT-PCR, and it showed 50% higher sensitivity than the traditional colloidal gold-based rapid diagnostic test using the same mAbs pair. In conclusion, our rapid diagnostic method can be utilized for the early detection of H5Nx 2.3.4.4 HPAIVs in avian fecal samples from poultry farms or for influenza surveillance in wild migratory birds.

Introduction: The avian influenza (AI) virus causes a highly contagious disease which is common in wild and domestic birds and sporadic in humans. Mutations and genetic reassortments among the 8 negative-sense RNA segments of the viral genome alter its pathogenic potential, demanding well-targeted, active surveillance for infection control. Methods: Wild duck fecal samples were collected during the 2018 bird health annual surveillance in South Korea for tracking variations of the AI virus. One low-pathogenic avian influenza H5N3 reassortment virus (A/mallard duck/South Korea/KNU18-91/2018 [H5N3]) was isolated and genomically characterized by phylogenetic and molecular analyses in this study. Results: It was devoid of polybasic amino acids at the hemagglutinin (HA) cleavage site and exhibited a stalk region without deletion in the neuraminidase (NA) gene and NA inhibitor resistance-linked E/D627K/N and D701N marker mutations in the PB2 gene, suggesting its low-pathogenic AI. It showed a potential of a reassortment where only HA originated from the H5N3 poultry virus of China and other genes were derived from Mongolia. In phylogenetic analysis, HA was different from that of the isolate of H5N3 in Korea, 2015. In addition, this novel virus showed adaptation in Madin-Darby canine kidney cells, with 8.05 ± 0.14 log10 50% tissue culture infectious dose (TCID50) /mL at 36 h postinfection. However, it could not replicate in mice well, showing positive growth at 3 days postinfection (dpi) (2.1 ± 0.13 log10 TCID50/mL) but not at 6 dpi. Conclusions: The HA antigenic relationship of A/mallard duck/South Korea/KNU18-91/2018 (H5N3) showed differences toward one of the old low-pathogenic H5N3 viruses in Korea. These results indicated that a novel reassortment low-pathogenic avian influenza H5N3 subtype virus emerged in South Korea in 2018 via novel multiple reassortments with Eurasian viruses, rather than one of old Korean H5N3 strains.

Several novel highly pathogenic avian influenza (HPAIVs) A(H5N6) viruses were reported in Mongolia in 2020, some of which included host-specific markers associated with mammalian infection. However, their pathogenicity has not yet been investigated. Here, we isolated and evaluate two novel genotypes of A(H5N6) subtype in Mongolia during 2018–2019 (A/wildDuck/MN/H5N6/2018-19). Their evolution pattern and molecular characteristics were evaluated using gene sequencing and their pathogenicity was determined using a mouse model. We also compared their antigenicity with previous H5 Clade 2.3.4.4 human isolates by cross-hemagglutination inhibition (HI). Our data suggests that A/wildDuck/MN/H5N6/2018-19 belongs to clade 2.3.4.4h, and maintains several residues associated with mammal adaptation. In addition, our evaluations revealed that their isolates are less virulent in mice than the previously identified H5 human isolates. However, their antigenicity is distinct from other HPAIVs H5 clade 2.3.4.4, thus supporting their continued evaluation as potential infection risks and the preparation of novel candidate vaccines for their neutralization.

Influenza A virus subtype H1N1 has caused global pandemics like the “Spanish flu” in 1918 and the 2009 H1N1 pandemic several times. H1N1 remains in circulation and survives in multiple animal sources, including wild birds. Surveillance during the winter of 2018–2019 in Korea revealed two H1N1 isolates in samples collected from wild bird feces: KNU18-64 (A/Greater white-fronted goose/South Korea/KNU18-64/2018(H1N1)) and WKU19-4 (A/wild bird/South Korea/WKU19-4/2019(H1N1)). Phylogenetic analysis indicated that M gene of KNU18-64(H1N1) isolate resembles that of the Alaskan avian influenza virus, whereas WKU19-4(H1N1) appears to be closer to the Mongolian virus. Molecular characterization revealed that they harbor the amino acid sequence PSIQRS↓GLF and are low-pathogenicity influenza viruses. In particular, the two isolates harbored three different mutation sites, indicating that they have different virulence characteristics. The mutations in the PB1-F2 and PA protein of WKU19-4(H1N1) indicate increasing polymerase activity. These results corroborate the kinetic growth data for WKU19-4 in MDCK cells: a dramatic increase in the viral titer after 12 h post-inoculation compared with that in the control group H1N1 (CA/04/09(pdm09)). The KNU18-64(H1N1) isolate carries mutations indicating an increase in mammal adaptation; this characterization was confirmed by the animal study in mice. The KNU18-64(H1N1) group showed the presence of viruses in the lungs at days 3 and 6 post-infection, with titers of 2.71 ± 0.16 and 3.71 ± 0.25 log10(TCID50/mL), respectively, whereas the virus was only detected in the WKU19-4(H1N1) group at day 6 post-infection, with a lower titer of 2.75 ± 0.51 log10(TCID50/mL). The present study supports the theory that there is a relationship between Korea and America with regard to reassortment to produce novel viral strains. Therefore, there is a need for increased surveillance of influenza virus circulation in free-flying and wild land-based birds in Korea, particularly with regard to Alaskan and Asian strains.