Since October 2020, H5N1 clade 2.3.4.4b high pathogenicity avian influenza (HPAI) viruses have spread to many countries. Although these viruses evolved from Eurasian ancestors, reassortant with other LPAI viruses has generated various genotypes. Here, we identified three H5N1 HPAI viruses belonging to clade 2.3.4.4b; these viruses were isolated from mandarin duck, common teal, and domestic breeder ducks in October 2022 during an avian influenza (AI) active surveillance program. Two of the H5N1 viruses (MD/WA496 and BD/H493) have been found sporadically in China, Russia, and Korea. It is presumed that two viruses with a similar gene constellation isolated in Russia, China, and Korea were introduced into the breeding area during the spring migration, and were introduced newly to Korea during the autumn migration. Due to international bird migration, the other virus (CT/WA537) is most similar (99.3–99.8%) to a virus detected in North Dakota, USA in April 2022. These results suggest that H5N1 viruses with at least two genotypes were introduced at the same time into Korea during the autumn of 2022, and that they originated from Eurasian breeding grounds and North America. Thus, multiple 2.3.4.4b H5N1 viruses were introduced into Korea during the autumn season of 2022.
Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens’ tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNβ, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.
The introduction of novel highly pathogenic (HPAI) viruses into Korea has been attributed to recombination events occurring at breeding sites in the Northern Hemisphere. This has increased interest in monitoring and genetically analyzing avian influenza viruses (AIVs) in northern regions, such as Mongolia, which share migratory bird flyways with Korea. AIVs in Mongolia were monitored by analyzing 10,149 fecal samples freshly collected from wild birds from April to October in 2021 to 2023. The prevalence of AIVs in wild birds was 1.01%, with a total of 77 AIVs isolated during these 3 years. These 77 AIVs included hemagglutinin (HA) subtypes H1, H2, H3, H4, H6, H10 and H13 and neuraminidase (NA) subtypes N1, N2, N3, N6, N7 and N8. The most frequently detected subtype combinations were H3N8 (39.0%) and H4N6 (19.5%), although HPAI viruses were not detected. Genetic analysis indicated that theses AIVs isolated from Mongolian samples were closely related to AIVs in wild birds in Korea, including those of Eurasian lineage. These findings indicate the necessity of continuous AIV surveillance and monitoring, as HPAI viruses introduced into Korea may derive from strains in Mongolia.