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High pathogenicity H5N1 avian influenza viruses pose a threat to both animal and human health worldwide. In late 2020, outbreaks of H5 high pathogenicity avian influenza viruses belonging to clade 2.3.4.4b emerged in Europe, following on from outbreaks in East Asia in earlier years. However, very recent studies show that clade 2.3.4.4b H5N1, rather than 2.3.4.4b H5N8, has become predominant in wild birds and has infected poultry in several countries. In this study, we describe isolation of a novel H5N1 virus from a captured mandarin duck in South Korea, and another H5N1 virus from a quail farm. We performed genetic analysis of these two viruses to identify their origin and to determine their relationship with the clade 2.3.4.4b H5N1 viruses currently circulating in Europe. Based on our results, it is presumed that the novel H5N1 virus isolated in Korea originated from an unknown reassortant between clade 2.3.4.4b H5N8 viruses circulating from 2020 and other Eurasian viruses, with additional reassortment of genes and point mutations that discriminate them from the recently reported H5N1 virus in Europe.

Virus-like particles (VLPs) are recognized as an alternative vaccine platform that provide effective protection against various highly pathogenic avian influenza viruses (HPAIVs). Here, we developed multi-clade VLPs expressing two HAs (a chimera of clade 2.3.2.1c and clade 2.3.4.4c HA) within a single vector. We then compared its protective efficacy with that of a monovalent VLP and evaluated its potency against each homologous strain. Chickens vaccinated with the multi-clade VLP shed less virus and were better protected against challenge than birds receiving monovalent vaccines. Single vaccination with a multi-clade VLP resulted in 100% survival, with no clinical symptoms and high levels of pre-challenge protective immunity (7.6–8.5 log2). Moreover, the multi-clade VLP showed high productivity (128–256 HAU) both in the laboratory and on a large scale, making it cheaper than whole inactivated vaccines produced in eggs. However, the PD50 (protective dose 50%) of the multi-clade VLP against clades 2.3.2.1c and 2.3.4.4c was < 50 PD50 (28 and 42 PD50, respectively), and effective antibody response was maintained for 2–3 months. This multi-clade VLP protects against both clades of HPAI viruses and can be produced in high amounts at low cost. Thus, the vaccine has potential as a pandemic preparedness vaccine.

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