Characterization of highly pathogenic avian influenza A (H5N1) viruses isolated from cats in South Korea, 2023

Influenza A viruses have frequently crossed species barriers and continually pose a threat to the health of birds, mammals, and humans. Highly pathogenic avian influenza virus (HPAIV), distinguished from low pathogenic avian influenza virus (LPAIV) by polybasic hemagglutinin (HA) cleavage site, extensively spread among wild birds and poultry since its initial discovery in Guangdong, China, in 1996 (A/goose/Guangdong/1/1996; Gs/GD) [1]. Numerous mutations have accumulated in the HA gene since the emergence of the Gs/GD lineage, further dividing HPAI into various clades, among which the HPAI H5N1 clade 2.3.4.4b emerged as the predominant strain on a global scale in 2020 [1]. Subsequently, HPAI H5N1 clade 2.3.4.4b was further classified into diverse genotypes via genetic reassortment among avian influenza viruses (AIVs) for eight gene segments [1–3]. Genotypes of H5N1 clade 2.3.4.4b were classified as G1–G16 from Chinese avian isolates in 2021–2022. The South Korean avian isolates reported in November 2022 were classified into South Korea genotypes I and II, both of which were derived from the G10 genotype [2,3].

However, a striking paradigm shift in the global epidemiology of HPAI H5N1 clade 2.3.4.4b emerged during the winter of 2021–2022 [4]. Interspecies transmission of HPAIVs has been increased, in addition to conventional infections in poultry and wild bird populations [4,5]. These cross-species transmissions have affected over 43 mammalian species spanning Europe, North America, South America, and Asia [5]. Besides these instances in wild mammals, it was also reported in cats in France, the United States, Italy, and Poland between 2022 and 2023 [5]. In June and July 2023, feline AIV infections, confirmed to be caused by H5N1, occurred at two different shelters in Seoul, South Korea, with no confirmed human cases involved [6]. The source of infection in the first case remains unclear; in the second case, the infection originated from infected poultry feed. This was the first case of mammalian infections caused by HPAI H5N1 in South Korea. This study covers the first case, whereas the second case is currently being analysed by the Ministry of Agriculture, Food and Rural Affairs. We describe the potential origin and genetic characteristics of HPAI H5N1 viruses from cats based on whole genome sequencing and phylogenetic analysis.

Three out of 40 cats housed in a non-profit private shelter in Seoul, South Korea between 24 and 27 June 2023 died of high fever and anorexia. Subsequent mortality persisted at 1–2-day intervals, and 38 of the 40 animals finally died; however, the exact cause of death was not revealed. The feline inhabitants of the shelter were originally stray cats and resided in separate rooms (four to five animals in each room) without being confined in individual cages. The closed structure of the shelter prevented cats from contact with the outside environment, including wild birds. Additionally, there were no poultry farms within a 10 km radius. Between July 4 and 6, two cats showing respiratory and neurological symptoms were transported to a veterinary hospital but died within two days. A private diagnostic centre detected influenza A virus in nasal swab samples collected from the dead cats. These two nasal swab samples were subsequently sent to our laboratory for further analysis.

The H5N1 subtype of AIVs was confirmed using real-time reverse transcription polymerase chain reaction after isolating them via inoculation into embryonated chicken eggs. Whole genome sequencing resulted in the designation of the two isolates as A/feline/South Korea/SNU-01/2023 (SNU-01) and A/feline/South Korea/SNU-02/2023. Their sequences were identical; therefore, subsequent analyses were conducted using only the SNU-01 isolate. The nucleotide sequences of these strains were deposited at GISAID (https://gisaid.org) (accession numbers EPI_ISL_1812700 and EPI_ISL_18102701).

Phylogenetic analysis of the HA gene confirmed that SNU-01 belonged to clade 2.3.4.4b of H5N1 viruses (Figure S1). Further examination of the HA gene from SNU-01 revealed the presence of a polybasic amino acid sequence at the cleavage site, classifying them as HPAI (PLREKRRKR/G). Each of the eight gene segments in SNU-01 shared a high degree of sequence identities, ranging from 99.59% to 100%, with the HPAI H5N1 virus clade 2.3.4.4b isolated from birds in Japan between November 2022 and April 2023, according to GISAID BLAST (https://blast.ncbi.nlm.nih.gov) (Table S1).

Phylogenetic analysis of all eight gene fragments consistently placed SNU-01 within the same cluster as the Japanese avian isolates, reaffirming their close genetic relationship and high nucleotide identities observed earlier (Figure S1–S8). Notably, the HA, neuraminidase (NA), and matrix genes of SNU-01 originated from G10 genotype avian isolates previously identified in 2022 from China and South Korea [2,7] (Figure 1(A)). In contrast, polymerase basic (PB) 2, PB1, polymerase acidic (PA), nucleoprotein, and non-structural protein genes had their origins in LPAIV of the Eurasian lineage that circulated in China, Russia, and South Korea between 2019 and 2022. The specific source of individual gene segments appeared different, although this reassortment pattern resembled that of the South Korean genotypes I and II reported in 2022 [3]. Therefore, we classified our cat isolates as South Korean genotype III.

Figure 1. Schematic diagrams of the (A) genetic reassortment of and (B) amino acid changes in cat isolates (SNU-01) from South Korea, 2023. (A) The origins of the low pathogenic avian influenza virus (LPAIV) Eurasian gene segments of South Korean genotypes I, II, and III were different, but the origins of the highly pathogenic avian influenza virus (HPAIV) gene fragments were the same. HPAIV origin gene segments are highlighted with red bars, whereas blue and green bars are LPAIV origin gene segments. (B) Amino acid mutations in cat and avian isolates are colour-coded: red text for mutations exclusively to SNU-01, blue text for mutations found in genetically close avian isolates with SNU-01, and green text for mutations common in most current Asian avian isolates. In other words, SNU-01 exhibits red, blue, and green mutations, closely related avian isolates with SNU-01 show blue and green mutations, and the majority of current Asian avian isolates display green mutations. CD, cytoplasmic domain; TD, transmembrane domain.

SNU-01 has shown multiple mammalian adaptations in several gene segments that enhance the polymerase activity of AIVs in mammalian hosts [3,5,8–14] (Figure 1(B)). Interestingly, except for D701N, which significantly contributes to mammalian adaptation, the remaining mammalian adaptive mutations were not limited to SNU-01; they were also observed in avian isolates. These mutations were prevalent among most avian isolates currently circulating in Eurasia, and additional mutations were identified in bird isolates that share a common ancestry with SNU-01 within phylogenetic trees (Figure S1, S4–6, Table S2-5). The D701N mutation has not been detected in avian isolates; however, its presence in wild birds cannot be ruled out, given recent cases in Chile [15]. Meanwhile, the function of the unique mutations in PA and NA gene segments, found exclusively in SNU-01 requires further study to understand their implications.

HPAI H5N1 cases in mammals continue to be reported globally [5], and our study described the first case of HPAI H5N1 in cats in South Korea. We discovered novel genotypes and identified the accumulation of diverse mammalian adaptations in avian and cat isolates. Our findings strongly indicated that the viruses responsible for infecting cats likely originated from migratory birds that travelled from Japan and South Korea during the previous winter. Additionally, an increase in genetic diversity of H5Nx clade 2.3.4.4b was confirmed owing to reassortment among AIVs [1–5]. Several mammalian adaptive mutations already accumulated within avian populations, and these changes may play an important role in facilitating the interspecies spread of AIVs to mammals [3,5,8–14]. Ongoing monitoring is imperative to track the potential spread of these multiple mammalian adaptations within avian populations and across various regions. The shelters where feline influenza occurred lacked veterinary control, which restricted the scope of sampling and information gathering for epidemiological investigations. The scarcity of sequence data for South Korean avian isolates from the 2022 to 2023 winter season posed constraints on genetic analysis with domestic isolates.

The discovery of the HPAI H5N1 clade 2.3.4.4b virus in cats and its potential origin from avian sources underscores the dynamic nature of cross-species transmission of AIVs. Active reassortment and the accumulation of numerous mammalian adaptations in currently circulating viruses may further facilitate interspecies transmission. The shelter environment where susceptible animal hosts live closely together increases the likelihood of infectious disease transmission compared to typical indoor settings. Therefore, it is crucial to enhance active surveillance in densely populated animal living areas and proactively identify and mitigate factors facilitating cross-species contact.

Acknowledgements

We appreciate our colleagues worldwide for their valuable laboratory contributions, which were shared via the GISAID database.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Seoul National University (grant number 550-20220059) and Evaluation for Technology in Food, Agriculture and Forestry (IPET) (grant number 122018-2).