Emerg. Microbes & Inf.: Characterization of HPAI A (H5N1) Viruses isolated from Cats in South Korea, 2023


Last July, while we were watching an outbreak of HPAI H5N1 in cats in Poland (see WHO Update WHO Update & Risk Assessment On H5N1 In Cats - Poland), word of a similar outbreak emerged from South Korea, where cats at two different shelters tested positive for the virus.
Unlike from Poland, where information has been slow to emerge, South Korea quickly determined that the outbreak at the second shelter was due to contaminated cat food (see South Korea: MAFRA Statement On Detection Of H5 Contaminated Cat Feed).

The first outbreak occurred at a private shelter, which has hampered the epidemiological investigation. Two samples (out of 40 cats), however, were delivered to the College of Veterinary Medicine, in Seoul, South Korea for analysis.

As we've discussed often, the H5N1 threat isn't from a single viral entity, but rather from a diverse array of genetically similar viruses that continually evolve. The H5N1 virus circulating in Egypt or Vietnam today is genetically distinct from the H5N1 viruses circulating in Peru, Japan, or Canada.

Influenza viruses are broadly categorized by their two surface glycoproteins - hemagglutinin (HA) and neuraminidase (NA) - hence we often talk about seasonal H1N1 or H3N2, or avian H5N1 or H9N2 viral subtypes.

But each subtype is further divided into clades; broad categories based on the genetic makeup of the HA gene. Over time, dozens of clades can emerge for a given subtype, and while some are supplanted by more `biologically fit' strains, multiple clades can co-circulate.

Clades can be further divided into sub-clades, and subclades (e.g. into genotypes (and variants within genotypes), producing a myriad of quasi-similar viruses - sometimes with differing characteristics (see Differences In Virulence Between Closely Related H5N1 Strains) - but still often referred to (outside of scientific journals) as a single subtype.

In 2020 a new clade H5N1 virus became dominant, and it - and its variants - have shown an enhanced ability to spill over into mammals around the globe (see here, here, here,
and here).

Today we've a letter to the editor, published in Emerging Microbes & Infections prior to final editing, from researchers in South Korea who analyzed the two samples gathered from the first H5N1 infected animal shelter.

While their report doesn't fully answer how the virus made it into the shelter, it does classify this virus as a new genotype (South Korea genotype III) , and identifies a number of mammalian adaptions (see graphic below).

(B) Amino acid mutations in cat and avian isolates are color-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.

I've only posted some excerpts from the letter, so follow the link to read (or listen to) the report in its entirety. I'll have a postscript after the break.

Characterization of Highly Pathogenic Avian Influenza A (H5N1) Viruses isolated from Cats in South Korea, 2023

Kyungmoon Lee, Minjoo Yeom, Thi Thu Hang Vu, Hai-Quynh Do, Woonsung Na, Mikyung Lee, show all
Article: 2290835 | Accepted author version posted online: 04 Dec 2023



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 June 24 and 27, 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 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 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 1A). 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.

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 1B). 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). 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 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 2022–2023 winter season posed constraints on genetic analysis with domestic isolates.

The discovery of the HPAI H5N1 clade 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.

(Continue . . . )

Long chains of infections - such as you'd see in an animal shelter (or on a fur farm) - provide avian viruses with additional opportunities to adapt to a mammalian host, as demonstrated by the classic serial passage experiment shown below.

While we don't know exactly what combination of changes are needed to turn HPAI H5N1 from primarily an avian threat to a well-adapted mammalian virus - or even if that combination is even possible - the genetic dice are being tossed millions of times each day in birds and mammals around the globe.

While there may be some as-yet unknown species barrier that protects us, given the stakes, it really isn't something we should be counting on.​

#17,799 Last July, while we were watching an outbreak of HPAI H5N1 in cats in Poland (see WHO Update WHO Update & Risk Assessment On H5N1 In...