Avian Flu Diary: Preprint: Infection of Ratites with Clade 2.3.4.4b HPAIV H5N1: Potential Implications for Zoonotic Risk

#18,869

As the months-long legal battle (see CFIA Update On H5N1 In a B.C. Ostrich Farm) over the fate of roughly 400 ostriches in British Columbia continues, overnight the Canadian Press reported the CFIA recently filed an affidavit with the courts calling the strain isolated in B.C. `among the most virulent' ever tested at Canada's National Microbiology Laboratory.

Over the years we've seen growing evidence that Ratites (large flightless birds which include Ostriches, Reas, and Emus) are particularly susceptible to avian flu viruses, including this early (Dec. 2007) report describing the emergency culling of 36K ostriches in Riyadh, Saudi Arabia for HPAI H5N1.

We've seen reports of both HPAI and LPAI AIV viruses in Ratites, with many displaying mammalian adaptations. Including 2016's Susceptibility and Status of Avian Influenza in Ostriches, which looked at > 70 outbreaks in South Africa, and included the following tidbit:

Seventeen of 27 (63%) ostrich viruses contained the polymerase basic 2 (PB2) E627K marker, and 2 of the ostrich isolates that lacked E627K contained the compensatory Q591K mutation, whereas a third virus had a D701N mutation.

PB2-627K is often associated with enhanced replication and pathogenicity in mammals, and is one of the most important mutations that H5Nx is thought to need in order to spread more efficiently in mammals (see A rapid review of the avian influenza PB2 E627K mutation in human infection studies).

There are others, of course (PB2 D701N, PB2 Q591K, HA Q226L, etc.) - each providing the virus with unique advantages - but if you want to kickstart mammalian transmission, PB2-E627K would be at or near the top of your list.

Luckily, these mammalian adaptations tend to exact a `fitness penalty' in avian hosts, making their spread via birds far less likely. An exception, however, is an emerging PB2-627V, which appears to be making inroads in Chinese poultry.

All of which brings us to the above mentioned preprint, where researchers from the UK's APHA investigated a (Dec 2024) outbreak among captive Rheas (n=5) infected with HPAI H5N1 (clade 2.3.4.4b) genotype DI.2. in the UK.

As we've seen previously, the PB2-E627K mutation was observed in most of the sequenced samples from the Rheas, with one containing both 627E (in the brain) and 627K (in the oropharynx) suggesting within-host viral diversity.

Unexpectedly, some of the infected co-housed chickens were also found to have the 627K mutation, presumably acquired from the Rheas. As the authors point out:

Understanding the extent that avian species can maintain mammalian adaptative mutations, is crucial for determine AIV evolution and zoonotic risk.

This study also makes note of the unexpected widespread strong vascular tropism of the HPAI H5N1 virus in Rheas, and its ability to cause severe multifocal necrotising inflammation.

I've reproduced the abstract, and some excerpts from the preprint, but you'll want to follow the link to read it in its entirety.

Infection of ratites with clade 2.3.4.4b HPAIV H5N1: Potential implications for zoonotic risk
Holly A. Coombes, Jacob Terrey, Audra-Lynne Schlachter, Phoebe McCarter, Isabella Regina, Richard Hepple, Natalie McGinn, James Seekings, Jayne Cooper, Benjamin Clifton, Benjamin C. Mollett, Marco Falchieri, Alejandro Nunez, Scott M. Reid, Joe James, Ashley C. Banyard
doi: https://doi.org/10.1101/2025.09.08.674895
This article is a preprint and has not been certified by peer review

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Abstract

We detected H5N1 high pathogenicity avian influenza in captive Greater Rhea (Rhea americana). Viral genetic analysis revealed the mammalian associated PB2-E627K mutation, indicating selection of mammalian-relevant mutations in ratites. Pathologic investigation of available tissues demonstrated severe multifocal necrotising inflammation, and a strong vasculotropism.

(SNIP)

To effectively replicate and transmit in mammalian cells, AIVs must overcome multiple host barriers. However, adaptation of AIVs to different avian hosts and variation in host factors is still poorly understood. A key viral adaptation for successful mammalian replication is restored binding of the viral polymerase to host factor acidic nuclear phosphoprotein 32 family member A(ANP32A) [9].

An amino acid change at residue 627 in the PB2 protein, from a glutamate (E) to a lysine (K) is frequently found in mammalian viral sequences [10]. Ratites, along with mammals, lack a 33 amino acid insertion in their ANP32A receptor, typically seen in other avian species, leading to a weaker interaction between the receptor and the viral polymerase [9].

The 627K mutation appears to compensate for this weakened interaction, restoring viral polymerase activity and replication in mammalian cell lines (ref for cell lines) [9]. This may explain why ratites appear to select for 627K mutations, as demonstrated by this study.

Epidemiological and clinical data suggests viral transmission from rheas to co-located chickens, supported by the 627K mutation being present in most of the chicken viral sequences. Although exact transmission chains could not be determined from the sequence data, the persistence of 627K in chickens, despite their avian-like ANP32A, indicates potential maintenance of mammalian adaptive mutations in avian species.

Understanding the extent that avian species can maintain mammalian adaptative mutations, is crucial for determine AIV evolution and zoonotic risk. One rhea contained both 627E and 627K viral variants, suggesting within-host viral diversity and viral trophism.

This is the first description of pathologic changes in ratites infected with HPAIV H5N1.

Virus induced endothelial damage, vascular inflammation and thrombosis is a known consequence of HPAI H5N1 infection, previously described in cats [11], wild carnivores [12], wild birds [13] and mice [14] in the brain, lungs and eyes.

However, the frequent leukocytoclastic inflammation observed in the walls of small to medium vessels, the resultant necrotising inflammation and the abundance of viral antigen detected in most tissues sampled in these birds was unexpected. Viral characteristics (e.g. viral strain, infection route and dose) or host specific factors such as species, age, duration of infection, concomitant disease and immune status likely influenced lesion severity and antigen expression [15].

Despite a limited selection of tissues and small sample size, findings suggest a widespread strong vascular tropism in this species.

Overall, the viral changes detected in the rhea demonstrated early adaptive events following infection of a novel host, including a key viral adaptation that is often associated with increased zoonotic risk. Assessment of viral evolution in microenvironments where unrelated species are co-housed can provide important evidence regarding adaptation to novel hosts.