Preprint: Polymerase Mutations Underlie Adaptation of H5N1 Influenza Virus to Dairy Cattle And Other Mammals

[Michael Coston]

Preprint: Polymerase Mutations Underlie Adaptation of H5N1 Influenza Virus to Dairy Cattle And Other Mammals

#18,533

The revelation - barely 9 months ago - that Texas cattle had been infected with HPAI H5N1 was surprising, but not entirely unexpected.

In 2008 researchers at Germany's FLI successfully infected four calves with an older clade of HPAI H5N1 (see EID Journal Experimental Infection of Cattle with HPAI H5N1), but reports of natural infection have been rare.​

What was surprising was how well adapted HPAI H5 had become to a (lactating) bovine host, and how far that virus has spread in cattle (>900 herds across 16 states & counting).

Instead of this being an incidental finding, this signified a radical change in the virus's behavior. At the same time, we've seen numerous spillbacks from infected cattle to other mammals (including humans), wild birds, and poultry.​

While 40+ human infections from this `bovine' B3.13 genotype have been uncharacteristically mild, the virus continues to evolve, and its future course and public health impact are unknown.

Like all of the (many) iterations of HPAI H5 influenza, genotype B3.13 is very much a work in progress.​

All of which brings us to a preprint, published yesterday on the bioRxiv server, which warns that the continued spread of HPAI H5N1 in dairy cattle affords the virus more opportunities to evolve and adapt, thereby increasing its zoonotic potential.

Specifically, they cite the PB2 M631L mutation, which is found in all cattle sequences, and PA K497R, which is found in the majority. Both appear to co-opt the bovine ANP32 cell protein, which is key to enabling efficient replication in a mammalian host.

• In 2016 Virologist Wendy Barclay et al. identified ANP32 as an important limiting factor in the replication of avian viruses in mammalian hosts (see Species difference in ANP32A underlies influenza A virus polymerase host restriction) and the role of specific PB2 mutations.

• In 2019, Barclay et al. returned with ANP32 Proteins Are Essential for Influenza Virus Replication in Human Cells

I've only reproduced the abstract, and some excerpts from the discussion, so you'll want to follow the link to read it in its entirety. I'll have more when you return.

Polymerase mutations underlie adaptation of H5N1 influenza virus to dairy cattle and other mammals.
Vidhi Dholakia, Jessica L Quantrill, Samuel Richardson, Nunticha Pankaew, Maryn D. Brown, Jiayun Yang, Fernando Capelastegui, Tereza Masonou, Katie-Marie Case, Jila Aejian, Ecco Staller, Carol M. Sheppard, Ian H Brown, Claire M. Smith, Munir Iqbal, Paul Digard, Wendy S. Barclay, Rute M Pinto, Thomas P. Peacock, Daniel H. Goldhill
doi: https://doi.org/10.1101/2025.01.06.631435
This article is a preprint and has not been certified by peer review

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Abstract

In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA. The epidemic remains uncontrolled, with spillbacks into poultry, wild birds and other mammals including humans.​

Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells, as well as cells of other mammalian species including humans and pigs.​

We find evidence of several mammalian adaptations gained early in the evolution of these viruses in cattle including PB2 M631L, which is found in all cattle sequences, and PA K497R, which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication. We show this mutation adapts the virus to co-opt bovine ANP32 proteins and thereby enhances virus replication in bovine and primary human airway cells.​

Importantly, we show that ongoing evolution during 2024 in the PB2 gene, including a convergently arising D740N substitution, further increases polymerase activity in a range of mammalian cells. Thus, the continued circulation of H5N1 in dairy cattle allows virus adaption improving replicative ability in cattle and increasing zoonotic spillover risk.​

(SNIP)

What is the current risk to humans posed by the cattle virus?​

Our results show that the cattle virus containing mammalian adaptations can replicate better in mammalian cells than the minimal avian-like precursor virus, and is therefore a clear risk to mammals which have consumed infected milk such as cats and raccoons13 . Thus far, these cattle viruses show inefficient airborne transmissibility in ferret experiments30,44 and have not adapted to transmit via respiratory route or to efficiently use human receptors (α-2,6-linked sialic acids)45-48 341.

Nonetheless, frequent exposure of an antigenically novel, highly pathogenic influenza virus that can replicate well in human cells is concerning. Our data suggest that the emerged cattle influenza virus is also capable of efficiently infecting avian and swine cells, further increasing the risk of spill over from cattle to other species.

In fact, the mammalian-adapted virus appeared to have no fitness cost in avian cells, explaining the high propensity of this virus to spillover into poultry, and suggesting such a virus could maintain its mammalian adaptations if it spilt back into wild birds.

With each human infection and increased polymerase activity leading to higher levels of replication, there is a danger of further evolution changing viral receptor properties. Additionally, a reassortment event with human seasonal influenza viruses could lead to a novel virus, particularly during the Northern hemisphere winter influenza season.

Recent experimental infections in cows suggest that the ability of the North American B3.13 cattle influenza virus to spread via milk is not unique and that other mammalian viruses are capable of transmitting via this route16 . Phylogenetic data suggests that this originated from a single spillover event from wild birds to cattle and such spillovers must be rare as IAV has rarely been recorded in cattle.​

Nonetheless, in the absence of an effective control strategy, the high pathogenicity H5N1 virus may now become endemic in US dairy cattle, requiring continuous monitoring even in the absence of overt disease.​

Moreover, many other clades and strains of H5N1 virus continue to emerge through reassortment, causing zoonotic infections. For example, the emergence of the D1 genotype of H5N1 virus in North America has caused a recent uptick in spillover from birds to mammals including humans and swine49.​

Urgent development and testing of broadly reactive H5 influenza vaccines for both animals and humans is a priority.​

(Continue . . . )




While it is still possible that the H5N1 virus is incapable of sparking a human pandemic (we won't know if it can, until it happens), it won't fail for a lack of opportunity.

Our unwillingness to aggressively contain the virus - by testing and quarantining herds - has given the virus valuable months to spread and evolve.​

Even if `bovine' H5 never directly sparks a pandemic, adaptations the virus picks up in cattle could help it jump into other mammals - like swine - where it could make even greater strides.

We have a long history of underestimating viruses, but they have evolved over ten of millions of years with only one purpose; to survive.​

And they've gotten exceedingly good at that.
​

https://afludiary.blogspot.com/2025/01/preprint-polymerase-mutations-underlie.html

 

[sharon sanders]

response of hogvet51 to the above blog -


Ongoing H5N1 B3.13 Polymerase Mutations

I want to devote the balance of this blog to a Michael Coston post I initially overlooked: Avian Flu Diary: Preprint: Polymerase Mutations Underlie Adaptation of H5N1 Influenza Virus to Dairy Cattle And Other Mammals. I reread it this weekend and more fully considered the true impact of the research findings. Here is the paper in full:

Polymerase mutations underlie early adaptation of H5N1 influenza virus to dairy cattle and other mammals | bioRxiv

In the introduction the authors’ state:

…Avian-origin influenza viruses require polymerase mutations to utilise the shorter mammalian ANP32A/B proteins[SUP]23[/SUP]. The most widespread, and best characterised adaptive mutations are those in the PB2 subunit of the polymerase including PB2 E627K[SUP]22[/SUP], Q591R/K[SUP]24[/SUP][SUP],[/SUP][SUP]25[/SUP], and D701N[SUP]25[/SUP][SUP],[/SUP][SUP]26[/SUP]; all of which have been detected during other mammalian H5N1 outbreaks[SUP]5[/SUP][SUP]–[/SUP][SUP]11[/SUP] but are absent in USA dairy cattle viruses. However, alternative adaptive mutations in the influenza polymerase that enable the virus to co-opt suboptimal ANP32 proteins are possible[SUP]27[/SUP][SUP]–[/SUP][SUP]29[/SUP]. Here, we investigated potential mammalian adaptations in the polymerase of cattle-adapted H5N1 viruses and identify two key mutations that allow the B3.13 virus to replicate in cells from cattle and other mammalian species, including humans.

The initial issue for researchers has been that the B3.13 “dairy” strain has lacked traditional PB2 subunit mutations to allow mammalian adaptation. So they set out through phylogenetic analyses to identify alternative common mutations among sequenced isolates to explain successful adaptation.


Abstract


In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA. The epidemic remains uncontrolled, with spillbacks into poultry, wild birds and other mammals including humans. Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells, as well as cells of other mammalian species including humans and pigs. We find evidence of several mammalian adaptations gained early in the evolution of these viruses in cattle including PB2 M631L, which is found in all cattle sequences, and PA K497R, which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication. We show this mutation adapts the virus to co-opt bovine ANP32 proteins and thereby enhances virus replication in bovine and primary human airway cells. Importantly, we show that ongoing evolution during 2024 in the PB2 gene, including a convergently arising D740N substitution, further increases polymerase activity in a range of mammalian cells. Thus, the continued circulation of H5N1 in dairy cattle allows virus adaption improving replicative ability in cattle and increasing zoonotic spillover risk.

I have made the mistaken assumption based on “no news” and relatively homogenous phylogenetic trees that no significant changes are occurring in B3.13 virus as infections proceed over time. However, this paper shows that “ongoing evolution during 2024 in the PB2 gene, including a convergently arising D740N substitution, further increases polymerase activity in a range of mammalian cells”. A convergent substitution can be defined as a random mutational change that provides an organism a competitive advantage. Thus, as the same mutation “succeeds” in multiple infections, it converges as a common change in the phylogeny of the virus in multiple sequences.

What really caught my eye in rereading this paper was the conclusion of the


Discussion section of the paper:

What is the current risk to humans posed by the cattle virus? Our results show that the cattle virus containing mammalian adaptations can replicate better in mammalian cells than the minimal avian-like precursor virus and is therefore a clear risk to mammals which have consumed infected milk such as cats and raccoons[SUP]13[/SUP]. Thus far, these cattle viruses show inefficient airborne transmissibility in ferret experiments[SUP]30[/SUP][SUP],[/SUP][SUP]44[/SUP] and have not adapted to transmit via respiratory route or to efficiently use human receptors (α-2,6-linked sialic acids)[SUP]45[/SUP][SUP]–[/SUP][SUP]48[/SUP]. Nonetheless, frequent exposure of an antigenically novel, highly pathogenic influenza virus that can replicate well in human cells is concerning. Our data suggest that the emerged cattle influenza virus is also capable of efficiently infecting avian and swine cells, further increasing the risk of spill over from cattle to other species. In fact, the mammalian-adapted virus appeared to have no fitness cost in avian cells, explaining the high propensity of this virus to spillover into poultry, and suggesting such a virus could maintain its mammalian adaptations if it spilt back into wild birds. With each human infection and increased polymerase activity leading to higher levels of replication, there is a danger of further evolution changing viral receptor properties. Additionally, a reassortment event with human seasonal influenza viruses could lead to a novel virus, particularly during the Northern hemisphere winter influenza season.

Recent experimental infections in cows suggest that the ability of the North American B3.13 cattle influenza virus to spread via milk is not unique and that other mammalian viruses are capable of transmitting via this route[SUP]16[/SUP]. Phylogenetic data suggests that this originated from a single spillover event from wild birds to cattle and such spillovers must be rare as IAV has rarely been recorded in cattle. Nonetheless, in the absence of an effective control strategy, the high pathogenicity H5N1 virus may now become endemic in US dairy cattle, requiring continuous monitoring even in the absence of overt disease. Moreover, many other clades and strains of H5N1 virus continue to emerge through reassortment, causing zoonotic infections. For example, the emergence of the D1 genotype of H5N1 virus in North America has caused a recent uptick in spillover from birds to mammals including humans and swine[SUP]49[/SUP]. Urgent development and testing of broadly reactive H5 influenza vaccines for both animals and humans is a priority.

H5N1 B3.13 and D1.X have proven their transmissibility and genomic flexibility. The big California dairy epidemic headlines may be ebbing, but this virus is still around. Michigan had a positive bulk tank again in late December, and we’ll likely find some more as National testing ramps up. Time will tell whether partial herd immunity prevents large acute herd outbreaks; however, it’s a long way from a “quiet herd” to a fully negative herd with influenza virus in an extremely movement-dependent industry.
​
https://hogvet51.substack.com/p/fmd-in-germany-news-flash-then-back?utm_source=post-email-title&publication_id=2578829&post_id=154156865&utm _campaign=email-post-title&isFreemail=true&r=4qjp7k&triedRedirect=true& utm_medium=email