🦠 microbiology

Infection of the bovine mammary gland by avian H5N1 subclade 2.3.4.4b influenza viruses

This study demonstrates that mammary tissue from three major cattle breeds (Aberdeen Angus, Holstein Friesian, and Limousin) is permissive to infection by avian H5N1 2.3.4.4b viruses, highlighting the potential for cattle to serve as a mixing vessel for viral reassortment and underscoring the critical need to include them in H5N1 surveillance frameworks.

Original authors: Ross, R. A., Walsh, S. K., Montgomery, H., Chen, H., Hutchinson, E., Murcia, P. R.

Published 2026-04-16

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CC BY 4.0

Original authors: Ross, R. A., Walsh, S. K., Montgomery, H., Chen, H., Hutchinson, E., Murcia, P. R.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The Big Picture: A New "Mixing Bowl" for Flu Viruses

Imagine the influenza virus as a master thief that usually breaks into specific houses (birds). Recently, this thief has started breaking into a new type of house: dairy cows. This is a big deal because it means the virus is changing its habits, and there's a fear it might learn how to break into human houses too.

This study asks a simple question: Is this new virus thief only interested in dairy cows (the ones that give milk), or can it also break into beef cows (the ones raised for meat)?

The Experiment: The "Tissue Test Kitchen"

Instead of infecting live cows (which would be unethical and dangerous), the scientists took a slice of the "kitchen" where milk is made—the mammary gland—from six different cows right after they were slaughtered. They used three popular breeds:

Holstein Friesian: The classic black-and-white dairy cow.
Aberdeen Angus: A famous beef breed.
Limousin: Another popular beef breed.

They treated these tissue slices like little test tubes in a lab. They dropped different types of flu viruses onto the tissue to see if the cells would let the virus in and start multiplying.

The Viruses: The "Guests" at the Party

The scientists tested three different "guests" (viruses):

The Bird Guests (H5N1): Three versions of the bird flu that have been causing trouble globally. These are the ones that usually only like bird cells.
The Human Guests (Seasonal Flu): Regular human flu viruses (H1N1 and H3N2) to see if the cow tissue could also be infected by viruses meant for people.
The Control: A harmless virus to make sure the experiment worked.

The Findings: Everyone Was Invited

Here is what happened when the viruses knocked on the door:

1. The "Do Not Enter" Signs Were Missing
Usually, bird flu viruses can't get into mammal cells because the "locks" on the cell doors are different. Bird cells have Type A locks, and human cells have Type B locks. Bird flu keys only fit Type A.

However, the scientists found that the cow mammary glands have both types of locks (Type A and Type B) on the surface of their cells.

Analogy: Imagine a cow's udder is like a house with a front door that has two different keyholes. A bird virus has a key for the left hole, and a human virus has a key for the right hole. The cow's door has both holes, so any flu virus can get in.

2. Beef Cows Are Just as Vulnerable as Dairy Cows
The virus didn't just infect the dairy cows. It successfully infected the beef cows (Angus and Limousin) too.

The Takeaway: It doesn't matter if the cow is raised for milk or for a burger. If a bird flu virus lands on a cow's udder, it can get in and start replicating.

3. The "Mixing Bowl" Effect
Because the cow's udder has both types of locks, it acts like a mixing bowl.

The Danger: If a cow gets infected by a bird flu virus and a human flu virus at the same time, the viruses can swap parts inside the cow's cells. This is called reassortment. It's like two different recipes being mixed together to create a brand new, potentially dangerous dish. This new "super-virus" could be one that spreads easily between humans.

Why This Matters

Surveillance is Key: We can't just watch dairy farms anymore. We need to watch beef farms too, because the virus can hide and change there.
The "Spillover" Risk: Since the virus can now live and multiply in cows, and cows are everywhere (not just in the US, but globally), the chance of the virus jumping from a cow to a human is higher than we thought.
The "Silent" Spread: Cows might get sick (lower milk production, mastitis) but not show obvious flu symptoms like coughing. This means the virus could be spreading quietly through herds without us noticing.

The Bottom Line

The cow's udder is a dangerous new playground for flu viruses. It's a place where bird viruses and human viruses can meet, shake hands, and potentially swap genetic material to create something new. The study shows that all common cattle breeds are vulnerable to this, not just the dairy cows we've been worried about.

In short: The virus has found a new, very welcoming home in cattle. We need to keep a very close eye on all cows to stop the next pandemic before it starts.

1. Problem Statement

The emergence of the H5N1 subclade 2.3.4.4b highly pathogenic avian influenza (HPAI) virus has significantly expanded the host range of Influenza A Viruses (IAVs). Since 2024, the virus has established sustained circulation in U.S. dairy cattle, causing mastitis, reduced milk yield, and high viral loads in milk, leading to over 40 human spillover cases. While the susceptibility of dairy cattle (specifically Holstein Friesians) is documented, the susceptibility of beef cattle breeds and other common dairy breeds in Europe, the Americas, and Oceania remains largely unexplored. Furthermore, the specific tissue tropism for the mammary gland and the potential for this tissue to act as a "mixing vessel" for viral reassortment between avian and mammalian strains require further investigation to assess global agricultural and public health risks.

2. Methodology

The study utilized an ex vivo bovine mammary explant model to assess viral permissiveness and receptor distribution without using live animals.

Tissue Collection: Mammary tissue (teat and gland cistern) was collected from six slaughtered female cattle representing three breeds: Aberdeen Angus (beef), Limousin (beef), and Holstein Friesian (dairy). All donors were free of mastitis and antibiotic treatment.
Viral Strains: The researchers used attenuated reassortant viruses generated via reverse genetics to ensure biosafety (BSL-2). These viruses possessed:
- Internal genes: From the attenuated vaccine strain A/Puerto Rico/8/34 (PR8, H1N1).
- Surface glycoproteins (HA/NA): From three avian H5N1 clade 2.3.4.4b isolates (AIV07, AIV09, AIV48) predating the U.S. cattle outbreak, and two human seasonal strains (H1N1 and H3N2).
- Safety Note: The polybasic cleavage site in the H5 HA was removed to prevent high pathogenicity.
Experimental Infection: Tissue explants were inoculated with the viruses (final dose: $5 \times 10^6$ genome copies). After 24 hours of incubation, tissues were fixed, embedded in paraffin (FFPE), and sectioned.
Detection & Analysis:
- Immunohistochemistry (IHC): Used a sheep anti-IAV-NP antibody to detect viral antigen in epithelial cells.
- Lectin Staining: Used MAL-I (for $\alpha2,3$ -linked sialic acids, avian receptor preference) and SNA (for $\alpha2,6$ -linked sialic acids, mammalian/human receptor preference) to map receptor distribution.
- Phylogenetics: Maximum likelihood phylogeny was constructed to contextualize the H5N1 isolates used against global circulating strains.

3. Key Contributions

Cross-Breed Susceptibility: This is the first study to demonstrate that beef cattle breeds (Aberdeen Angus and Limousin) are permissive to H5N1 infection in mammary tissue, challenging the assumption that susceptibility is limited to dairy breeds.
Receptor Profiling: The study provides definitive evidence that bovine mammary epithelium expresses both $\alpha2,3$ -linked and $\alpha2,6$ -linked sialic acids, a prerequisite for the infection of both avian-adapted and human-adapted influenza viruses.
Mixing Vessel Hypothesis: The findings support the hypothesis that the bovine mammary gland is a unique multicellular niche capable of supporting mixed infections, potentially facilitating viral reassortment and adaptation.

4. Results

Receptor Expression: Lectin staining confirmed widespread expression of both $\alpha2,3$ - and $\alpha2,6$ -linked sialic acids in the epithelial cells of the teat and gland cisterns across all three breeds. There were no qualitative differences in receptor distribution between beef and dairy breeds, though significant variation existed between individual donors.
Viral Permissiveness:
- H5N1 Infection: All three breeds were permissive to infection by at least one of the H5N1 strains (AIV09 and AIV48 were the most infectious). Viral antigen was detected in epithelial cells of both the teat and gland cisterns.
- Breed Comparison: No breed-specific resistance was observed. The Holstein Friesian donor (D5) was highly permissive to both H5N1 strains in both tissue types. Beef donors (Aberdeen Angus and Limousin) also showed infection, particularly in the teat cistern, though infection in the gland cistern was more variable.
- Human Strains: Susceptibility to human seasonal H1N1 and H3N2 was observed but was generally more limited and donor-dependent compared to the H5N1 strains.
Tissue Tropism: Infection was generally more robust in the teat cistern compared to the gland cistern, though both sites supported viral replication.
Phylogenetic Context: The H5N1 isolates used (AIV07, AIV09, AIV48) clustered within the 2.3.4.4b clade, distinct from the specific U.S. dairy outbreak strains but evolutionarily related.

5. Significance

Global Risk Assessment: The findings suggest that H5N1 susceptibility is not restricted to specific dairy breeds or geographic regions. Given that the tested breeds (Aberdeen Angus, Limousin, Holstein Friesian) are globally distributed, the risk of H5N1 spillover into cattle herds worldwide is likely higher than previously estimated.
Surveillance Implications: The study argues for the inclusion of beef cattle in H5N1 surveillance frameworks. Current surveillance may be under-recognizing the extent of viral circulation in cattle populations.
Public Health & Evolutionary Risk: The co-expression of avian and mammalian receptors in the mammary gland creates a "mixing vessel" environment. This increases the risk of:
1. Reassortment: If a cow is co-infected with avian and human/swine influenza strains.
2. Adaptation: The selective pressure of the mammary gland may drive H5N1 to adapt to better bind $\alpha2,6$ receptors, potentially increasing its transmissibility to humans.
Policy Recommendation: The authors conclude that cattle must be integrated into One Health preparedness planning and risk-assessment frameworks, particularly during poultry outbreaks or wild bird migration seasons.