There is no ethical way to experimentally infect a red panda with influenza. The same goes for dama gazelles, Goeldi's monkeys and most of the wild species that virologists would like to understand as potential flu hosts. Experimental infection studies -- the gold standard for assessing susceptibility -- require deliberately sickening animals, and for endangered or protected species that is off the table. This leaves a hole in pandemic preparedness exactly where it matters. We do not know which wild animals can catch and spread the viruses we worry about most.
A team led by researchers at the Centre de Recerca en Sanitat Animal, an animal health research institute in Barcelona, has found a way around the problem. In a study published in Emerging Microbes & Infections, Ferran Tarrés-Freixas, Gerardo Ceada and colleagues report that they grew airway organoids from ten wildlife and livestock species using a single standardized protocol. These are tiny, self-organizing clusters of respiratory tissue. Cells extracted from postmortem lung or tracheal samples are embedded in a gel matrix, where they spontaneously assemble into three-dimensional structures that mirror a living airway. No animal needs to be deliberately infected or harmed. The tissue comes from animals that died or were euthanized for unrelated reasons.
The team infected these miniature airways with two influenza A strains and watched what happened.
The results varied a lot from species to species. Iberian wolf organoids lost more than 90% of their cells within three days of exposure to either virus, and dama gazelle cultures were similarly devastated. Chicken organoids were hammered by the avian H5N1 strain but barely affected by the mammalian pandemic H1N1, while pig cultures, consistent with decades of in vivo data, were hit roughly equally by both. Red panda organoids were strongly susceptible to pandemic H1N1. Alpaca and Goeldi's monkey cultures, by contrast, showed little cell death even though the virus still replicated in them. Every species tested, from buzzard to wolf, produced infectious viral particles.
The organoid panel did more than rank species by susceptibility. It revealed something about how influenza adapts. When the researchers sequenced the virus after three days of replication in each host's cells, they found mutations emerging in a host-dependent pattern. The most striking case was the Goeldi's monkey, a small New World primate. A mutation at position 190 in the hemagglutinin protein, which the virus uses to enter cells, became dominant in both replicates. That position helps determine which host cells the virus can latch onto. The particular substitution, D190E, appears to enhance the virus's compatibility with avian-type receptors.
This is a strange result for a mammalian host, and one with real implications. The authors suggest that certain mammals could serve as "gateway" species in which a human-adapted virus begins acquiring affinity for avian receptors, or vice versa. A virus that picks up such mutations while replicating in a tolerant mammalian host could emerge better equipped to jump to birds, or from birds back to mammals. The Goeldi's monkey cultures, it turned out, also expressed unusually high levels of the avian-type sialic acid receptor, which may explain why the virus shifted in that direction.
Sialic acid receptors are the standard explanation for why some species catch certain flu strains and others do not. Mammalian respiratory tracts tend to display alpha-2,6-linked sialic acids, which the pandemic H1N1 prefers. Birds tend to display alpha-2,3-linked versions, which avian H5N1 targets. The organoid panel confirmed these broad trends. Species with high alpha-2,6 expression tended to be more susceptible to pH1N1, and those with high alpha-2,3 levels were more susceptible to H5N1.
But the correlations were loose. Receptor expression alone left too much variation unexplained, suggesting that downstream factors matter at least as much -- host proteases that activate the virus, innate immune responses that contain it and intracellular machinery that helps or hinders replication. Organoids capture all of these. A receptor study, by design, cannot.
The team has already expanded the approach to more than 20 additional species, including wallabies, sea lions, Sumatran tigers, iguanas and African elephants. The protocol works across mammals and birds, and the organoids can be passaged and frozen for future use.
The applications go beyond cataloguing which species are susceptible. When a new flu variant emerges, a frozen organoid library could be thawed and screened within days to identify which animals are at risk. The same system could be used to probe why individuals or species differ in their vulnerability, teasing apart how receptors, immune responses and cellular machinery each shape the outcome. With H5N1 clade 2.3.4.4b circulating in dairy cattle, wild birds and sporadic human cases across the Americas, that kind of rapid, ethical screening is exactly what pandemic preparedness has lacked.