New insights on how bird flu crosses the species barrier

In recent years, public health measures, surveillance, and vaccination have helped bring about significant progress in reducing the impact of seasonal flu epidemics, caused by human influenza viruses A and B. However, a possible outbreak of avian influenza A (commonly known as 'bird flu') in mammals, including humans, poses a significant threat to public health.

The Cusack group at EMBL Grenoble studies the replication process of influenza viruses. A new study from this group sheds light on the different mutations that the avian influenza virus can undergo to be able to replicate in mammalian cells.

Some avian influenza strains can cause severe disease and mortality. Fortunately, significant biological differences between birds and mammals normally prevent avian influenza from spreading from birds to other species. To infect mammals, the avian influenza virus must mutate to overcome two main barriers: the ability to enter the cell and to replicate within that cell. To cause an epidemic or pandemic, it must also acquire the ability to be transmitted between humans.

However, sporadic contamination of wild and domestic mammals by bird flu is becoming increasingly common. Of particular concern is the recent unexpected infection of dairy cows in the USA by an avian H5N1 strain, which risks becoming endemic in cattle. This might facilitate adaptation to humans, and indeed, a few cases of transmission to humans have been reported, so far resulting in only mild symptoms.

At the heart of this process is the polymerase, an enzyme that orchestrates the virus's replication inside host cells. This flexible protein can rearrange itself according to the different functions it performs during infection. These include transcription -- copying the viral RNA into messenger RNA to make viral proteins -- and replication -- making copies of the viral RNA to package into new viruses.

Viral replication is a complex process to study because it involves two viral polymerases and a host cell protein -- ANP32. Together, these three proteins form the replication complex, a molecular machine that carries out replication. ANP32 is known as a 'chaperone', meaning that it acts as a stabiliser for certain cellular proteins. It can do this thanks to a key structure -- its long acidic tail. In 2015, it was discovered that ANP32 is critical for influenza virus replication, but its function was not fully understood.

The results of the new study, published in the journal Nature Communications, show that ANP32 acts as a bridge between the two viral polymerases -- called replicase and encapsidase. The names reflect the two distinct conformations taken up by the polymerases to perform two different functions -- creating copies of the viral RNA (replicase) and packaging the copy inside a protective coating with ANP32's help (encapsidase).

Through its tail, ANP32 acts as a stabiliser for the replication complex, allowing it to form within the host cell. Interestingly, the ANP32 tail differs between birds and mammals, although the core of the protein remains very similar. This biological difference explains why the avian influenza virus does not replicate easily in mammals and humans.

"The key difference between avian and human ANP32 is a 33-amino-acid insertion in the avian tail, and the polymerase has to adapt to this difference," explained Benoît Arragain, a postdoctoral fellow in the Cusack group and first author of the publication. "For the avian-adapted polymerase to replicate in human cells, it must acquire certain mutations to be able to use human ANP32."

To better understand this process, Arragain and his collaborators obtained the structure of the replicase and encapsidase conformations of a human-adapted avian influenza polymerase (from strain H7N9) while they were interacting with human ANP32. This structure gives detailed information about which amino acids are important in forming the replication complex and which mutations could allow the avian influenza polymerase to adapt to mammalian cells.

To obtain these results, Arragain carried out in vitro experiments at EMBL Grenoble, using the Eukaryotic Expression Facility, the ISBG biophysical platform, and the cryo-electron microscopy platform available through the Partnership for Structural Biology. "We also collaborated with the Naffakh group at the Institut Pasteur, who carried out cellularexperiments," added Arragain. "In addition, we obtained the structure of the human type B influenza replication complex, which is similar to that of influenza A. The cellular experiments confirmed our structural data."

These new insights into the influenza replication complex can be used to study polymerase mutations in other similar strains of the avian influenza virus. It is therefore possible to use the structure obtained from the H7N9 strain and adapt it to other strains such as H5N1.

"The threat of a new pandemic caused by highly pathogenic, human-adapted avian influenza strains with a high mortality rate needs to be taken seriously," said Stephen Cusack, EMBL Grenoble Senior Scientist who led the study and has been studying influenza viruses for 30 years. "One of the key responses to this threat includes monitoring mutations in the virus in the field. Knowing this structure allows us to interpret these mutations and assess if a strain is on the path of adaptation to infect and transmit between mammals."

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Bird flu associated with hundreds of seal deaths in New England in 2022

Researchers at Cummings School of Veterinary Medicine at Tufts University found that an outbreak of highly pathogenic avian influenza (HPAI) was associated with the deaths of more than 330 New England harbor and gray seals along the North Atlantic coast in June and July 2022, and the outbreak was connected to a wave of avian influenza in birds in the region.

The study was published on March 15 in the journal Emerging Infectious Disease.

HPAI is more commonly known as bird flu, and the H5N1 strain has been responsible for about 60 million poultry deaths in the U.S. since October 2020, with similar numbers in Europe. The virus was known to have spilled over from birds into mammals, such as mink, foxes, skunk, and bears, but those were mostly small, localized events. This study is among the first to directly connect HPAI to a larger scale mortality event in wild mammals.

The co-first authors on the paper -- virologist and senior scientist Wendy Puryear and post-doctoral researcher Kaitlin Sawatzki, who both work in the Runstadler Lab at Cummings School -- have been researching viruses in seals for years. They credit their findings in the new study to a unique and robust data set made possible by a collaboration with wildlife clinics and rehabilitation and response organizations in the region, in particular with Tufts Wildlife Clinic and director Maureen Murray, V03, associate clinical professor at Cummings School, and an author on the paper.

"We have a better resolution and greater depth of detail on this virus than before because we were able to sequence it and detect changes almost in real time," said Puryear. "And we have pairings of samples, sometimes literally from a bird and a seal on the same beach."

The clinic has been conducting avian influenza surveillance on birds and some mammals since January 2022, shortly after this strain of avian influenza took a trans-Atlantic journey from Europe into the U.S. Through this testing, the team found a wide range of flu viruses, including at least three strains that crossed the Atlantic, and they witnessed consistent waves of infection in birds.

At the same time, in collaboration with NOAA's Greater Atlantic Region Marine Mammal Stranding Network, they were able to screen nearly all seals that came through the network, whether or not the animal appeared sick. The stranding network is composed of experts from state and federal wildlife and fisheries agencies, non-profit rehabilitation and response facilities, aquariums, and academic institutions who respond to strandings.

"Because of the genetic data that we gathered, we were the first to see a strain of the virus that's unique to New England. The data set will allow us to more meaningfully address questions of which animals are passing the virus to which animals and how the virus is changing," said Sawatzki.

How HPAI Is Transmitted

In addition to poultry, H5N1 also has had a huge impact on wild birds, especially sea birds. Multiple locations around the globe have experienced large die-offs, such as recently in Peru, where the virus killed 60,000 pelicans, penguins, and gulls.

At the time of the seal mortality event in New England, the virus was hitting gulls particularly hard, the researchers found. There are lots of ways gulls and other birds may transmit the virus to seals, they said. Seals and sea birds are coastal animals living in the same areas that have environmental contact, if not direct contact, since they share the same water and shoreline. A seal may contract the virus if it comes in contact with a sick bird's excrement or water contaminated by that excrement, or if it preys upon an infected bird.

The accepted knowledge is that H5N1 is nearly 100% fatal for domestic and wild birds other than waterfowl, and the same is proving true when it comes to spillover in wild mammals. All the seals that tested positive for HPAI were deceased at the time of sampling or succumbed shortly after. None of the animals that tested positive recovered. However, it's possible some asymptomatic or recovered cases never came into the stranding networks.

In addition to the seal mortality event in New England, which was the first time H5N1 was detected in marine mammals in the wild, other locations have lost marine mammals to the virus. Peru announced about 3,500 sea lions died from the virus, Canada reported a seal mortality event along the St. Lawrence Estuary, and there was a similar event with seals in the Caspian Sea, according to news reports from Russia.

A hotly debated topic among scientists is whether there has been mammal-to-mammal transmission of HPAI between seals.

"It's not surprising that you might have transmission between the seals, because it has happened with low pathogenic avian influenza," said Puryear. "However, we can't say definitively whether or not there has been mammal-to-mammal transmission of HPAI."

"To get strong evidence of mammal-to-mammal transmission, you need two things: lots of infected animals and time," explained Sawatzki. "Time for the virus to mutate, and time for the mutated virus to be transmitted to another seal. As the virus acquires mutations, we can see shared mutations in the sequences that are specific only to mammals and that haven't been seen in a bird before. We had the numbers, but this outbreak didn't last long enough to provide evidence for seal-to-seal transmission."

The research team found evidence that the virus mutated in a small number of seals. But fortunately, they have not seen a case of bird flu in seals along the Atlantic coast since the end of last summer. However, stranding season is about to start for harbor seals and gray seals, so they are bracing themselves for what might happen.

Prevention and Risk to Humans

The risk to the public remains low, according to the Centers for Disease Control and Prevention. Since December 2021, less than 10 human cases of H5N1 have been reported globally, and those cases occurred in people with direct exposure to infected poultry. There are no documented cases of human transmission for this variant.

However, there is the possibility it could become a larger issue for human health. Avian influenza emerged in 1996, and since 2003, 868 cases of human infection with H5N1 have been reported worldwide, according to the World Health Organization. Of those, 457 were fatal, roughly a 50% fatality rate.

"And that's why people get nervous about it," Puryear said.

There is a single-dose vaccine available for poultry, but it's not currently administered on a large scale -- in part because of cost and logistics, and in part because there's some concern it may make future surveillance of the virus more difficult. There's not much that can be done in terms of responding to the virus for wildlife, particularly given the scale at which infection is occurring.

Biosecurity is important in limiting the ways in which the virus can spread between and within species, the researchers said. For example, wild birds should be kept separate from domestic birds, such as backyard chickens. In addition, thorough and timely surveillance of domestic animals and wildlife is key to understanding how the virus is evolving to prepare the best possible vaccines and treatments.

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