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Disease and treatment

Mapping the influenza viruses that can be transmitted from animals to people

When a virus suddenly spreads from animals to people, this can herald the start of a worldwide pandemic that can kill thousands of people – just as we see it now with COVID-19. An international research team based in Denmark is determining which influenza viruses originating in pigs can cause major problems for people in the future.

On 17 April 2009, doctors in southern California discovered a new type of influenza virus that had infected two children who had been brought to the local hospital.

This virus, today called influenza A(H1H1)pdm09 virus, influenza A virus or influenza A virus subtype H1N1, contained a unique combination of genes not seen before in either humans or pigs anywhere in the world. However, all the genome segments had been detected in pigs but not in this combination.

This combination proved to be potent.

The virus spread rapidly, and by late April the World Health Organization (WHO) had declared the situation to be a Public Health Emergency of International Concern.

In June, WHO stopped keeping track of the number of infected people after the influenza A(H1H1)pdm09 virus outbreak had become a global pandemic.

In the past decade, thousands of people have died from this virus.

“Sequencing of the influenza A(H1H1)pdm09 virus showed that all eight gene segments had previously been detected in pigs, but the actual virus had not been detected in pigs before jumping to people. In addition, the virus infects both ways, so it first jumped from pigs to people, probably in Mexico, but since then people have infected pigs globally with this virus,” explains Lars Erik Larsen, Professor, Department of Veterinary and Animal Sciences, University of Copenhagen.

The Novo Nordisk Foundation recently awarded Lars Erik Larsen a Challenge Programme grant to reveal the mechanisms behind the ability of influenza viruses to be transmitted from animals to people over the next 6 years.

Several hundred swine-origin influenza viruses can potentially infect people

April 2009 also marked a turning point for influenza researchers worldwide.

The overview of the circulating influenza viruses was clearly inadequate, especially in pigs, including their potential for transmission to people. This was like holding a closed box without knowing whether the box contains dynamite or firecrackers.

Since 2009, researchers have therefore screened pigs globally and mapped hundreds of influenza viruses to determine what is circulating and to determine the capabilities of these swine-origin influenza viruses that can make them so problematic for people.

This research has shown that influenza A(H1H1)pdm09 virus excels at mixing genes with other viruses and reassorting them, which it can use to outmanoeuvre the immune systems of both pigs and people.

This research has also shown that pigs have a very wide range of influenza viruses. More than 20 types have been found in Denmark, 35 in Germany and 135 in China. They all contain different combinations of genes that regulate their infection potential and how ill people become.

The latest research finding since 2009 is that swine-origin influenza viruses very often contains some genes from human influenza viruses.

“We recently detected a virus in pigs in which seven of the eight genesegments originated from strains known to infect human, even though this virus never has been detected in humans. The virus was presumably reassorted in pigs. We havealso seen that since 2009, the two types of swine-origin influenza viruses in people and pigs have diverged. They have evolved and adapted to the different hosts so that they no longer are as similar as they were before,” explains Lars Erik Larsen.

Vaccines can be ready before a pandemic emerges

This major research project headed by Lars Erik Larsen is striving to determine the mechanisms that enable viruses to jump from animals to people.

“Many viruses are circulating out there, and they contain genes that can enable them to both infect people and create pandemics. But we cannot cry wolf every time we find a new virus strain. We therefore need tools to predict whether a given virus has the potential to be transmitted to people or not,” says Lars Erik Larsen.

Lars Erik Larsen believes that understanding viruses in pigs better could form the basis for developing vaccines against the potential threats.

The vaccines would not be used immediately but would be stored for rapid deployment if a virus develops the ability to be transmitted from animals to people (zoonosis).

“We want to rate all these viruses based on their transmission potential. But this requires knowing the signals for this potential – finding markers for these zoonotic risk factors,” explains Lars Erik Larsen.

Hard to determine why viruses transmit between people and animals

Lars Erik Larsen is not the first researcher to try to identify markers for zoonotic risk factors in swine-origin influenza viruses.

Unfortunately, researchers have not yet managed to find specific genetic markers that enable certain viruses to be more transmissible than others.

This is why Lars Erik Larsen also believes that the problem should be viewed from several angles.

“We believe that both genetic markers in the viruses and factors in the human immune response determine the risk of transmission,” he says.

The new research project is therefore striving to find these viral genetic risk factors and link them with the risk factors in people’s immune response.

“This is probably related to the innate immune response, which is activated within the first hours and days of infection. The innate immune response determines whether a virus can survive in a human cell or not and whether the immune system activates a more specific response. But the viruses must avoid innate mechanisms to get established in humans,” explains Lars Erik Larsen.

Avian-origin and swine-origin influenza viruses infect people differently

The differences between human, avian-origin and swine-origin influenza viruses can provide an idea about the differences in immune systems and the interaction between the viruses and the host.

Both swine-origin and human influenza viruses penetrate cells of the upper respiratory tract through the same receptors on the cell surface. Examining pigs in the search for the next new threat to people therefore makes good sense.

In contrast, avian-origin influenza viruses use another receptor to penetrate the cells, and although these receptors are located on the surface of the cells of the upper respiratory tract of birds, they only exist on the surface of the cells deep in the lungs of pigs and people.

Swine-origin influenza viruses can therefore penetrate the cells in people’s upper respiratory tract much more easily than avian-origin influenza viruses, and the avian viruses do not easily transmit between people since the receptors are located in the lower respiratory tract.

“People who are infected with avian-origin influenza viruses have very close interaction with birds, such as on an infected chicken farm. They are exposed to so much of the virus that some inevitably enters the lungs, where it can penetrate the cells. But swine-origin influenza viruses can much more easily be transmitted to people because they can use the receptors located in the upper respiratory tract,” says Lars Erik Larsen.

Viruses circumvent the immune response

However, the fact that swine-origin and human influenza viruses use the same receptor to penetrate the cells of the lungs does not explain why some swine adapted influenza viruses can transmit from pigs to people and others cannot.

Something more is required.

Once the swine-origin influenza virus has penetrated cells in people’s upper respiratory tract, it must circumvent the immune system so it is not suppressed immediately.

This is the moment when viruses must possess genetic variants that enable them to circumvent the immune system, and the cells’ immune response must enable the virus to jump from pigs to people.

“If viruses just had one or two relevant genetic markers, we would have found them already, but something more is clearly needed. We hypothesize that a combination of the markers and how the human cells respond to these viruses determines transmission,” explains Lars Erik Larsen.

Five phases of infection risk

WHO divides the risk of emerging zoonotic diseases into five phases:

• phase 1 – the virus only infects animals;

• phase 2 – the virus sporadically transmits to people;

• phase 3 – the virus can replicate in people;

• phase 4 – the virus has sustained community-level transmission from person to person (epidemic); and

• phase 5/6 – the virus transmits globally (pandemic).

“The novel coronavirus, 2019-nCoV, is now in phase 6. We would like to be able to detect and determine what happens to viruses in the transition to phases 2 and 3. The problem is also the same for the coronaviruses. These are widely prevalent in animals and especially bats, but only a few can transmit to people. We also lack valid genetic markers for these,” says Lars Erik Larsen.

Experts assigned to reveal the secrets of influenza viruses

In the new research project, Lars Erik Larsen has assembled an international team of experts in all the fields needed to carry out the research.

These include experts in the human as well as swine immune response. There are also experts in characterizing the swine-origin influenza viruses and human influenza viruses.

The researchers will initially try to identify the gene variants that are required for swine-origin influenza viruses to infect humans. The researchers will then identify the relevant mechanisms in the human immune response, which must also be present.

The researchers will try to transmit various naturally occurring swine-origin and human-origin strains of influenza A(H1H1)pdm09 virus to pigs and ferrets, which are model animals for humans.

Inserting transmission markers into other viruses

Influenza A(H1H1)pdm09 virus has mutated significantly since 2009, and the researchers will try to find differences in the transmission patterns between ferrets and pigs and hope to identify the underlying causes related to both the viruses and the immune responses.

“The goal is to determine whether some mechanisms in pigs versus humans and ferrets are prerequisites for transmission,” says Lars Erik Larsen.

Once the researchers have identified markers for transmission potential, they will try to change viruses to either remove their ability to infect humans or insert this ability into other viruses to see whether they can also infect people or ferrets.

“If we change the genes and the viruses can infect people, we have found the right genes. We already know that some genes in viruses are designed to circumvent the immune response, and we will try toexplore the specific mechanism behind this,” says Lars Erik Larsen.

Testing viruses on human lung tissue

Infecting ferrets as model animals for humans is one thing, but viruses transmitting to people is completely different.

The researchers will therefore follow up on the initial research by testing their findings in the lung tissue of people who have died.

The researchers will extract the lung tissue, cultivate it in the laboratory and investigate what happens when they try to infect it with a swine-origin influenza virus.

“This has the dual advantages of testing the viruses in human tissue and using fewer test animals,” says Lars Erik Larsen.

Discoveries may lead to better vaccines and treatment

Lars Erik Larsen says that researchers hope to identify the genetic markers before the project ends.

The researchers also want to develop the assays that make it easy to quickly analyse any swine-origin influenza virus and rapidly determine its potential for transmission to humans.

Conversely, the researchers have also allocated money to produce transgenic pigs, in which they will change the genetic markers discovered in the immune system and determine whether this affects the risk of transmission to the pigs.

If it does, the transgenic pigs could form the basis for herds of pigs that are not susceptible to the most common and deadly forms of influenza viruses.

“A completely independent goal is to contribute to the basic scientific understanding of the immune response to viral infections. This can be useful in developing vaccines and medicine to counter influenza viruses. We might discover new treatment targets for viral infections that can be used pharmaceutically,” explains Lars Erik Larsen.

In 2019, the Novo Nordisk Foundation awarded a Challenge Programme grant to Lars Erik Larsen for the project Viral and Host Factors of Zoonotic and Pandemic Influenza A Viruses (FLUZOOMARK).

Lars Erik Larsen
Professor
Influenza A virus is a severe pathogen of human and animals, which continues to kill millions of people. There is a constant exchange of influenza viruses between human and pigs, which lead to new viruses with pandemic potential. Pigs are regarded as a melting pot for new influenza virus, and the most recent pandemic in 2009 was caused by a swine virus. Denmark produces more than 30 million pigs yearly, and thousands of people are in daily close contact with swine. This makes Denmark a hotspot for evolution of new influenza viruses, and provides a perfect setting for the study of the exchange of viruses. This project will combine leading researchers with unique access to influenza isolates, laboratory techniques and data. Human and animal viruses will be characterized, with the aim of identifying both viral and host factors needed to cross the species barrier. The project outputs are groundbreaking new tools for prevention of future influenza pandemics and design of improved vaccines.