What needs to be done to make the H5N1 avian influenza virus a pandemic

In recent months, there has been growing concern about the spread of the H5N1 avian influenza virus.

It is increasingly isolated from more birds of different species and en masse, for longer periods, out of season, with greater geographic extent. In addition, it is isolated from an increasing number of different mammalian species, and among some of them, such as mink and sea lions, it has begun to multiply and be transmitted.

However, in humans, cases have so far been very sporadic. The first of these occurred in 1999 in China, and since then, about 900 human cases have been described, always in people who work in poultry farms or deal with birds. This virus is not transmitted between people. However, the lethality of the virus in humans is very high and can reach 50%.

For the H5N1 avian influenza virus to become a pandemic, it needs to improve its ability to spread through the air between mammals, become more able to enter and replicate in human cells, and be able to evade the human immune system.

At first glance, it seems that there are too many changes at once to worry about. But let’s see what the research says about this.

The ferrets managed to become infected by airborne droplets

First, for H5N1 to cause a pandemic, mutations are needed to make the virus more airborne between mammals.

In this sense, more than a decade ago, a couple of articles were published on the creation of mutant strains of the H5N1 virus that can be transmitted between ferrets through the air. One of them, published in NatureThe researchers built a chimera: the new virus mixes seven genes from the human influenza virus H1N1 A/California/04/2009 and the hemagglutinin gene, the key to cell entry, from the mutant avian influenza virus H5N1 A/Vietnam/1203/2004.

Previously, they have obtained several mutants of the H5N1 virus with modifications to the hemagglutinin gene to increase its stability and facilitate binding to human cell receptors and better replication in human cells. Thus, this new recombinant virus carried all the genes of the human H1N1 influenza virus, with the exception of the hemagglutinin gene, which was derived from the H5N1 virus.

Ferrets have been used to demonstrate whether this new virus is airborne. Ferrets are used as animal models for experiments because they are susceptible to infection with human and avian influenza viruses and develop influenza very similar to ours.

For the experiment, healthy ferrets were placed in cramped cages along with ferrets infected with the new viruses. Within a few days, they were able to confirm the infection and the presence of the virus in healthy ferrets, showing that it was transmitted by airborne droplets. The researchers concluded that four modifications of H5 hemagglutinin were enough to allow airborne transmission of the virus in ferrets.

In another work published in The scienceInstead of creating a new chimeric virus by recombination, the researchers genetically modified the H5N1 virus (specifically the A/Indonesia/5/2005 strain isolated from humans) using site-directed mutagenesis techniques. Subsequently, they subjected her to several successive passages between ferrets. After the passage, the virus acquired the necessary mutations that allowed it to be transmitted between ferrets through the air.

In this case, the viruses had four mutations in the hemagglutinin gene and one in the RNA polymerase 2 (PB2) gene, showing that with only 5 mutations, the H5N1 virus could be airborne between ferrets. And all this without the need for recombination between viruses: only with mutation mechanisms.

Both studies also concluded that the new viruses, while easily transmitted, were not virulent in animals and none of the ferrets died.

This confirms that transmissibility and virulence are very different things and that a virus can be made more transmissible, but it does not mean that it is more virulent, and vice versa.

Cell Entry Refinement

In addition to these mutations, mutations would be needed that would make the virus easily enter human cells.

Influenza virus enters human cells by binding hemagglutinin on the surface of the virus to a cell receptor, in particular alpha-2-6-sialic acid on cell membranes. In the case of avian cells, the receptor for the virus is alpha 2-3 sialic acid. Therefore, avian viruses cannot effectively enter human cells.

However, at least a couple of mutations in the hemagglutinin of the virus at positions Q226L and G228S are known to facilitate binding of H5N1 to the human receptor.

The changes don’t end there. For example, the H5N1 virus isolated from mammals has been found to have a mutation in the PB2 RNA polymerase gene, in particular the E627K mutation (glutamine lysine substitution at position 627). This change appears to make the virus replicate better inside mammalian cells.

This mutation was found in virus samples from foxes infected in the Netherlands in late 2021 and early 2022, and from a New England seal last year.

Similarly, in October 2022, an H5N1 outbreak was detected in Galicia on a mink farm and nearly 50,000 animals had to be euthanized. The virus also had a mutation in the same PB2 polymerase gene, but in a different position, which could facilitate its replication in mammals.

Evade the immune system

Finally, it is known that there is an intracellular protein with antiviral function, MxA, which detects the virus NP protein and activates the immune system against the influenza virus. Therefore, the NP protein gene must be mutated in order to evade the human immune system.

Getting all this right combination of different mutations isn’t easy, but it’s not impossible either.

The H5N1 virus has been “warning” us for a long time, it is getting closer and closer. The fact that more and more species of mammals are isolating and starting to be transmitted between them is not good news. Because the more times it happens, the more likely it is that the changes he needs will have a massive impact on people.

Pigs are one of the mammalian species of concern for infection with this virus. Pig cells have on their surface receptors for human influenza virus (alpha 2-6 sialic acid) and bird flu (alpha 2-3 sialic acid). This means that pigs are susceptible to infection with both types of virus.

Therefore, pigs act as “test tubes” where both types of viruses can penetrate and mix. Infection of pigs with the H5N1 virus may have contributed to the mutation and recombination of the virus and its possible “transition” to humans. Therefore, pig farms need to be closely monitored.

The original version of this article was published on the microBIO author’s blog.

Ignacio López-Goni, Member of the SEM (Spanish Society for Microbiology) and Professor of Microbiology, University of Navarra

This article was originally published on The Conversation. Read the original.