Background

Influenza A viruses are negative-sense RNA viruses that infect a wide-range of species, including birds and humans. The classification of these viruses depends on the variations in surface glycoproteins hemagglutinin and neuraminidase.

Some strains of zoonotic influenza viruses are capable of infecting and replicating in humans, causing mild to fatal diseases. Most human influenza viruses carry genetic sequences from avian influenza viruses that encode hemagglutinin, neuraminidase, and polymerase basic-1. Genetic mutations in these viral proteins are responsible for bird-to-human transmission of avian influenza viruses.

In the current study, scientists have characterized the mutations in viral neuraminidase that facilitate zoonotic transmission of influenza viruses. They have analyzed all neuraminidase sequences of H5N1 influenza viruses of human- and bird-origin from Eurasia and Africa to identify the causative mutations.

Furthermore, they have characterized the functions of identified mutations by infecting chickens, mice, and ferrets with several recombinant viruses carrying these mutations.

Important observations

The analysis of neuraminidase genetic sequences identified A46D, L204M, S319F, and S430G mutations that act synergistically to improve H5N1 viral fitness and adaptability in humans. The scientists determined the functions of these mutations in both in vitro and in vivo (chickens, mice, and ferrets) setups.    

The analysis of neuraminidase activity revealed that human-like H5N1 viruses have significantly lower activity compared to bird-like H5N1 viruses. Such reduced activity might be needed to maintain an optimal receptor binding/destroying balance. Moreover, low activity might be required for the selection and adaptation of avian influenza viruses in humans.

Specifically, it was observed in the study that the reduced neuraminidase activity of human H5N1 viruses is due to potential conformational changes in the enzyme, as well as due to a low amount of enzyme incorporation into viral particles.

Regarding the incorporation of neuraminidase into viral particles, the study found that the level of RNA replication and transcription of human-like neuraminidase is lower than that of bird-like neuraminidase. The findings of co-transfection experiments revealed that the abundance of bird-like neuraminidase favors its selection over homologous human-like neuraminidase. This explains the relatively lower incorporation of human-like neuraminidase into viral particles.  

Regarding receptor binding activity of neuraminidase, the study found that the S430G mutation is primarily responsible for increasing the binding affinity of human-like neuraminidase for human-like receptors.

The head domain of neuraminidase has sialidase activity, which helps cleave sialic acid (SA) to release progeny viral particles in the respiratory tract for systemic viral transmission. The analysis of human-type 2,6-SA receptors and bird-type 2,3-SA receptors revealed that mutated human-like neuraminidase removes 2,3-SA during viral entry, leading to a reduction in bird-type receptors. This might reduce the adherence of hemagglutinin of progeny viral particles to 2,3-SA during viral release.

As speculated by the scientists, the abundance of 2,6-SA during viral release might act as the main driver for the adaptation of hemagglutinin and neuraminidase of avian influenza viruses to human-type receptors.

The findings of the mutational analysis revealed that all identified mutations in human-like neuraminidase act synergistically to increase viral replication in the human airway epithelium. These mutations also conferred moderate virulence and longer survival of H5N1 avian influenza virus in mice and ferrets, while not affecting the high virulence of the virus in chickens.  

Study significance

The study identifies four mutations in neuraminidase of human-like H5N1 influenza viruses that act synergistically to increase viral replication and induce moderate virulence in mammals, while maintaining high virulence in birds.

Collectively, these mutations reduce neuraminidase activity, expression, and incorporation in viral particles and increase the binding affinity of H5N1 viruses to human-like receptors.   

As mentioned by the scientists, these findings are vital to understand the genetic changes that improve the fitness of avian influenza viruses in humans. The scientists highlight the requirement of continuous genomic surveillance to monitor avian virus replication in mammals. This is particularly needed for the early detection of emerging viruses that have the potential to induce a pandemic condition.