Highly pathogenic avian influenza H5 viruses have been a public health concern since 1997. Prior to 2014, H5 viruses were primarily of the H5N1 subtype but evolved into different genetic clades. Since 2014, Clade 2.3.4.4 ‘H5Nx’ viruses, where the H5 haemagglutinin (HA) has reassorted with at least 4 different neuraminidase (NA) subtypes (N2, N5, N6 and N8), have spread around the world. Reassortment is a major mechanism by which influenza viruses acquire new properties. Due to the lack of population immunity in humans and ongoing evolution of the virus, there is a risk that clade 2.3.4.4 H5 viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. This project seeks to identify the molecular mechanism(s) that allowed the change in the ability of H5 HA to reassort with multiple NAs.
We have compared the reassortment potential of 2014 H5Nx viruses with a 2005 H5N1 virus using two complementary models; competitive transfection to evaluate preferential gene incorporation, and co-infection of WT/VAR viruses to study reassortment in an unbiased way.
Nine-plasmid transfections revealed almost exclusive co-selection of 2005 HA and NA genes when competed on a 2005 H5N1 genetic background, consistent with prevalence of H5N1 viruses prior to 2014. In contrast, no HA preference was observed on the genetic background of 2014 viruses. Coinfection experiments showed that 2014 N6 NA and 2005 N1 NA genes were incorporated into 2005 viruses with equivalent frequency after a single round of infection but virions expressing N1 NA genes predominated after multicycle infection.
There was no genetic incompatibility of 2014 N6 genes with the 2005 H5 HA at the level of packaging, rather we observed functional incompatibilities after multicycle infection. The role internal genes of 2014 H5Nx viruses play in lifting constraints on HA and NA selection has been investigated.