Genetic incompatibilities and reduced transmission in chickens may limit the evolution of reassortants between H9N2 and panzootic H5N8 clade 2.3.4.4 avian influenza virus showing high virulence for mammals

Abstract

The unprecedented spread of H5N8- and H9N2-subtype avian influenza virus (AIV) in birds across Asia, Europe, Africa and North America poses a serious public health threat with a permanent risk of reassortment and the possible emergence of novel virus variants with high virulence in mammals. To gain information on this risk we studied the potential for reassortment between two contemporary H9N2 and H5N8 viruses. While the replacement of the PB2, PA and NS genes of highly pathogenic H5N8 by homologous segments from H9N2 produced infectious H5N8 progeny, PB1 and NP of H9N2 were not able to replace the respective segments from H5N8 due to residues outside the packaging region. Furthermore, exchange of the PB2, PA and NS segments of H5N8 by those of H9N2 increased replication, polymerase activity and interferon antagonism of the H5N8 reassortants in human cells. Notably, H5N8 reassortants carrying the H9N2-subtype PB2 segment and to lesser extent the PA or NS segments showed remarkably increased virulence in mice as indicated by rapid onset of mortality, reduced mean time to death and increased body weight loss. Simultaneously, we observed that in chickens the H5N8 reassortants, particularly with the H9N2 NS segment, demonstrated significantly reduced transmission to co-housed chickens. Together, while the limited capacity for reassortment between co-circulating H9N2 and H5N8 viruses and the reduced bird-to-bird transmission of possible H5N8 reassortants in chickens may limit the evolution of such reassortant viruses, they show a higher replication potential in human cells and increased virulence in mammals.

The C-terminus of non-structural protein 1 (NS1) in H5N8 clade 2.3.4.4 avian influenza virus affects virus fitness in human cells and virulence in mice

“…A/turkey/ Germany-MV/AR2487/2014 (H5N8) (hereafter designated A_NS237) and A/tufted duck/Germany/8444/ 2016 (H5N8) (hereafter designated B_NS217), belonging to clades 2.3.4.4A and 2.3.4.4B, respectively, were kindly provided by Timm C. Harder (National Reference Laboratory for Avian Influenza Virus, Friedrich-Loeffler-Institut (FLI)). Recombinant A_NS237 and B_NS217 viruses were previously generated [33]. The NS segments of these viruses were modified using site-directed mutagenesis to generate A_NS230 and A_NS217 from the A_NS237 plasmid, and similarly B_NS230 and B_NS237 were generated from B_NS217, by changing the stop codons in the CTE as described in a recent study [22] (Figure 1).…”

The Phage Nucleus and PhuZ Spindle: Defining Features of the Subcellular Organization and Speciation of Nucleus-Forming Jumbo Phages

“…Consisting of 8 individual segments, the influenza A genome is shuffled during replication. When two viruses co-infect the same cell, shuffling can result in genetic incompatibilities when a capsid packages incompatible genome segments from different parental viruses (such as H5N8 and H9N2) ( Mostafa et al, 2020 ), resulting in the production of fewer functional progeny. Although the underlying mechanisms are not yet understood, this evidence suggests that Virogenesis Incompatibility is also widespread.…”

Preferential selection and contribution of non-structural protein 1 (NS1) to the efficient transmission of the panzootic avian influenza H5N8 2.3.4.4 clades A and B viruses in chickens and ducks

“…PB1, NP and NA) contributed to the high virulence of an American HPAIV H5N2 clade 2.3.4.4A in chickens (35). Moreover, previous studies have shown that NS1 V149A contributed to the virulence of an H5N1 virus in chickens (36) and replacing the NS segment of the current H5N8-B with that of an H9N2 reduced transmission and replication of H5N8-B in chickens (22). In ducks, several studies found that the HA alone is not sufficient for high virulence (37-42) and we recently found that H5N8-B NS segment, in addition to the HA and NP, increased virulence and transmission of H5N8-A in Pekin ducks (Scheibner et al in preparation) indicating a significant role for NS in H5N8-B virulence and bird-to-bird transmission efficiency.…”

“…All viruses were handled in biosafety level 3 (BSL3) facilities at the FLI. Recombinant B_NS217 virus was generated in a previous study (22). To generate a recombinant A_NS237 virus, viral RNA was extracted from allantoic fluid using Trizol reagent (Thermo Fischer, Germany) and Qiagen RNeasy Kit (Qiagen, Germany) following the manufacturers’ guidelines.…”

“…H5N8-A viruses were avirulent in Pekin ducks, while H5N8-B viruses were highly virulent (21). We have recently shown that NS1 is a major virulence determinant of an H5N8-B virus in ducks (Scheibner et al in preparation) and swapping NS gene segment of H5N8-B and LPAIV H9N2 reduced virus transmission in chickens (22). Little is known about the global evolution of NS1 gene of clade 2.3.4.4 viruses overtime and the role of CTE on virus fitness in gallinaceous birds and waterfowls.…”