Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential.

An H7N9 low-pathogenicity avian influenza virus (LPAIV) emerged in 2013 through genetic reassortment between H9N2 and other LPAIVs circulating in birds in China. This virus causes inapparent clinical disease in chickens, but zoonotic transmission results in severe and fatal disease in humans. To examine a natural reassortment scenario between H7N9 and G1 lineage H9N2 viruses predominant in the Indian subcontinent, we performed an experimental coinfection of chickens with A/Anhui/1/2013/H7N9 (Anhui/13) virus and A/Chicken/Pakistan/UDL-01/2008/H9N2 (UDL/08) virus. Plaque purification and genotyping of the reassortant viruses shed via the oropharynx of contact chickens showed H9N2 and H9N9 as predominant subtypes. The reassortant viruses shed by contact chickens also showed selective enrichment of polymerase genes from H9N2 virus. The viable "6+2" reassortant H9N9 (having nucleoprotein [NP] and neuraminidase [NA] from H7N9 and the remaining genes from H9N2) was successfully shed from the oropharynx of contact chickens, plus it showed an increased replication rate in human A549 cells and a significantly higher receptor binding to α2,6 and α2,3 sialoglycans compared to H9N2. The reassortant H9N9 virus also had a lower fusion pH, replicated in directly infected ferrets at similar levels compared to H7N9 and transmitted via direct contact. Ferrets exposed to H9N9 via aerosol contact were also found to be seropositive, compared to H7N9 aerosol contact ferrets. To the best of our knowledge, this is the first study demonstrating that cocirculation of H7N9 and G1 lineage H9N2 viruses could represent a threat for the generation of novel reassortant H9N9 viruses with greater virulence in poultry and a zoonotic potential. IMPORTANCE We evaluated the consequences of reassortment between the H7N9 and the contemporary H9N2 viruses of the G1 lineage that are enzootic in poultry across the Indian subcontinent and the Middle East. Coinfection of chickens with these viruses resulted in the emergence of novel reassortant H9N9 viruses with genes derived from both H9N2 and H7N9 viruses. The "6+2" reassortant H9N9 (having NP and NA from H7N9) virus was shed from contact chickens in a significantly higher proportion compared to most of the reassortant viruses, showed significantly increased replication fitness in human A549 cells, receptor binding toward human (α2,6) and avian (α2,3) sialic acid receptor analogues, and the potential to transmit via contact among ferrets. This study demonstrated the ability of viruses that already exist in nature to exchange genetic material, highlighting the potential emergence of viruses from these subtypes with zoonotic potential.

Adsorptive mutation and N-linked glycosylation modulate influenza virus antigenicity and fitness.

Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is "fit" and able to overcome vaccine pressure.