Avian influenza H9N2 haemagglutinin: molecular basis of antigenicity and receptor binding

Abstract

H9N2 avian influenza viruses are enzootic in poultry across much of Asia and North Africa. These viruses are also zoonotic and are considered potential pandemic candidates. Vaccination of poultry is employed to try and reduce the disease impact; however vaccine effectiveness is regularly compromised due to antigenic drift. The focus of this thesis, therefore, is to evaluate molecular markers in the H9N2 major antigen, haemagglutinin (HA), which influence virus antigenicity and zoonotic potential. Initially, the antigenic variability, as well as zoonotic potential, of field H9N2 viruses was assessed. Similarly to human influenza viruses, H9N2 viruses exhibit a large variability in their antigenic cross-reactivity. It was found that circulating H9N2 HAs have receptor binding and a pH of fusion similar to that of pandemic influenza HAs indicating these lineages may pose a particular zoonotic risk. Mapping of the antigenic sites of the H9N2 HA was undertaken using a variety of different methods. These revealed novel residues responsible for the antigenic variability of H9N2 viruses that mostly fell into two discrete antigenic sites designated ‘H9-A’ and ‘H9-B’. Additionally, the molecular basis of receptor binding preference was investigated revealing several residues responsible for virus binding to different receptor analogues. Finally, a group of mutants with amino acid deletions in their receptor binding site were assessed for their zoonotic potential and replicative fitness in vitro and in vivo. It was concluded that these viruses retained the ability to infect birds, could potentially arise in nature due to immune pressure and could pose a greater zoonotic threat than the parental virus. Overall, these results will help guide future vaccine seed strain selection, surveillance for vaccine breakdown and the emergence of novel, more zoonotic variants, as well as give a better understanding of the basic biology of these viruses.