Identification and characterization of host specificity factors of a lethal human influenza H5N1 isolate

Influenza A viruses are major human and avian pathogens. Despite a species barrier, subtypes of influenza A can transmit from the avian reservoir to humans and widely spread in the population. Since H5N1 viruses circulate in the avian reservoir and cause high lethality rates when transmitted to humans, infections with the H5N1 subtype pose an ongoing threat. Although human-to-human transmission is a rare event, rapid evolution of the virus might result in a strain, which gains the ability to spread in the human population, leading to high morbidity and mortality. Advanced surveillance by understanding the mechanisms by which influenza viruses acquire the ability to cross the species barrier from birds to humans and new strategies to improve current vaccines are needed to control future pandemics. In this study, the fatal human case isolate A/Thailand/1(KAN-1)/2004 (H5N1) (KAN-1) was analyzed to examine mechanisms of H5N1 viruses to overcome host range restriction. • We were able to identify an adaptive mutation in KAN-1 hemagglutinin (HA). A polymorphism leading to an amino acid change in the HA that was previously reported to be positively selected during replication in humans altered the organ tropism of KAN- 1 in mice and ferrets. In the mouse model we found an increased replication of the selected variant in the lung. • In a genetic analysis of the KAN-1 virus, we identified further mutations crucial for adaptation to the mammalian host. Interestingly, the KAN-1 polymerase was poorly adapted to human cells, in contrast to other H5N1 viruses isolated from humans. We identified the NEP protein as a new pathogenicity factor of H5N1 viruses in humans, which is able to overcome this incomplete adaptation of the KAN-1 and avian H5N1 polymerases in human cells. Furthermore, functional studies revealed that the restriction of avian influenza polymerases in mammals is due to a general defect in RNA-replication and not transcription. • Since the human MxA GTPase is an important factor in the immune response against influenza viruses, we analyzed its antiviral activity against KAN-1. KAN-1 proved to be sensitive, while an isolate of the 2009 pandemic was relatively resistant to MxA. We were able to determine the viral nucleoprotein (NP) as the determinant for MxA sensitivity in vitro and in vivo. In addition, we identified mutations in NP responsible for resistance against MxA and could draw conclusions about the evolution of NP. • Based on our knowledge about protein-protein interactions in the polymerase complex, we developed a new strategy to create polymerase assembly mutants as a basis for live attenuated vaccines against H5N1. Vaccination of mice with these mutants showed protection against homologous and heterologous challenge with lethal doses of H5N1 viruses including KAN-1, therefore providing new options for live attenuated vaccine design