The PB1 gene from H9N2 avian influenza virus showed high compatibility and increased mutation rate after reassorting with a human H1N1 influenza virus.

BACKGROUND: Reassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes. METHODS: The polymerase activity of the viral ribonucleoprotein (RNP) complex as combinations of polymerase-related gene segments derived from different reassortment events was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics. Gene segments of the human WSN-H1N1 virus (A/WSN/1933) were replaced by gene segments of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011), which were both the Hemagglutinin (HA) and Neuraminidase (NA) gene segments in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next-Generation Sequencing (NGS). RESULTS: We discovered that the avian PB1 gene of H9N2 increased the polymerase activity of the RNP complex in backbone of H1N1. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice. CONCLUSIONS: The higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene of H9N2 may drive the evolution and adaptation of reassortant viruses to the human host. This study provides novel insights in the characteristics of viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of the reassortant viruses in humans is important for pandemic preparedness.

Pathogenicity of reassortant H9 influenza viruses with different NA genes in mice and chickens.

To better understand the influence of different NA genes on pathogenicity of H9 viruses, three reassortant H9 viruses (rH9N1, H9N2 and rH9N3) were generated and characterized. All three viruses replicated efficiently in eggs and MDCK cells, whereas the rH9N1 and rH9N3 replicated more efficiently than H9N2 in A549 cells. The rH9N3 replicated more efficiently than rH9N1 and H9N2 viruses in mice, however, rH9N3 replicated and shed less efficiently than the H9N2 virus in chickens. Further studies indicate that N3 had higher NA activity and released virus from erythrocytes faster, which may improve the adaptation of H9 influenza virus to mammals.