Full Factorial Analysis of Mammalian and Avian Influenza Polymerase Subunits Suggests a Role of an Efficient Polymerase for Virus Adaptation

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern

Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule

Viral reassortment as an information exchange between viral segments

Viruses have an extraordinary ability to diversify and evolve. For segmented viruses, reassortment can introduce drastic genomic and phenotypic changes by allowing a direct exchange of genetic material between coinfecting strains. For instance, multiple influenza pandemics were caused by reassortments of viruses typically found in separate hosts. What is unclear, however, are the underlying mechanisms driving these events and the level of intrinsic bias in the diversity of strains that emerge from coinfection. To address this problem, previous experiments looked for correlations between segments of strains that coinfect cells in vitro. Here, we present an information theory approach as the natural mathematical framework for this question. We study, for influenza and other segmented viruses, the extent to which a virus’s segments can communicate strain information across an infection and among one another. Our approach goes beyond previous association studies and quantifies how much the diversity of emerging strains is altered by patterns in reassortment, whether biases are consistent across multiple strains and cell types, and if significant information is shared among more than two segments. We apply our approach to a new experiment that examines reassortment patterns between the 2009 H1N1 pandemic and seasonal H1N1 strains, contextualizing its segmental information sharing by comparison with previously reported strain reassortments. We find evolutionary patterns across classes of experiments and previously unobserved higher-level structures. Finally, we show how this approach can be combined with virulence potentials to assess pandemic threats