A statistical strategy to identify recombinant viral ribonucleoprotein of avian, human, and swine influenza A viruses with elevated polymerase activity

Objectives Reassortment of influenza A viruses can give rise to viral ribonucleoproteins (vRNPs) with elevated polymerase activity and the previous three pandemic influenza viruses contained reassorted vRNPs of different origins. These suggest that reassorted vRNP may be one of the factors leading to a pandemic virus. In this study, we reconstituted chimeric vRNPs with three different viral strains isolated from avian, human and swine hosts. We applied a statistical strategy to identify the effect that the origin of a single vRNP protein subunit or the interactions between these subunits on polymerase activity. Design Eighty one chimeric vRNPs were reconstituted in 293T cells at different temperatures. Polymerase activity was determined by luciferase reporter assay and the results were analysed by multiway ANOVA and other statistical methods . Results It was found that PB2, PB1, NP, PB2-PB1 interaction, PB2-PA interaction and PB1-NP interaction had significant effect on polymerase activity at 37 degrees C and several single subunits and interactions were identified to lead to elevation of polymerase activity. Furthermore, we studied 27 out of these 81 different chimieric vRNPs in different combinations via fractional factorial design approach. Our results suggested that the approach can identify the major single subunit or interaction factors that affect the polymerase activity without the need to experimentally reproduce all possible vRNP combinations. Conclusions Statistical approach and fractional factorial design are useful to identify the major single subunit or interaction factors that can modulate viral polymerase activity.