title: A statistical strategy to identify recombinant viral ribonucleoprotein of avian, human, and swine influenza A viruses with elevated polymerase activity.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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°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.
authors: Centre of Influenza Research & School of Public Health, The University of Hong Kong, Hong Kong.
authors: Alex W H
authors: AW
authors: Chin
authors: Benjamin D
authors: BD
authors: Greenbaum
authors: Olive T W
authors: OT
authors: Li
authors: Richard J
authors: RJ
authors: Webby
authors: Leo L M
authors: LL
authors: Poon
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D017930:Genes, Reporter; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059386:Reverse Genetics; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D008156:Luciferases; D012324:RNA-Dependent RNA Polymerase
keywords: Fractional factorial design; influenza virus; polymerase; reassortment
references: J Virol. 2012 Feb;86(3):1750-7
references: 22090127
references: Emerg Infect Dis. 2005 Jul;11(7):1036-41
references: 16022777
references: N Engl J Med. 2009 Jun 18;360(25):2605-15
references: 19423869
references: J Gen Virol. 2008 Dec;89(Pt 12):2923-2932
references: 19008377
references: J Mol Genet Med. 2008 Nov 27;3(1):158-66
references: 19565018
references: PLoS Pathog. 2008 Feb 29;4(2):e1000012
references: 18463694
references: Nature. 2006 Apr 27;440(7088):E8; discussion E9-10
references: 16641948
references: J Gen Virol. 2012 Mar;93(Pt 3):531-540
references: 22090209
references: PLoS One. 2009 Jul 28;4(7):e6402
references: 19636415
references: Cell. 1977 Jan;10(1):1-10
references: 837439
references: J Virol. 1989 Nov;63(11):4603-8
references: 2795713
references: PLoS One. 2012;7(3):e33383
references: 22438920
references: Nature. 2009 Jun 25;459(7250):1122-5
references: 19516283
references: PLoS One. 2009 May 21;4(5):e5658
references: 19462010
references: Nature. 2011 May 26;473(7348):519-22
references: 21614079
references: J Virol. 1998 Jul;72(7):5493-501
references: 9621005
references: Nature. 2005 Oct 6;437(7060):889-93
references: 16208372
references: Arch Virol. 2011 Jun;156(6):987-94
references: 21302122
references: Nature. 2006 Apr 27;440(7088):E9; discussion E9-10
references: 16641950
references: Influenza Other Respir Viruses. 2013 Nov;7(6):969-78
references: 23634764
references: Mol Phylogenet Evol. 2008 Jun;47(3):1100-10
references: 18353690
references: Virology. 2004 Oct 10;328(1):101-19
references: 15380362