Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses - PubMed (original) (raw)

. 2011 Jul;85(13):6275-86.

doi: 10.1128/JVI.02125-10. Epub 2011 Apr 20.

Philippe Noriel Q Pascua, Jun Han Lee, Yun Hee Baek, Kuk Jin Park, Hyeok-il Kwon, Su-Jin Park, Chul-Joong Kim, Hyunggee Kim, Richard J Webby, Robert G Webster, Young Ki Choi

Affiliations

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Min-Suk Song et al. J Virol. 2011 Jul.

Abstract

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.

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Figures

Fig. 1.

Fig. 1.

In vitro characterization of reverse genetic-generated polymerase mutant pandemic (H1N1) 2009 influenza viruses expressing different virulence markers. (A) Polymerase activity [relative to that of WT CA04 (H1N1) virus] of the reconstituted viral polymerase complexes (PB2, PB1, and PA genes) of the indicated polymerase mutants. Polymerase activity was measured as luciferase activity in a minigenome reporter gene assay; values are the means (standard deviations) of at least three assays. *, P < 0.05; **, P < 0.005 (two-tailed paired t test). (B) Growth kinetics of polymerase mutant viruses in MDCK cells inoculated at an MOI of 10−4. Viral titers were determined at the indicated time points.

Fig. 2.

Fig. 2.

Polymerase activity of reassortant viral polymerase complexes. The replication and transcription activity of reconstituted viral polymerase complexes in which the PB2, PB1, and/or PA genes of CA04 (H1N1) were replaced with cognate polymerase subunits from human, avian, and swine influenza viruses. (A) Single-gene reassortants; (B) double-gene reassortants; (C) triple-gene reassortants. Luciferase-based minigenome reporter assays were used to measure polymerase activity in the background of a WT CA04 (H1N1) NP plasmid. Activity values shown are the means from at least three assays. The first row at the bottom lists the recombinants by number (“r” denotes reverse genetics). Shaded reassortants were efficiently rescued. Asterisks indicate P values (*, P < 0.05; **, P < 0.005) that represent a statistically significant difference from the polymerase activity of the reconstituted viral polymerase of the homogeneous WT CA04 (H1N1) complex (100%), the baseline for comparison (as determined by two-tailed paired t tests). Error bars indicate standard deviations of the means. Virus abbreviations: A/California/04/09, CA04; A/Puerto Rico/8/34, PR8; A/Sw/Korea/CAN01/04, CAN01; A/Cheongju/H407/08, H407; A/Sw/Korea/JNS06/04, JNS06; A/Dk/Korea/LPM91/06, L91; A/Em/Korea/W150/06, W150; A/Ab/Korea/W81/05, W81; A/Ab/Korea/ma81/07, ma81; A/Ab/Korea/ma81K/07, ma81K; A/Ab/Korea/W44/05, W44; A/Ab/Korea/ma44/07, ma44; A/Md/Korea/6L/07, 6L; A/Ck/Korea/04116/04, 04116; A/Ck/Korea/04164/04, 04164.

Fig. 3.

Fig. 3.

Survival of mice inoculated with reassortant viruses that have pathogenicity equal to or greater than that of the CA04 (H1N1) virus. Groups of 10 mice were inoculated with 104.5 TCID50 of each virus. Survival was monitored daily for 13 days. An asterisk indicates viruses that were more pathogenic than Rg CA04 (H1N1) in vitro. A dagger indicates viruses that were as pathogenic as Rg CA04 (H1N1) in vitro.

Fig. 4.

Fig. 4.

Phylogenetic trees of the viral polymerase genes used in this study. Virulence-related properties are indicated. The nucleotide sequences were aligned, and the phylograms were generated by the neighbor-joining method using the tree-drawing program Clustal X (1) and were viewed by using NJ plot (49). The scale bar represents the number of substitutions per nucleotide. Branch labels indicate the stability of the branches for 100 bootstrap replicates. Only those with bootstrap values of ≥60% are shown. Boldface entries represent viruses used in this study. Shaded viruses are pandemic (H1N1) 2009 influenza virus strains. Ab, aquatic bird; AGT, American green-winged teal; Av, avian; Em, environment; Hu, human; Sw, swine. An asterisk indicates viruses that induced polymerase activity at a higher level than that of Rg CA04 (H1N1). A dagger indicates viruses that resulted in replicative ability higher than that of Rg CA04 (H1N1). A double dagger indicates viruses that conferred virulence higher than that of parental Rg CA04 (H1N1) virus.

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References

    1. Aiyar A. 2000. The use of CLUSTAL W and CLUSTAL X for multiple sequence alignment. Methods Mol. Biol. 132:221–241 - PubMed
    1. Baek Y. H., et al. 2009. Molecular characterization and phylogenetic analysis of H3N2 human influenza A viruses in Cheongju, South Korea. J. Microbiol. 47:91–100 - PubMed
    1. Baek Y. H., et al. 2010. Surveillance and characterization of low pathogenic H5 avian influenza viruses isolated from wild migratory birds in Korea. Virus Res. 150:119–128 - PubMed
    1. Brockwell-Staats C., Webster R. G., Webby R. J. 2009. Diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza A (H1N1). Influenza Other Respi. Viruses 3:207–213 - PMC - PubMed
    1. Bussey K. A., Bousse T. L., Desmet E. A., Kim B., Takimoto T. 2010. PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. J. Virol. 84:4395–4406 - PMC - PubMed

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