Adaptation of pandemic H1N1 influenza viruses in mice

Abstract

The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to alpha2,3 together with decreasing binding to alpha2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals.

FIG. 1.

Multistep growth curves of wild-type A/CA/04/09 (H1N1) (A) and A/TN/1-560/09 (H1N1) (B) viruses and their respective mouse-adapted variants in MDCK cells. Confluent MDCK cells were infected with H1N1 viruses at an MOI of 0.005 PFU/cell. Virus yields (log₁₀ TCID₅₀/ml) at 12, 24, 36, 48, 60, and 72 h after infection were titrated in MDCK cells. Each data point represents the mean virus yield from three individually infected wells ± the standard deviation. *, P < 0.05, and °, P < 0.01, compared with the value for the wild-type virus (shown in color) or compared with the value for the respective mouse-adapted virus (shown in black) (one-way ANOVA).

FIG. 2.

Survival of BALB/c mice infected with 5,000 PFU/mouse (each in 50 μl) of wild-type A/CA/04/09, A/CA/04/09-MA1, or A/CA/04/09-MA2 virus (A) and wild-type A/TN/1-560/09, A/TN/1-560/09-MA1, or A/TN/1-560/09-MA2 virus (B).

FIG. 3.

Receptor specificity of wild-type and mouse-adapted H1N1 influenza viruses. Association constants (K_ass) of virus complexes with sialylglycopolymers conjugated to fetuin (containing α2,3- and α2,6-linked sialyl receptors), 6′SLN (“human-like” α2,6-containing receptor), and 3′SL (“avian-like” α2,3-containing receptor). Higher K_ass values indicate stronger binding. Values are the means of three independent experiments ± standard deviations. *, P < 0.05, and °, P < 0.01, compared with the value for the wild-type virus (shown in color) or compared with the value for the respective mouse-adapted virus (shown in black) (one-way ANOVA).

FIG. 4.

Total protein expression and viral polymerase activity of RNP complexes. (A and B) Total expression of wild-type and mutant PB2 and NP proteins of A/CA/04/09 (H1N1) (A) and A/TN/1-560/09 (H1N1) (B) viruses in 293T cells. 293T cells expressing wild-type and mutant proteins were processed 24 h posttransfection and analyzed by Western blotting with rabbit polyclonal antibodies raised against PB2 and NP proteins of H1N1 influenza A virus (GenScript, Piscataway, NJ). *, P < 0.01 compared with the value for the wild-type virus (one-way ANOVA). (C and D) Polymerase activity of RNP complexes reconstituted from PB2, PB1, PA, and NP plasmids of wild-type A/CA/04/09 (H1N1) (C) and A/TN/1-560/09 (H1N1) (D) viruses and of their single point mutants. The polymerase activities were determined by a dual luciferase reporter assay in three independent experiments. *, P < 0.01 compared with the value for the wild-type virus (one-way ANOVA).