PB2 and hemagglutinin mutations are major determinants of host range and virulence in mouse-adapted influenza A virus

Abstract

Serial mouse lung passage of a human influenza A virus, A/Hong Kong/1/68 (H3N2) (HK-wt), produced a mouse-adapted variant, MA, with nine mutations that was >10(3.8)-fold more virulent. In this study, we demonstrate that MA mutations of the PB2 (D701N) and hemagglutinin (HA) (G218W in HA1 and T156N in HA2) genes were the most adaptive genetic determinants for increased growth and virulence in the mouse model. Recombinant viruses expressing each of the mutated MA genome segments on the HK-wt backbone showed significantly increased disease severity, whereas only the mouse-adapted PB2 gene increased virulence, as determined by the 50% lethal dose ([LD(50)] >10(1.4)-fold). The converse comparisons of recombinant MA viruses expressing each of the HK-wt genome segments showed the greatest decrease in virulence due to the HA gene (10(2)-fold), with lesser decreases due to the M1, NS1, NA, and PB1 genes (10(0.3)- to 10(0.8)-fold), and undetectable effects on the LD(50) for the PB2 and NP genes. The HK PB2 gene did, however, attenuate MA infection, as measured by weight loss and time to death. Replication of adaptive mutations in vivo and in vitro showed both viral gene backbone and host range effects. Minigenome transcription assays showed that PB1 and PB2 mutations increased polymerase activity and that the PB2 D701N mutation was comparable in effect to the mammalian adaptive PB2 E627K mutation. Our results demonstrate that host range and virulence are controlled by multiple genes, with major roles for mutations in PB2 and HA.

FIG. 1.

Genetic and biological characterization of recombinant HK and MA viruses in mice. (A) Mouse adaptation increases virulence in CD-1 mice. Groups of five mice were infected intranasally with 10⁵ PFU of HK-wt (HK) and HK-MA (MA) viruses. Mortality was monitored daily for 14 dpi. (B) Mouse adaptation increased lung pathology in CD-1 mice. Groups of three mice were inoculated intranasally with 10⁶ PFU of HK and MA viruses. Lungs were collected at 6 dpi for staining and imaging with H&E. Magnification, ×100. (C) Virulence of the recombinant viruses (r-HK and r-MA) in mice. HK genome segments were exchanged on the MA backbone and vice versa. Gene segments derived from HK and MA viruses are shown in blue and pink, respectively. The MLD₅₀ was determined by inoculating groups of five CD-1 mice with 10-fold serial dilutions of the stock recombinant viruses in a 50-μl volume. In some instances the MLD₅₀ value was greater than the highest dose tested (indicated by >). The MLD₅₀ was calculated by using the Karber-Spearman method (32). (D) Body weight loss of mice infected with single gene reassortants in the r-HK background. (E) Body weight loss of mice infected with single gene reassortants in the r-MA background. For panels D and E, groups of five mice were intranasally infected with 10⁵ PFU of each of the recombinant viruses. Body weight loss was monitored for 14 dpi.

FIG. 2.

Survival and body weight loss in CD-1 mice infected with recombinant viruses possessing combinations of PB2 and HA from HK and MA on the alternate backbone. Groups of five mice were infected intranasally with 10⁵ PFU of each of the recombinant viruses as indicated in the figure. Mortality and morbidity were monitored daily for 14 dpi. (A) Survival curve. (B) Body weight loss curve.

FIG. 3.

Immunofluorescent staining of mouse lungs infected with HA and PB2 recombinant viruses. CD-1 mice were infected with 10⁵ PFU of the recombinant viruses, and lungs were collected at 2 dpi. Viral antigens were detected by staining lung sections with anti-HK primary antibody followed by Cy3-conjugated secondary antibody (red). Nuclei were stained with Hoechst (blue), and images were taken using a 20× objective.

FIG. 4.

Replication kinetics of HA and PB2 recombinants viruses in vivo and in vitro. (A) Replication kinetics of the MA reassortants on the r-HK background in mouse lungs. (B) Replication kinetics of the HK reassortants on the r-MA background in mouse lungs. Groups of 12 CD-1 mice were infected intranasally with 5 × 10³ PFU. Lungs were collected at 1, 3, 5, and 7 dpi and then homogenized, and virus titers were assessed by plaque assay. (C to H) Replication kinetics of the HA and PB2 reassortants in vitro. Monolayers of MDCK, A549, or M1 cells were infected in triplicate with each of recombinant viruses at an MOI of 0.01 in the presence of TPCK-trypsin. Supernatants were collected at 12, 24, 48, and 72 h p.i. and titrated by plaque assay.

FIG. 5.

Role of MA HA G218W^HA1 and T156N^HA2 mutations in virulence and replication. (A) Viral growth in CD-1 mice. Groups of 12 CD-1 mice were infected intranasally with 5 × 10³ PFU of MA backbone viruses that possessed either or both HA mutations. Lungs were collected on days 1, 3, 5, and 7 p.i. and then homogenized, and virus titers were assessed by plaque assay. (B) Body weight loss in CD-1 mice. Groups of five CD-1 mice were intranasally infected with 10⁵ PFU of each of the recombinant viruses. Body weight loss was monitored for 14 dpi. (C, D, and E) Replication kinetics of the reassortants in vitro. Monolayers of MDCK, A549, or M1 cells were infected with each of recombinant viruses at an MOI of 0.01 in the presence of TPCK-trypsin. Supernatants were collected at 12, 24, 48, and 72 hpi, and virus titers were assessed by plaque assay. (F) Plaque phenotypes of r-HK and r-MA recombinant viruses that differed due to PB2 and individual HA mutations. Plaque assays were produced in MDCK cells under standard conditions and stained with crystal violet. (G) Average plaque diameter for each recombinant virus. The diameter of 10 random plaques was measured for each virus (**, P < 0.01; ***, P < 0.001). The horizontal dashed line represents the mean plaque diameter of HK-wt.

FIG. 6.

Polymerase activity of RNP complex genes of HK and MA in human, mouse, and chicken cell lines. Human 293T, mouse B82, and avian DF1 cells were transfected with the human (H) or mouse-adapted (M) polymerase subunits and NP, with the appropriate influenza virus-like minigenome carrying the firefly luciferase gene. Each luciferase activity value is the average of three independent experiments. (A) 293T cells. (B) B82 cells. (C) DF1 cells (*, P < 0.05). The MA-PA has no mutations relative to HK-wt PA (indicated by dashes). The horizontal dashed lines on the graphs represent the baseline polymerase activity of the HK-wt polymerase complex.

FIG. 7.

Assessment of the relative roles of PB2 E627K and D701N mutations in MA virus. (A) Viral growth in CD-1 mice. Groups of 12 CD-1 mice were infected intranasally with 5 × 10³ PFU of the indicated MA mutants. Lungs were collected at days 1, 3, 5, and 7 p.i. and assessed by plaque assay. (B) Body weight loss in CD-1 mice. Groups of five CD-1 mice were intranasally infected with 10⁵ PFU of each of the recombinant viruses. Body weight loss was monitored for 14 dpi. (C) Replication kinetics of the reassortants in vitro. Monolayers of MDCK, A549, or M1 cells were infected with each of recombinant viruses at an MOI of 0.01 in the presence of TPCK-trypsin. Supernatants were collected at 12, 24, 48, and 72 hpi, and virus titers were assessed by plaque assay. (D, E, and F). Polymerase activity of reconstituted RNP complexes with mutations of PB2 residues 627 and 701 in 293T, B82, and DF1 cell lines. Human 293T, mouse B82, and avian DF1 cells were transfected with human (H) or MA (M) polymerase subunits (note that the PA sequence is the same for H and M viruses), NP, and the appropriate influenza virus-like minigenome. Each luciferase activity value is the average of three independent experiments (*, P < 0.05; **P < 0.01). The horizontal dashed lines represent the baseline polymerase activity of the HK-wt polymerase complex.