The PB2-E627K mutation attenuates viruses containing the 2009 H1N1 influenza pandemic polymerase

Abstract

The swine-origin H1N1 influenza A virus emerged in early 2009 and caused the first influenza pandemic in 41 years. The virus has spread efficiently to both the Northern and the Southern Hemispheres and has been associated with over 16,000 deaths. Given the virus's recent zoonotic origin, there is concern that the virus could acquire signature mutations associated with the enhanced pathogenicity of previous pandemic viruses or H5N1 viruses with pandemic potential. We tested the hypothesis that mutations in the polymerase PB2 gene at residues 627 and 701 would enhance virulence but found that influenza viruses containing these mutations in the context of the pandemic virus polymerase complex are attenuated in cell culture and mice.

FIG 1

Viral replication kinetics in A549 cells at 37°C. Cells were infected at a 0.01 multiplicity of infection. Supernatants from infected A549 cells were collected at 12-h intervals, and viral titer was determined by a plaque assay. Cultures and measurements were performed in triplicate. Titers are expressed as numbers of PFU per milliliter. Error bars represent standard errors of the means (SEM). See the key for the different viruses evaluated.

FIG 2

Clinical course of infection in mice as measured by weight loss. Mean percentage body weight loss from mean baseline weight of mice in each group inoculated with rescued A/New York/312/2001 (H1N1) (referred to as rNY312) or chimeric viruses containing the four RNP gene segments of different influenza viruses on the NY312 background (see key) from 0 to 14 days postinoculation (dpi). Groups of five 8- to 10-week-old female BALB/c mice were inoculated intranasally with 2 × 10⁵ PFU of virus. Error bars represent SEM.

FIG 3

Viral titer in mouse lung tissues. Mean viral titer in mouse lung on days 3 and 5 postinfection was determined by a plaque assay. Data were collected from three mice in each group. Titers are expressed as numbers of PFU per gram of lung tissue. Error bars represent SEM. See the key for the different viruses evaluated.

FIG 4

Pathology and immunohistochemistry of influenza virus-infected mouse lung tissue. Photomicrographs of hematoxylin-and-eosin-stained tissue sections and immunohistochemically stained sections for detection of influenza viral antigen from mice infected with different influenza virus constructs at day 5 postinfection. Viral antigen is stained reddish brown on a hematoxylin-stained background. Arrows and arrowheads show examples of positive cells. (A, B) Sections from an animal infected with the rNY312 virus showing no pathological changes in the lung and no viral antigen (original magnifications, ×100). (C, D) Sections from an animal infected with the 1918RNP virus showing marked necrotizing bronchiolitis with a marked transmural inflammatory cell infiltrate. Prominent viral antigen staining was observed in bronchiolar epithelial cells (arrow) and in necrotic intraluminal debris (arrowhead) (original magnifications, ×200). (E, F) Sections from an animal infected with the CA09RNP virus showing focal bronchiolitis with a minimal inflammatory cell infiltrate. Viral antigen staining was observed in bronchiolar epithelial cells (arrow) and in necrotic intraluminal debris (arrowhead) (original magnifications, ×100). (G, H) Sections from an animal infected with the CA09RNP-E627K virus showing no pathological changes in the lung and no viral antigen (original magnifications, ×100).

FIG 5

Pathology and immunohistochemistry of influenza virus-infected mouse lung tissue. Photomicrographs of hematoxylin-and-eosin-stained tissue sections and immunohistochemically stained sections for detection of influenza viral antigen from mice infected with different influenza virus constructs at day 5 postinfection (except for the VN1203RNP virus, which is shown at day 3 postinfection). Viral antigen is stained reddish brown on a hematoxylin-stained background. Arrows show examples of positive cells. (A, B) Sections from an animal infected with the S09RNP virus showing a focus of moderate alveolitis. Focal viral antigen is seen in bronchiolar epithelial cells (arrow) and in alveolar macrophages (original magnifications, ×100). (C, D) Sections from an animal infected with the S09RNP-E627K virus showing necrotizing bronchiolitis with a transmural inflammatory cell infiltrate. Viral antigen staining was observed in bronchiolar epithelial cells (arrow) and in alveolar macrophages (original magnifications, ×100). (E, F) Sections from an animal infected with the VN1203RNP virus at day 3 postinfection showing marked necrotizing bronchiolitis with a marked transmural inflammatory cell infiltrate. Prominent viral antigen staining was observed in bronchiolar epithelial cells (arrow) (original magnifications, ×100). Similar histopathological changes were observed at day 5, but only limited viral antigen staining was observed (data not shown). (G, H) Sections from an animal infected with the 1918RNP-K627E virus showing no pathological changes in the lung but some viral antigen staining in bronchiolar epithelial cells (arrow) in the absence of inflammation (original magnifications, ×100). (I, J) Sections from an animal infected with the CA09RNP-D701N virus showing no pathological changes in the lung and no viral antigen (original magnification, ×100).