The role of interferon in influenza virus tissue tropism

Abstract

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1-/- animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1-/- mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT-/- mice, and mice lacking gamma interferon (IFNgamma-/- mice) or the IFN-alpha receptor (IFNalphaR-/- mice). Influenza A/WSN/33 virus replicates to high titers in STAT1-/- or IFNalphaR-/- fibroblasts, while cells derived from WT or IFNgamma-/- animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.

FIG. 1

Determination of lung virus titers over a 9-day period for WT (■) and STAT1−/− (▵) CD1 mice inoculated i.n. with WSN and PR8 viruses. (A) Animals of each genotype were inoculated i.n. with 1,000 PFU (1 LD₅₀ for a WT animal) of WSN virus in 50 μl of PBS. Four to five WT and STAT1−/− animals were sacrificed at each time point. Results from two experiments are included. Viral titers in lung homogenates from each animal (PFU/gram of tissue) were determined by plaque assay in MDCK cells. (B) Fifteen animals of each genotype were inoculated i.n. with 10 TCID (1 LD₅₀ for a WT animal) of PR8 virus in 100 μl of PBS. Five WT and five STAT1−/− animals were sacrificed at days 3, 6, and 9 p.i. Viral titers in lung homogenates from each animal were determined as TCID/milliliter.

FIG. 2

Virus titers following i.p. WSN virus inoculation of STAT1−/− mice. Four STAT1−/− and three WT mice were injected i.p. with 10⁷ PFU of WSN virus. Animals were sacrificed at day 4 postinoculation when the mutant animals showed signs of illness. Viral titers of tissue homogenates were determined by plaque assay in MDCK cells and are expressed as PFU/gram of tissue. Virus titers for each tissue are shown for each of the STAT1−/− animals. No virus could be detected in samples obtained from three WT animals.

FIG. 3

Pathology of hepatic and central nervous system lesions of STAT1−/− mice following i.p. injection with WSN virus. (A) Hematoxylin-eosin-stained section of liver showing multiple aggregates of inflammatory cells within the hepatic parenchyma. Magnification, ×98. (B [magnification, ×197] and C [magnification, ×394]) Sections reacted with polyclonal antibody against influenza A virus. Infected cells are bright red. Only a fraction of inflammatory cells constituting the liver lesions show positive staining for influenza virus antigens. Multiple hepatocytes stain strongly, some showing only nuclear staining, indicating an early phase of virus replication. In other hepatocytes, viral proteins are also present in the cytoplasm. (D) Section of brain with a focus staining positively for the presence of viral antigen (magnification, ×98). In panel E (magnification, ×394), it can be appreciated that many of the red-staining cells are neurons. Glial cells within the focus also stain positively. (F) Single infected ependymal cell (magnification, ×394).

FIG. 4

Comparison of tissue virus titers in WT (A), STAT1−/− (B), and IFNαR−/− (C) mice at different days after i.n. inoculation with WSN virus. (A and B) Eight to 10 WT (A) or STAT1−/− (B) mice were infected i.n. with 1,000 PFU of WSN virus, and viral titers in the indicated tissues at days 3 (■) and 6 (□) p.i. were determined by plaque assay in MDCK cells. Results from two experiments are expressed as PFU/gram of tissue. (C) Six IFNαR−/− mice were infected i.n. with 1,000 PFU of WSN virus, and viral titers at days 4 (■) and 8 (□) p.i. were determined by plaque assay in MDCK cells. Results are expressed as PFU/gram of tissue.

FIG. 5

Viral RNA and protein expression levels in MEFs derived from WT (STAT+/+) and STAT−/− CD1 mice. (A) STAT+/+ and STAT−/− MEFs were infected with WSN virus at an MOI of 5, and vRNA levels specific for the NA and NS genes were determined by PAGE analysis of primer extension products at different times p.i. (B) STAT+/+ and STAT−/− MEFs were infected with WSN virus at an MOI of 2 and ³⁵S labeled at the indicated time points, and total amount of viral proteins was immunoprecipitated with a polyclonal antiserum against WSN virus. Immunoprecipitated products were analyzed by SDS-PAGE.

FIG. 6

Immunofluorescence analysis of NP expression in WT and STAT1−/− MEFs infected with WSN virus. Cells were infected with WSN virus (MOI = 2) and stained with a monoclonal antibody against NP 14 h p.i. (A and D) Different-magnification fields of STAT1−/− cells. The majority of the cells showed a cytoplasmic NP staining, indicative of a late phase of virus replication. (B and C) Low magnification of two different fields of WT-infected cells. Although cell densities were roughly similar between the WT and STAT1−/− samples, only a few WT cells showed positive NP staining. (E and F) Higher magnification of individual positive-stained WT cells. Note that the majority of the NP staining is nuclear, which indicates a delayed or abortive viral replication.