Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs

Abstract

It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-alpha/beta) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-alpha/beta synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-alpha/beta induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.

FIG. 1.

Generation of plasmid-derived Sw/Tx/98 influenza viruses with mutated NS1 proteins. (A) Schematic diagram of the wild-type and mutated NS influenza virus gene segments. The NS gene segment was modified to create mutated NS1 genes encoding 73, 99, and 126 aa, respectively. NS1 mutations did not affect the sequence of the NEP. Underlined sequences were introduced to generate stop codons (in bold). (B) RT-PCR analysis of the rWT and NS1 deletion mutant viruses. Influenza viral RNA segments were amplified using primers specific for the noncoding regions of all eight influenza virus segments. Arrows indicate the decreasing size of the NS segment. Dots indicate a product corresponding to the 3′ end of the HA gene segment due to internal binding of the forward primer. Size markers (in nucleotides) are indicated on the left.

FIG. 2.

Characterization of plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses in tissue culture. (A) Multicycle growth curves in PK-15 cells. (B) Single-cycle growth curves in PK-15 cells. (C) Plaque size in MDCK cells at 3 days p.i.

FIG. 3.

Induction of IFN-α/β in PK-15 cells infected with plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses. (A) Schematic representation of the IFN bioassay. (B) Time course of IFN-α/β synthesis. Fresh PK-15 cells were treated for 24 h with UV-inactivated supernatants (harvested at different times p.i. as indicated) from PK-15 cells which were infected with the indicated viruses, followed by VSV-GFP infection. Sixteen hours p.i., cells expressing GFP were visualized by fluorescence microscopy. (C) RT-PCR analysis of TNF-α-, IFN-β-, and β-actin-specific mRNAs in virus-infected PK-15 cells.

FIG. 4.

Levels of rWT and mutant NS1 TX/98 proteins determined by metabolic labeling and Western blotting. (A) Metabolic labeling with [³⁵S]Met-Cys of PK-15 cells infected with the rWT and deleted mutants (MOI = 2), from 8 to 10 h p.i. (B) Immunoblot with antibodies specific for NS1 and NP in PK-15 cells infected with rWT and the different mutants at 10 h p.i.

FIG. 5.

Infection of 4-week-old pigs with plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses on day 5 p.i. (A) Mean percentage (± standard error of the mean [SEM]) of lung surface with macroscopic lesions. (B) Microscopic lesions (± SEM) in the medium-sized bronchioles. (C) Virus titers (± SEM) in BALF.

FIG. 6.

Histopathological examination of lungs from pigs infected with plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses. Magnification, ×200. (Mock) Medium-sized bronchiole from the lung of a mock-infected control pig. (rWT) Severe acute necrotizing bronchiolitis and interstitial pneumonia characteristic of the widespread lesion induced by infection with rWT TX/98 virus. (1-99) One normal bronchiole and one affected bronchiole in early recovery from infection with deletion mutant 1-99 virus. (1-126) Normal bronchiole and surrounding alveoli from the lung of a pig inoculated with the 1-126 virus. Bar, 50 μm.

FIG. 7.

Histopathological examination of lungs from pigs infected with plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses. Magnification, ×500. (Mock) Epithelial lining of a larger bronchiole from the lung of a mock-infected control pig. (rWT) Epithelial lining from a larger bronchiole from the lung of a pig infected with TX/98 rWT virus. (1-99) Large bronchiole and smaller branching airway from the lung of a pig infected with deletion mutant 1-99 virus. (1-126) Normal epithelial lining from the lung of a pig inoculated with 1-126 virus. Bar, 200 μm.