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. 2012 Oct;93(Pt 10):2204-2214.
doi: 10.1099/vir.0.045005-0. Epub 2012 Jul 18.

Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine

Lindomar Pena  1   2 , Amy L Vincent  3 , Crystal L Loving  3 , Jamie N Henningson  3 , Kelly M Lager  3 , Weizhong Li  1   2 , Daniel R Perez  1   2
Affiliations

Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine

Lindomar Pena et al. J Gen Virol. 2012 Oct.

Abstract

The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.

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Figures

Fig. 1.
Fig. 1.
Infectivity of PAMs with PB1-F2 viruses. Virus titres at different times p.i. from PAMs previously infected with virus at an m.o.i. of 2. (a) Sw/99 strains: Sw/99 WT (▪), Sw/99 KO (□) Sw/99 N66S (♦). (b) Ty/04 strains: Ty/04 WT (•), Ty/04 KO (○). One-way ANOVA followed by Dunnett’s post-hoc test was used to determine significant differences between the WT and PB1-F2 modified viruses (dashed brackets, P<0.05). Means±sd of five independent experiments are shown.
Fig. 2.
Fig. 2.
Viability of PAMs after infection with influenza viruses. (a) Sw/99 strains. (b) Ty/04 strains. PAMs were mock-infected or infected with PB1-F2 recombinant viruses at an m.o.i. of 10 and cell viability was measured by the XTT assay at 24, 48 and 72 h p.i. Each data point represents the mean±sd of three independent experiments. Different letters above data points indicate a statistically significant difference (P<0.05). (c) PAMs infected at an m.o.i. of 2 with the indicated viruses and assayed for apoptotic cell death using a TUNEL assay at 72 h p.i. Dashed brackets indicate a statistically significant difference (P<0.05).
Fig. 3.
Fig. 3.
Replication of PB1-F2 viruses in porcine respiratory explants. NTs (a, e), tracheal (b, f), proximal lung (c, g) and distal lung (d, h) explants were infected with 106 TCID50 of each virus and titrated by TCID50. Values shown correspond to virus titre [log10(TCID50 ml−1)] from each replicate. Data are presented as means±sd of three independent experiments. Dashed brackets indicate a statistically significant difference (P<0.05).
Fig. 4.
Fig. 4.
Replication and pathology induced by PB1-F2 recombinant viruses in swine. Pigs (n = 10 per group) were infected with 105 TCID50 virus and nasal swabs were collected from days 1 to 3 p.i. to measure virus shedding. Lungs were collected at 1 and 3 days p.i. (n = 5 day−1) for virus titration and pathological analysis. (a, b) Virus shedding in nasal secretions. (c, d) Pulmonary replication of PB1-F2 isogenic viruses in pigs (measured as virus titre in BALF). (e) Percentage of macroscopic lung lesions. (f) Lung histopathology scores. Data are presented as means±sd of five independent experiments. Dashed brackets indicate a statistically significant difference (P<0.05).
Fig. 5.
Fig. 5.
IHC of influenza viruses in swine lungs. (a) Anti-NP antigen IHC staining in lungs of infected pigs. Values are the mean±sd IHC scores based on the percentage of influenza-positive cells in the airway and lung interstitium, from five independent experiments. (b) Representative IHC slides depicting viral antigen primarily in airway epithelium at 1 and 3 days p.i.
Fig. 6.
Fig. 6.
PB1-F2 modulates the innate immune response in swine lungs. Data represent cytokine/chemokine levels (by ELISA) in BALF from each pig in the respective groups (▪, Sw/99 WT; □, Sw/99 KO; ⧫, Sw/99 N66S; •, Ty/04 WT; ○, Ty/04 KO). (a) IFN-α; (b) IL-1β; (c) IL-6; (d) IL-8; (e) IFN-γ. Data are presented as means±sd of five independent experiments. Dashed brackets indicate a statistically significant difference (P<0.05).
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