Restored PB1-F2 in the 2009 pandemic H1N1 influenza virus has minimal effects in swine

Abstract

PB1-F2 is an 87- to 90-amino-acid-long protein expressed by certain influenza A viruses. Previous studies have shown that PB1-F2 contributes to virulence in the mouse model; however, its role in natural hosts-pigs, humans, or birds-remains largely unknown. Outbreaks of domestic pigs infected with the 2009 pandemic H1N1 influenza virus (pH1N1) have been detected worldwide. Unlike previous pandemic strains, pH1N1 viruses do not encode a functional PB1-F2 due to the presence of three stop codons resulting in premature truncation after codon 11. However, pH1N1s have the potential to acquire the full-length form of PB1-F2 through mutation or reassortment. In this study, we assessed whether restoring the full-length PB1-F2 open reading frame (ORF) in the pH1N1 background would have an effect on virus replication and virulence in pigs. Restoring the PB1-F2 ORF resulted in upregulation of viral polymerase activity at early time points in vitro and enhanced virus yields in porcine respiratory explants and in the lungs of infected pigs. There was an increase in the severity of pneumonia in pigs infected with isogenic virus expressing PB1-F2 compared to the wild-type (WT) pH1N1. The extent of microscopic pneumonia correlated with increased pulmonary levels of alpha interferon and interleukin-1β in pigs infected with pH1N1 encoding a functional PB1-F2 but only early in the infection. Together, our results indicate that PB1-F2 in the context of pH1N1 moderately modulates viral replication, lung histopathology, and local cytokine response in pigs.

Fig 1

PB1-F2 upregulates early polymerase activity. (A) Minigenome assay. 293T cells were transfected with plasmids encoding the minimal components required for viral transcription and replication (PB2, PB1, and PA polymerase subunits, NP, and a vRNA influenza virus-driven luciferase reporter replicon expressing GLuc) and with pCMV/SEAP, a plasmid to normalize transfection efficiency, as previously described (49). At the indicated hours posttransfection (hpt), the supernatant was harvested and assayed for both luciferase and phosphatase activities. (B and C) Kinetics of polymerase activity after virus infection. MDCK cells were transfected with 1 μg of GLuc reporter under the control of the canine Pol I reporter, followed by infection with Ca/04 (WT or KI) virus at an MOI of 0.01 (B) or 1 (C). Gluc activity was determined as described in Materials and Methods at the indicated hours postinfection (hpi). Data are expressed as polymerase activities (mean ± standard error [SE]) determined from two independent experiments performed in quadruplicate. A two-tailed Student t test was used to determine significant differences between two treatment means for each data point. An asterisk and dashed brackets indicate statistically significant differences (P < 0.05).

Fig 2

Replication of PB1-F2 recombinant viruses in porcine respiratory explants. Explants from nasal turbinates (A), trachea (B), and proximal (C) and distal (D) lung were infected with 10⁶ TCID₅₀ of either Ca/04 WT or KI. The bathing medium was collected at the indicated time points and titrated by the TCID₅₀ method in MDCK cells. Values shown are the mean and range of virus titers (log₁₀ TCID₅₀/ml) obtained from triplicate explant preparations. A two-tailed Student t test was used to determine significant differences between two treatment means for each data point. Dashed brackets indicate statistically significant differences (P < 0.05).

Fig 3

Replication and immunohistochemical analysis of PB1-F2 recombinant viruses in swine lungs. Groups of pigs (n = 10) were infected with 10⁵ TCID₅₀ of PB1-F2 recombinant viruses, and five animals from each group were euthanized either at 1 or 3 dpi. The lungs were collected and processed for virus titration and immunohistochemical analysis. (A) Pulmonary replication of PB1-F2 isogenic viruses in pigs. Values are means and ranges of virus titers (log₁₀ TCID₅₀/ml) in bronchoalveolar lavage fluid (BALF) collected at the indicated time points. Two-way ANOVA was used to determine significant differences between two treatment groups. Dashed brackets indicate statistically significant differences (P < 0.05). (B) Immunohistochemical staining against influenza A virus nucleoprotein (NP) in the lungs of infected pigs. Values given are the mean ± the standard error of the mean IHC scores based on the percentage of influenza virus-positive cells in the airway and lung interstitium. (C) Representative IHC slides depicting viral antigen primarily in airway epithelium at 1 and 3 dpi. Scattered labeling in the interstitium at 3 dpi is present.

Fig 4

Viral shedding and macroscopic pneumonia are unaltered by PB1-F2 expression in swine. Groups of pigs (n = 10) were infected with 10⁵ TCID₅₀ of PB1-F2 recombinant viruses, and nasal swabs were collected from 1 to 3 dpi for measuring virus shedding. At 1 and 3 dpi, five animals from each group were euthanized, and the lungs were scored for gross pneumonia. (A) Viral shedding in nasal secretions of pigs. Viral titers in nasal swabs were determined by TCID₅₀ on MDCK cells. Values are shown as the mean and range and expressed as log₁₀TCID₅₀/ml. (B) Percentage of macroscopic lung lesions. Values are shown as the mean ± the standard error of the mean. The differences are not statistically significant (two-way ANOVA).

Fig 5

PB1-F2 exacerbates microscopic pneumonia in swine. Groups of pigs (n = 10) were infected with 10⁵ TCID₅₀ of PB1-F2 recombinant viruses. At 1 and 3 dpi, five animals from each group were euthanized, and the histopathological changes in the lower respiratory tract were evaluated. (A) Histopathologic scores in the lungs. The differences are statistically significant (two-way ANOVA, P < 0.05). (B) Histopathologic scores of trachea. The differences are not statistically significant (two-way ANOVA, P > 0.05). (C) Photomicrographs representing microscopic pneumonia in Ca/04 KI and Ca/04 WT virus. Dashed brackets indicate statistically significant differences (P < 0.05).

Fig 6

PB1-F2 exacerbates the pulmonary levels of IFN-α and IL-1β in swine. Groups of pigs (n = 10) were infected intratracheally with 10⁵ TCID₅₀ of PB1-F2 recombinant viruses. On days 1 and 3 postinfection, animals from each group were euthanized, and the cytokine/chemokine levels in BALF were determined by ELISA. Data are shown as the mean ± the standard error of the mean for five animals in each challenge group. Two-way ANOVA was used to determine significant differences between groups. When the interaction between factors (time and virus) was significant, a two-tailed Student t test was used, considering time and virus, to determine significant differences between two treatment means for each data point. Dashed brackets indicate statistically significant differences (P < 0.05).