
==== Front
Virus Res
Virus Res
Virus Research
0168-1702
1872-7492
Elsevier

S0168-1702(24)00137-0
10.1016/j.virusres.2024.199444
199444
Article
PB1-F2 of low pathogenicity H7N7 restricts apoptosis in avian cells
Hohensee Luise a1
Scheibner David bc
Luttermann Christine a
Shelton Holly d
Dorhoi Anca a
Abdelwhab Elsayed M. b
Blohm Ulrike Ulrike.blohm@fli.de
a⁎
a Institute of Immunology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, Greifswald, Insel Riems 17493, Germany
b Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, Greifswald, Insel Riems 17493, Germany
c Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, Greifswald, Insel Riems 17493, Germany
d The Pirbright Institute, Pirbright, Ash Road, Surrey GU24 0NF, United Kingdom
⁎ Corresponding author. Ulrike.blohm@fli.de
1 Present address: Infection Pathogenesis, TUM School of Life Sciences, Technische Universität München, Freising 85354, Germany.

23 8 2024
11 2024
23 8 2024
349 19944415 4 2024
26 7 2024
29 7 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• In chicken full-length PB1-F2 of LPAIV H7N7 prolonged survival of infected cells by limiting apoptotic cell death.

• PB1-F2 knockout LPAIV significantly decreased MHC-I expression on fibroblasts, delayed tissue healing and increased phagocytic uptake of infected cells.

• Taken together, the full-length PB1-F2 allows AIV to cause prolonged infections without serious harm to the avian host.

Avian influenza viruses (AIV) pose a continuous challenge to global health and economy. While countermeasures exist to control outbreaks in poultry, the persistent circulation of AIV in wild aquatic and shorebirds presents a significant challenge to effective disease prevention efforts. PB1-F2 is a non-structural protein expressed from a second open reading frame (+1) of the polymerase basic 1 (PB1) segment. The sequence and length of the PB1-F2 protein can vary depending on the host of origin. While avian isolates typically carry full-length PB1-F2, isolates from mammals, often express truncated forms. The selective advantage of the full-length PB1-F2 in avian isolates is not fully understood. Most research on the role of PB1-F2 in influenza virus replication has been conducted in mammalian systems, where PB1-F2 interfered with the host immune response and induced apoptosis. Here, we used Low Pathogenicity (LP) AIV H7N7 expressing full-length PB1-F2 as well as a knockout mutant. We found that the full-length PB1-F2 of LPAIV prolonged survival of infected cells by limiting apoptotic cell death. Furthermore, PB1-F2 knockout LPAIV significantly decreased MHC-I expression on fibroblasts, delayed tissue healing and increased phagocytic uptake of infected cells, whereas LPAIV expressing PB1-F2 has limited effects. These findings indicate that full-length PB1-F2 enables AIV to cause prolonged infections without severely harming the avian host. Our observations may explain maintenance of AIV in the natural bird reservoir in absence of severe clinical signs.

Graphical abstract

Image, graphical abstract

Keywords

Influenza
PB1-F2
Chicken
Avian
Apoptosis
Interferon
Phagocytosis
In vitro
==== Body
pmc1 Introduction

Avian influenza viruses (AIV) are pathogens of global importance (WOAH, 2023). They belong to the genus Alphainfluenzavirus, which, together with Beta-, Gamma- and Deltainfluenzaviruses make up the family of Orthomyxoviriridae (Krammer et al., 2018). Genetic variation of the surface glycoproteins is used to differentiate AIV into 16 hemagglutinin (HA or H) and 9 neuraminidase (NA or N) subtypes (Fouchier et al., 2005). Furthermore, AIV can be distinguished by pathotype into low pathogenicity (LP) and high pathogenicity (HP) AIV. HPAIV are mostly restricted to H5/H7 viruses (de Bruin et al., 2022).

AIVs are potentially zoonotic viruses. They pose a health threat to poultry and humans (Bosman et al., 2005). Outbreaks of avian influenza (AI) impose a significant economic burden on the poultry industry (Pasick et al., 2009; Villarreal, 2009). Therefore, numerous countries apply various containment and control strategies (Knobler et al., 2005; Capua and Marangon, 2006; Liu et al., 2020; Sagong et al., 2023). AIV is endemic in wild aquatic and shorebird populations (Webster et al., 1992). Despite successful eradication of the virus from affected poultry flocks there is a continuous risk of reintroduction from the natural reservoir. Therefore, for efficacious control of the disease outbreaks it is essential to understand pathophysiology of AIV, including its propensity to replicate in certain avian hosts, while causing few, if any, symptoms.

A key player in maintaining the virus-host equilibrium may be PB1-F2, a non-structural protein derived from a second open reading frame (ORF) of the PB1 polymerase segment (Krumbholz et al., 2011). PB1-F2 is only present in influenza A viruses (IAV). Influenza B viruses, which have a more limited host spectrum than IAV (van de Sandt et al., 2015), do not express PB1-F2 (Chen et al., 2001). Full-length PB1-F2 consists of at least 87 amino acids. However, mutations can eliminate or truncate the protein either by altering the start codon (Chen et al., 2001) or by introducing premature stop codons (Kamal et al., 2017). Extended forms (up to 106 aa) are rarely reported (DeLuca et al., 2011). PB1-F2 expression varies depending on the host of origin. While 93 % of examined avian IAV carry full-length PB1-F2, less than 50 % of mammalian IAV express the full-length protein (James et al., 2016). In mammals, PB1-F2 is thought to be under negative selection pressure. The newly emerging influenza viruses carry full-length PB1-F2, but in the transition from pandemic/epidemic virus to seasonal influenza the protein is often truncated or deleted (McAuley et al., 2010a). In mammalian cells, PB1-F2 has proven pro-apoptotic effects (Chang et al., 2015; Pasricha et al., 2018; Zamarin et al., 2005), mainly targeting immune cells (Chen et al., 2001). It has also been suggested that the protein can influence the success of a viral infection, by increasing the viral polymerase activity (Mazur et al., 2008) and by interfering with the host immune response e.g. by affecting type I IFN signalling (Dudek et al., 2011; Le Goffic et al., 2010). PB1-F2 is also associated with increased virulence of influenza viruses in mammals (Zamarin et al., 2006), which was attributed to development of immunopathology (McAuley et al., 2010a) as well as secondary bacterial infections (McAuley et al., 2007).

Despite its high prevalence in AIV, information about the impact of PB1-F2 on virus fitness in avian hosts is sparse and inconsistent. Considering that effects of PB1-F2 are highly strain- and host-specific, it remains questionable whether knowledge gained from mammalian isolates in mammalian cell culture can be extrapolated to birds. Here, we used LPAIV H7N7 expressing full-length PB1-F2 (hereafter referred to as wt) and a knockout mutant (hereafter referred to as ΔF2) to investigate the properties of PB1-F2 in avian cell culture. Determining its effects in vitro paves ways for further characterization of the role of PB1-F2 in maintaining AIV in the natural reservoir.

2 Materials and methods

2.1 Cells and eggs

We conducted our experiments in MDCK-II (Madin-Darby canine kidney type II cells), HEK293T (human embryo kidney 293T), DF-1 (chicken fibroblasts), HD11 (chicken monocytes/macrophages) and CEK (primary chicken embryo kidney) cells. The cell lines (MDCK-II, HEK293T, DF-1, HD11) were obtained from the Collection of Cell Lines in Veterinary Medicine (CCLV) at the Friedrich-Loeffler-Institut (FLI). The primary CEK cells were prepared as previously described (Choi et al., 2007). Briefly, chicken embryos from 18-to-19 day-old specific-pathogen-free (SPF) eggs were humanely killed, kidneys were harvested, minced, digested and strained, producing a cell suspension. Confluent cells were detached with trypsin and then resuspended in their respective media. For MDCK-II cells media containing 5.32 g MEM (Hank's salts), 4.76 g MEM (Earle's salts), 1.25 g NaHCO3 and 10 mL non-essential amino acids (1x) per litre (pH 7.2) was used. HEK293T, DF-1 and HD11 were cultured in Ham's F12 and IMDM 1:1. For all cell lines 10 % fetal bovine serum (FBS) (PAN biotech) and 1 % penicillin/streptomycin (P/S, 100X, Cytiva HyClone™) were added to the media. Media for HD11 cells was supplemented in addition with 2 % chicken serum (Sigma-Aldrich). CEK cells were seeded in MEM (Earle's salts) containing 10 % FBS, 1 % P/S and 0,1 % Amphotericin B (Biowest), in plates pre-treated with gelatine (Carl Roth). All cells were incubated at 37 °C and 5 % CO2.

SPF chicken eggs, that were used for virus propagation, harvesting of allantoic fluid (used as a trypsine alternative during LPAIV infections) and in ovo studies, were purchased from Valo BioMedia (Osterholz-Scharmbeck, Germany) and incubated on site.

2.2 Viruses

Recombinant LPAIV H7N7 A/chicken/Germany/AR915/2015 H7N7 (LPAIV H7N7 AR915/15) was used for all experiments. The field virus was isolated from commercially farmed laying-hens in the Emsland, Germany in 2015 as described by Dietze et al. (2018). The recombinant LP H7N7 virus expressing full-length PB1-F2 (wt) as well as the respective plasmids were previously described (Scheibner et al., 2019). The knockout virus (ΔF2) was created by introducing a point mutation (C138A) to PB1 using the QuikChange Site-directed mutagenesis kit (Invitrogen). The mutation has no effect on the amino acid (aa) sequence in the PB1 reading frame, but introduced a stop codon and thereby truncated PB1-F2 at a length of 12 aa, as previously done by James et al. (2016).

The recombinant viruses were rescued and propagated as previously reported (Hohensee et al., 2024). Embryonated chicken eggs were inoculated with supernatant, which was previously harvested from a transfected co-culture of HEK 293T and MDCK-II. The allantoic fluid was collected and checked for bacterial contamination. Sequences were checked by Sanger sequencing and analysed with Geneious.

A/turkey/Germany/AR534/2013 (H7N7), which was described by Abdelwhab et al. (2016), was utilized as a positive control in the polymerase assay.

2.3 Viral replication kinetics

Cells (DF-1, CEK, MDCK-II, HD11) were inoculated with virus suspension at the indicated MOI. After 1 h incubation at 37 °C, 5 % CO2 the supernatant was removed. Citric acid buffer (CBS, pH=3.0) was used to inactivate all non-intracellular virus particles. Subsequently cells were washed thrice with PBS. Since both DF-1 and HD11 cells detached after CBS treatment, the inactivation step was skipped and a thorough washing with PBS was performed. Afterwards media for infectious purposes containing 2 µg/ml TPCK trypsin was added (MEM (Hank's salts), MEM (Earle's salts), NEA and 0.56 % Bovine serum albumin (BSA) solution (35 %)). The cells were incubated at 37 °C, 5 % CO2. At 1, 8, 24 and 48 hpi cells and supernatant were collected using a cell scraper and immediately frozen at −70 °C for later titration.

2.4 Virus titration

Virus titration was performed using the FOCI-assay (Ma et al., 2010). Briefly, confluent MDCK-II cells were inoculated with 50 µl of ten-fold virus dilutions. At 1hpi the supernatant was discarded and Avicell medium (50 % Avicell stock (2,5 %), 33 % H2O, 10 % 10xMEM, 4 % NaHCO3 (7.5 %, pH = 9.0 −9.3), 1 % BSA, 1 % DEAE Dextran, 1 % P/S) with 2 µg/ml TPCK trypsin was added. After 20 to 24 h of incubation (37 °C, 5 % CO2) the supernatant was carefully removed by washing with PBS++ (PBS containing 1 mM MgCl2, 0.9 mM CaCl2) at least thrice. Subsequently the cells were fixated for 1 h with PBS++ containing 4 % PFA and 1 % 100x Triton. Focus forming units (FFU) were visualized by staining 1 h each with monoclonal mouse-anti NP antibody (HB65), goat-anti mouse horseradish peroxidase (HRP) antibody and AEC-staining solution. AEC-staining solution contains 95 % Acetate buffer (pH= 5), 4.8 % 20x AEC substrate (AEC in dimethyl formamide) and 0.2 % hydrogen peroxide (Roth, 30 %). Between each staining the plates were washed thrice with washing solution (10 % PBS++ and 0.05 % Tween 20). After washing and drying, the plates were counted microscopically and titres were calculated: Focus forming units (FFU)/ml = FFU x 20 x dilution factor (10x).

2.5 Plaque size assay

To determine cell-to-cell spread confluent MDCK-II were infected in tenfold dilutions. Briefly, cells were infected for 1 h at 37 °C, washed with PBS and plaque test media (50 % BactoAgar, 50 % media with MEM (H), MEM (E), NEA and BSA) was added. The cell plates were incubated for 4 days at 37 °C, 5 % CO2. Then the cells were fixed with crystal violet in formalin (0.1 %) for 24 h. After removing staining solution, the dried plates were analysed using a Nikon eclipse Ti microscope. The diameter of 275 and 338 plaques induced by wt and ΔF2, respectively were measured using the NIS Element BR software's 3-point measurement option.

2.6 Polymerase assay

We investigated the impact of PB1-F2 expression on the polymerase activity in a transfection/ infection system. Semiconfluent DF-1 cells were transfected with Lipofectamine™ 2000 (ThermoFisher) containing pCAGGS plasmid allowing expression of Nanoluciferase (NanoLuc) under the control of the AIV promoter. Firefly-Luciferase expressing pCAGGS plasmid was used as a transfection control. Four hours after transfection the cells were carefully washed and infected with the respective viruses or A/turkey/Germany/AR534/2013 (H7N7) as a positive control at MOI of 1. Minimal essential medium containing 10 % allantoic fluid, harvested from specific-pathogen-free embryonated chicken eggs, was used. Cells were harvested 24hpi using Reporter Lysis 5X Buffer (Promega), and diluted according to the manufacturer's instructions. After one freeze-thaw cycle firefly and NanoLuc reagents were added to the samples and luminescence was detected using a GloMax® Discover Microplate Reader. The NanoLuc expression was normalized to Firefly expression.

2.7 Flow cytometrical analysis of DF-1 cells

To characterize the phenotype of infected DF-1 cells, we infected confluent DF-1 cells at MOI 0.01. 24hpi the cells were detached, washed and resuspended in PBS. Staining against surface antigens was performed in various 20 min intervals. Between each step cells were washed with cell staining buffer (PBS with 0.1 % FBS, 0.4 % Natrium azide). For intracellular staining, cells were first permeabilized and fixated with intracellular staining buffer (Biolegend) according to manufacturer's instructions. Antibodies against surface antigens were diluted in cell staining buffer, those against intracellular antigens in PermWash (Biolegend). Measurements and analyses were carried out using the BD LSRFortessa™ Cell analyzer and the corresponding BD FACSDIVA™ software.

2.8 Antibodies

2.8.1 Primary antibodies

Antigen	Clone	Conjugate	Manufacturer	Dilution	
AIV NP	HB65		ATCC (2009, KatNr. HB-65)	1:200	
Aqua Zombie		V500	Biolegend	1:500	
Chicken MHC-I	F21–2	PE	Southern biotech	1:50	
Mouse aCas3	C92–605	PE	BD	7,5 µl/test	

2.8.2 Secondary antibodies

Mouse IgG2α	PerCP	dianova	1:100	

2.9 Cell viability

The assay was performed using a ready-to use kit (Cell Proliferation Reagent WST-1, CELLPRO-RO, Roche) according to the manufacturer's guidelines. The absorbance was measured using a TECAN plate reader (infinite M200 PRO).

2.10 IFN-induction inhibition

Semiconfluent DF-1 cells were subjected to Lipofectamine™ 2000 (ThermoFisher) based transfection of plasmids containing Firefly-Luciferase gene under control of chicken IFN-ß (IFN 2) promotor, and Renilla-Luciferase gene under control of a CMV promoter. The latter was added as a transfection control. 3 h post transfection the cells were carefully washed and infected at MOI 0.1 with the respective viruses. After incubation (1 h), cells were carefully washed and infection media with 10 % allantoic fluid instead of TPCK-treated trypsin was added, to limit cell damage. Since, the infection efficiency is lower in media with 10 % allantoic fluid than with trypsin, the MOI was adjusted to MOI 0.1, this corresponds with viral tires of 6.7 × 104 (wt) and 2.2 × 105 FFU/mL (ΔF2) at the time of cell harvest. 24 h post transfection cells were carefully washed and lyzed with PLBuffer according to the manufacture´s information (Dual Luciferase Kit, Promega). After one freeze-thaw cycle firefly and Renilla luciferase substrate was added to the samples and luminescence was detected with a Berthold Tristar2 reader. The Firefly luciferase expression was normalized against Renilla luciferase expression.

2.11 Phagocytosis assays

To model phagocytic elimination of infected cells, we first labelled naïve DF-1 cells with Tag-it violet (Biolegend) and naïve HD11 cells with carboxyfluorescein succinimidyl ester (CSFE, ebioscience) according to the manufacturer's instruction. The pre-stained fibroblasts were then infected at MOI 0.01 with the respective virus and harvested 24hpi. Subsequently, DF-1 were added to the pre-stained monocytes. After mixing carefully, the cultures were incubated at 37 °C for 1hpi, then cells were harvested on ice, inactivated with Fixation buffer (Biolegend) and analysed by flow cytometry.

To assess the phagocytic rate of infected phagocytes, we utilized fluorescein conjugated, Escherichia coli (K-12 strain) BioParticles™ (Invitrogen™, E2861, #12,090,126). Infected (MOI: 0.01) HD11 cells (chicken monocytes) were collected at 8hpi. E. coli bioparticles were added to the cell suspension. After incubating for 10 min at 37 °C and 350 rpm samples were stored on ice to stop the phagocytic uptake of the particles. Subsequently, cells were fixated with intracellular staining buffer (Biolegend) and processed by flow cytometry.

2.12 Scratch assay

To assess tissue healing we employed the scratch assay (Liang et al., 2007). Cells were incubated in media with 10 % allantoic fluid. A confluent layer of previously inoculated (MOI: 0.1) DF-1 cells was damaged by scratching with sterile pipette tips. The lesion size was measured at infliction and subsequently after incubating the cells for 16 h (S4). The difference of initial scratch width and width 17hpi was calculated.

2.13 Software & statistics

Flow cytometry data was processed using BD FACSDIVA™.

Statistical analyses were performed using GraphPad Prism. Data was first tested for normality (Shapiro-Wilk test and Kolmogorov-Smirnov-test). Based on this, appropriate statistical tests were selected: For polymerase activity and WST Assay Kruskal-Wallis test was used. Replication kinetics and DF-1 dose-finding were analysed by two-way ANOVA. Cell-to-cell-spread was evaluated using the Mann Whitney test. NP MFI and DF-1 living cells were analysed with unpaired T-test. All remaining data was analysed by one-way ANOVA. Significant differences were depicted according to the P-value as non-significant (ns) (P > 0.05), * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001), **** (P ≤ 0.0001).

3 Results

3.1 The knockout of PB1-F2 did not affect polymerase activity

We generated two recombinant viruses based on LPAIV H7N7 AR915/15. We aimed to knockout PB1-F2, without disrupting the PB1 open reading frame (Fig. 1A, S1) as previously reported (Hohensee et al., 2024; James et al., 2016). Sequencing of stock viruses revealed no unwanted mutations. We assessed the polymerase activity using a minigenome reporter assay in avian cells (chicken fibroblasts (DF-1) cell line) (Fig. 1B). We found no significant difference in the polymerase activity between wt and ΔF2 infected DF-1 cells. Furthermore, wt and ΔF2 could be propagated to similar titres in embryonated chicken eggs (not shown).Fig. 1 In vitro characterization of wt and ΔF2. A. A mutation (C138A) introduced a stop codon at aa position 12 of the secondary ORF (PB1-F2), while the polymerase frame stayed intact. B. DF-1 cells were transfected with plasmids containing H7 Nanoluciferase and Firefly (as transcription control), subsequently the cells were infected at multiplicity of infection (MOI) of 1. Luminescence of cells, lysed 24hpi, was analysed. Data is depicted as mean (SD), control (white), wt (blue), ΔF2 (red), statistical analysis was done as a Kruskal-Wallis test with Dunn's multiple comparisons test. C. Viral replication kinetics were performed in cell lines (MDCK-II, DF-1) and primary cell culture (CEK) at a MOI of 0.001. Cells and supernatant were collected and titrated by FOCI-assay. Results are shown as mean (SD), wt (blue), ΔF2 (red). Statistical analysis was calculated as two-way ANOVA with Šídák's multiple comparisons test. D. Cell-to-cell spread was investigated by measuring the diameter of at least 250 plaques. Statistical analysis was performed as a Mann-Whitney test.

Fig 1

3.2 PB1-F2 had limited effects on virus replication and spread

To assess the impact of PB1-F2 on viral fitness we performed viral replication kinetics in avian (DF-1 and CEK) and mammalian cell culture (MDCK-II) (Fig. 1C) and measured the cell-to-cell spread in MDCK-II cells (Fig. 1D). The replication curves of both viruses are nearly congruent in avian cells. In MDCK-II cells, however, expression of PB1-F2 gave a non-significant replication advantage at 24 hpi. However, since this benefit is only temporary, its biological relevance is questionable. Cell-to-cell spread was not affected by PB1-F2 expression (Fig. 1D).

3.3 Knockout of PB1-F2 decreased viability and ameliorated infection rates of DF-1 cells

We next evaluated infection rate and viability of avian cells using multiparametric flow cytometry. In initial experiments we determined the infection dose for subsequent experiments (Fig. S2). At 24 hpi cells were harvested and subjected to live-dead staining as well as staining for infected cells (IAV nucleoprotein (NP) +). While a MOI 0.001 is usually employed to ascertain viral replication kinetics, this MOI was not satisfactory for flow cytometrical analyses. We selected MOI 0.01, because this infectious dose resulted in homogenous infection of a large population of cells, while maintaining acceptable viability.

Surprisingly, although PB1-F2 did not influence viral replication in DF-1 cells at MOI 0.01 and 0.001 at 24 hpi (Fig. S2C, 1C), we found strong differences in the survival rates of infected cells. While this difference was apparent under all conditions tested (Fig. S2A), it was highly significant at the selected infection dose (Fig. 2A). In contrast to reports from mammalian cells (Chang et al., 2015; Zamarin et al., 2005), PB1-F2 was beneficial for cell survival. Infection with the wt virus slightly reduced cell survival compared to the naïve control, whereas ΔF2 decreased survival 0.6-fold. We confirmed these results using the WST assay (Fig. 2B), a viability assay, which evaluates the activity of mitochondrial dehydrogenases. Both viruses caused a reduction in cell viability, yet this was much more pronounced in ΔF2 infected cells (wt: P = 0.0107 vs. ΔF2 P > 0.0001). We observed also significantly higher infection rates (NP+ cells) in ΔF2 compared to wt infected cells (Fig. 2C) following an early replication advantage of ΔF2 infected DF-1 cells that plateaued at 24 and 48hpi (MOI 0.01; Fig. S2C). Not only did the knockout virus infect on average 33 % more cells than wt, but the expression of the IAV nucleoprotein, measured as mean fluorescence intensity, was also significantly increased in ΔF2 infected cells (Fig. 2D).Fig. 2 Knockout of PB1-F2 causes a more detrimental infection in DF-1 cells. All experiments were conducted with infected (MOI: 0.01) DF-1 cells, that were harvested 24hpi. Data is shown as mean (SD) (A, C, D) or median with interquartile range (B), control (white), wt (blue), ΔF2 (red). A. Flow cytometrical live-dead differentiation was performed using Zombie Aqua™ viability dye. Depicted are representative results from one of at least three individual experiments. Statistical analysis was performed as unpaired t-test. B. Cell viability was further investigated using the WST assay. Statistics were calculated as Kruskal-Wallis test with Dunn's multiple comparisons test. C, D. To distinguish infected from uninfected cells a monoclonal antibody against IAV nucleoprotein (NP) was used. Here, we show the infection rate in% of alive cells (C, one-way ANOVA with Tukey's multiple comparisons test) as well as the intracellular NP expression level as mean fluorescence intensity (MFI) (D, Mann-Whitney test) Data depicted is a representative of at least three individual experiments.

Fig 2

3.4 Knockout of PB1-F2 increased apoptosis and restricted IFN-β mRNA induction

Considering the higher infection rate of ΔF2, it could be assumed that this is a cause for the different survival rates (Fig. 2A) and viability (Fig. 2B). Furthermore, PB1-F2 is associated with apoptosis in mammalian cells (Chang et al., 2015). Therefore, we investigated its influence on the induction of apoptosis. We utilized an antibody against active Caspase 3 (aCas3) to identify apoptotic cells, as Caspase 3 is part of both the intrinsic and extrinsic apoptotic pathway. To exclude bias due to different infection rates, we focused only on NP+ cells for further analysis. Surprisingly, PB1-F2 limited the apoptotic cell death (Fig. 3A). While both viruses caused apoptosis compared to the naïve control, the percentage of aCas3+ cells was significantly higher (−14 %) in DF-1 cells infected with ΔF2 than those infected with wt.Fig. 3 PB1-F2 limits apoptotic processes. All experiments were conducted with infected (MOI: 0.01) DF-1 cell or MDCK-II cells, that were harvested 24hpi. Data is shown as mean (SD), control (white), wt (blue), ΔF2 (red). A-B. Apoptotic cells were identified using an antibody against active Caspase3 (aCas3). Zombie-negative, NP-positive DF-1 (A) or MDCK-II (B) cells were used for the analysis. Naïve, Zombie-negative cells were used as control. Statistics were performed as one-way ANOVA with Tukey's multiple comparisons test. The results shown are representative of at least three individual experiments. C. DF-1 were transfected with plasmids containing firefly luciferase gene under control of a chicken IFN-ß promoter and Renilla luciferase gene under control of a CMV promoter (as transfection control). Cells were subsequently infected with the respective viruses at MOI of 0.1 (naïve control (white), wt (blue), ΔF2 (red)). 24 h post transfection cells were harvested and luminescence measurement was performed. Here, we show results representative of two individual experiments as mean (SD). Statistical analysis was performed as one-way ANOVA with Tukey's multiple comparisons test. D. Tag-it violet stained, infected DF-1 cells (violet) were harvested 24hpi and introduced to CSFE-stained HD11 cells (green), phagocytic uptake of DF-1 cells was measured by flow cytometry. Depicted are exemplary FACS blots (right, phagocyted population (red)) as well as the graphical representation of three experimental rounds. Statistical analysis was performed as ordinary one-way ANOVA with Tukey's multiple comparisons test.

Fig 3

Since PB1-F2 has been described to have predominately pro-apoptotic properties in mammals (Chang et al., 2015), we tested whether the restriction of apoptosis caused by H7N7 PB1-F2 is limited to avian cells (DF-1). Hence, we infected and stained susceptible mammalian cells (MDCK-II) following the protocol established previously (Fig. 3B). We observed that both viruses induced apoptosis in mammalian cells. However, frequencies of apoptotic cells were lower (−5 %) in wt-infected MDCK-II cells compared to ΔF2-infected cells (P = 0.0468).

Apoptotic processes can be dependent on or independent of type I interferon (IFN) signalling (Knowlton et al., 2012; Kotredes and Gamero, 2013). Here, we investigated the impact of PB1-F2 on IFN-β induction 24hpi by infecting DF-1 cells, that were previously transfected with chicken IFN-β promoter reporter plasmids (Fig. 3C). Wt induced a significant type I IFN response, while ΔF2 infection did not. This suggests that ΔF2 was superior at blocking IFN type I signalling and inducing apoptosis, potentially independent of IFN-β.

3.5 Knockout of PB1-F2 did not directly affect phagocytic uptake of infected cells, but limited MHC-I expression

Macrophages and monocytes have been described to be most vulnerable to PB1-F2 induced apoptosis (Chen et al., 2001; Mitzner et al., 2009), delaying viral clearance (Zamarin et al., 2006), contributing to severe immunopathology in murine lungs (Le Goffic et al., 2011; McAuley et al., 2010a) and increasing the risk of bacterial superinfections (Iverson et al., 2011; McAuley et al., 2007). To investigate whether avian PB1-F2 affected phagocytic elimination of infected cells similarly, we harvested pre-stained, infected DF-1 cells (violet) and introduced them to naïve, CFSE pre-stained chicken monocytes (HD11). DF-1 cells infected with ΔF2 were phagocytized at significantly higher rates than wt infected cells (Fig. 3D).

Even though H7N7 could not successfully replicate in HD11 cells over longer periods of time, chicken monocytes were susceptible to infection (Fig. S3A). Therefore, we were interested if the differences in phagocytic uptake were caused by viral infection of the monocytes. While viral infection generally increased the phagocytosis rate, we observed no significant difference between wt and ΔF2 (Fig. S3B). Thus, expression of PB1-F2 did not directly influence phagocytic uptake. However, cells infected with ΔF2 were more likely to be eliminated by phagocytes than wt infected cells, likely due to their apoptotic state.

Antigen presentation via major histocompatibility complex (MHC-I and MHC-II) is another important part of the host defence machinery. By flow cytometry we could investigate cellular MHC-I expression under infection (Fig. 4A). While both viruses caused significant downregulation, MHC-I was more significantly decreased in ΔF2-infected DF-1 cells. This shows yet another way, in which ΔF2 might restrict host response.Fig. 4 Knockout of PB1-F2 obstructed host defence mechanisms and wound healing. A. Surface expression of MHC-I on inoculated DF-1 cells (MOI 0.01) was analysed by flow cytometry. The data (one-way ANOVA with Tukey's multiple comparisons test) shown is representative of at least three rounds of experiments. B-C. To investigate PB1-F2’s influence on wound healing, a confluent monolayer of inoculated (MOI 0.1) DF-1 cells was damaged (C., left). The repair of the lesion was measured microscopically (C., right). Results 17hpi (16 h post lesion, B.) are depicted here as mean (SD). Statistics were performed as one-way ANOVA with Tukey's multiple comparisons test.

Fig 4

Even though apoptosis does not typically cause inflammatory tissue damage (Haanen and Vermes, 1995), we investigated whether reduced cellularity could affect the healing capacity of a scratched cell sheet to mimic PB1-F2’s effect on wound healing. For this purpose, we damaged a confluent layer of infected DF-1 cells and analysed the lesion closure (Fig. 4B-C). While wt infected cells healed similarly to naïve cells, the closure was significantly delayed in ΔF2 infected cells. These results further indicate that ΔF2 infection is more detrimental to host cells by delaying repair mechanisms either directly or by eliminating viable cells, that could close the lesions.

4 Discussion

Several studies have investigated the impact of PB1-F2 on various aspects of AIV biology. The results suggest that the effects of PB1-F2 are not universal and can vary depending on the virus strain, cell type, and experimental conditions. Here, we demonstrated that expression of PB1-F2 has limited impact on LPAIV H7N7 replication and spread. This is in line with previous publications (James et al., 2016; Mazur et al., 2008; McAuley et al., 2010b). Our data (Fig. S2A-C) suggests a dose-dependent replication advantage for the knockout mutant independent of the polymerase activity. Previous reports on the impact of PB1-F2 on the viral polymerase activity are contradictory. PB1-F2 increased the polymerase activity of PR8/H1N1 through the interaction with PB1 (Mazur et al., 2008), whereas PB1-F2 of other less lab-adapted viruses (H1N1 (A/California/04/2009), H3N2 (A/Wuhan/359/95) or H5N1 (A/Vietnam/1203/04)) or H7N7 had negligible (Hai et al., 2010; McAuley et al., 2010b) or no (Hohensee et al., 2024) effect on the polymerase activity.

PB1-F2 is well-known for inducing apoptosis (Chen et al., 2010, 2001; Jaworska et al., 2014; Mitzner et al., 2009), but H7N7 PB1-F2 increased the viability of chicken fibroblasts by limiting apoptotic cell death. Pro-apoptotic effects of PB1-F2 may vary depending on virus strain and type of cell infected, hence comparison with previously published results is challenging due to varying experimental setups. While PB1-F2 of some IAV strains (LPAIV H2N3 (A/mallard duck/England/7277/06), recombinant HPAIV H5N1, LPAIV H6N1 (A/turkey/England/198/09)) has been found to increase apoptotic cell death in infected porcine macrophages, others (swine H1N1 (A/swine/England/117,316/1986), human pandemic H1N1 2009 (A/California/07/2009), human USSR H1N1 (A/USSR/77 [USSR]) had no effect (Chang et al., 2015). Further experiments with H1N1 viruses have shown that PB1-F2 of PR8 consistently triggered apoptosis (Chen et al., 2010; McAuley et al., 2010a), whereas H1N1–1918 PB1-F2 (A/Brevig Mission/1/18) did not induce apoptosis (McAuley et al., 2010a). Certain residues in the PB1-F2 protein, such as I68, L69, and V70, have been linked to cytotoxicity (Alymova et al., 2014a). However, H7N7 PB1-F2 shared neither motif (Fig. S1B), which may explain its limited apoptotic potential. Our observations are consistent with previous findings using HPAIV H7N7 PB1-F2 (Hohensee et al., 2024), and indicate that the restriction of apoptosis may be a common feature of this H7N7 virus regardless of the pathotype. Early replication advantage of ΔF2-infected DF-1 cells 8 hpi at MOI 0.01 (Fig. S2C) suggests the knockout of PB1-F2 increases early viral replication, while also promoting apoptosis. As apoptosis has been described to be beneficial to viral replication (Tran et al., 2013), it remains to be determined whether PB1-F2 limits apoptosis directly, thereby potentially restricting early viral replication, or causes an early replication disadvantage compared to ΔF2 that in attenuates the apoptotic response. Future studies should elucidate which mechanism are implicated in PB1-F2’s apoptosis restriction and thereby explain whether the restriction of apoptosis is limited to virus-induced apoptosis or extends to other proapoptotic stimuli. We also found that avian cells showed more pronounced protection from apoptosis than mammalian cells, suggesting this may be a co-evolutionary trait that facilitates effective viral circulation in avian species.

PB1-F2 has been reported to interfere with type I IFN signalling (James et al., 2019). However, reports on PB1-F2’s impact on type I IFN vary, with some showing decreased signalling (Conenello et al., 2011; Dudek et al., 2011; Yoshizumi et al., 2014) and reduced induction (Jaworska et al., 2014; Varga et al., 2012) during IAV infections, while others reported the opposite (Chang et al., 2015; Le Goffic et al., 2010; 2011). H7N7 PB1-F2 did not suppress type I IFN, and actually induced an IFN-β response, its lack of certain residues linked to IFN antagonism might contribute to this effect (Cheng et al., 2017; Conenello et al., 2011) (Fig. S1B). This induction of antiviral signalling might have caused the decreased infection rates in wt infected cells (Fig. 2C). It has been suggested that PB1-F2 may inhibit global IFN-inducing pathways via the RIG-I/MDA5-MAVS-pathway and TBK1 while increasing IFN-β production by interacting with CALCOCO2 and stimulating TRAF-6-mediated NF-κB activation (Leymarie et al., 2017).

However, other IAV proteins such as NS1 or PA-X are also potent IFN antagonists (Hao et al., 2020; Hayashi et al., 2015), therefore IFN-β downregulation may occur independently of PB1-F2 in both wt- and ΔF2-infected cells, while only the full-length protein induces IFN-β through its interaction with NF-κB in parallel or apoptotic processes in ΔF2-infected cells might be too swift and severe to allow IFN type I signalling. Alternatively, PB1-F2 was shown to interact with other IAV proteins (Mazur et al., 2008), suggesting that NS1’s interaction with PB1-F2 might affect its efficiency in blocking IFN induction.

PB1-F2 can influence the host's defence, including immune cell dynamics (Chen et al., 2001; Jaworska et al., 2014), and its virulence is partially attributed to immunopathogenicity (McAuley et al., 2010a). The protein decreases adaptive immunity by targeting immune cells (Chen et al., 2001) and hinders macrophage functions (Leymarie et al., 2013). These can result in an increase in secondary bacterial infections (Iverson et al., 2011; McAuley et al., 2007). However, LPAIV H7N7 PB1-F2 likely does not share the macrophage modulatory properties, as we observed no differences in phagocytic activity of infected macrophages. Instead, we propose that the difference in phagocytic uptake is likely caused by diverging apoptotic rates, as apoptotic cells emit signals that attract phagocytic cells to the site of apoptosis (Fogarty and Bergmann, 2015). However, whether this is a direct effect of PB1-F2 or due to replication advantages of the PB1-F2 knockout cannot be determined without follow-up experiments. The increased phagocytic uptake of ΔF2-infected cells might also explain the increased infection rate of ΔF2-inoculated HD11 cells (Fig. S3).We cannot exclude an impact of H7N7 PB1-F2 on the antimicrobial features of the macrophages (Leymarie et al., 2013), such as bacterial killing, or production of immune mediators such as TNF-α, IL-6 and IL-10 (Chang et al., 2015).

To account for possible differences in antigen presentation we investigated the expression of major histocompatibility complex I (MHC-I). Both tested H7N7 viruses reduced the MHC-I expression on DF-1 cells, with the decrease being more prominent in ΔF2-infected cells. Downregulation of MHC-I is a common feature of human influenza A and influenza B viruses (Koutsakos et al., 2019). This was also apparent in our experiments with LPAIV H7N7. In humans, IFN released by infected cells can facilitate an increase in MHC-I surface expression (Keskinen et al., 1997). Thus, it is conceivable that both wt and ΔF2 initially impacted MHC-I expression similarly. However, since only full-length PB1-F2 induced a heightened type I IFN response, MHC-I expression is observed to be higher in wt-infected cells at the investigated time point. To ensure that this is a direct effect of PB1-F2 and not due to differences in viral replication, kinetic experiments would be needed.

In our experiments the knockout of PB1-F2 negatively impacted tissue healing in an in vitro model, while wt-infected cells healed similarly to naïve ones. Even though in vivo wound healing and tissue repair are complex processes, our data allows us to hypothesize that LPAIV H7N7 PB1-F2 enables the host to maintain mostly functional tissues, likely due to either restriction of apoptotic damage or reduced viral replication compared to ΔF2.

Even though our study demonstrated that LPAIV H7N7 PB1-F2 does not share certain immunomodulatory properties, that were observed in other strains, expression of full-length PB1-F2 limited apoptotic cell death and prolonged survival of infected cells. Hence, we propose that PB1-F2 may play a role to ensure perpetuation of AIV in wild birds. However, since this study was limited to LPAIV H7N7 AR915/15, further research utilizing various strains, both LPAIV and HPAIV, is needed to confirm our findings and to fully understand PB1-F2’s complex interactions within the avian immune system and its effects in vivo.

Funding

No external funding to declare.

Authors statement

PB1-F2 of Low Pathogenicity H7N7 Restricts Apoptosis in Avian Cells.

Luise Hohensee, David Scheibner, Christine Luttermann, Holly Shelton, Anca Dorhoi, Elsayed M. Abdelwhab, Ulrike Blohm

CRediT authorship contribution statement

Luise Hohensee: Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Formal analysis, Data curation. David Scheibner: Investigation, Formal analysis. Christine Luttermann: Investigation, Formal analysis. Holly Shelton: Writing – review & editing, Conceptualization. Anca Dorhoi: Writing – review & editing, Supervision, Conceptualization. Elsayed M. Abdelwhab: Writing – review & editing, Validation, Supervision, Resources, Methodology, Data curation, Conceptualization. Ulrike Blohm: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Image, application 2

Image, application 3

Image, application 4

Image, application 5

Data availability

Data will be made available on request.

Acknowledgments

The authors thank Stefanie Knöfel and Dajana Helke for laboratory technical assistance.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2024.199444.
==== Refs
References

Abdelwhab E.M. Veits J. Ulrich R. Kasbohm E. Teifke J.P. Mettenleiter T.C. Composition of the hemagglutinin polybasic proteolytic cleavage motif mediates variable virulence of H7N7 avian influenza viruses Sci. Rep. 6 2016 39505 28004772
Alymova I.V. Samarasinghe A. Vogel P. Green A.M. Weinlich R. McCullers J.A. A novel cytotoxic sequence contributes to influenza A viral protein PB1-F2 pathogenicity and predisposition to secondary bacterial infection J. Virol. 88 2014 503 515 24173220
Bosman A. Meijer A. Koopmans M. Final analysis of Netherlands avian influenza outbreaks reveals much higher levels of transmission to humans than previously thought Euro Surveill. 10 2005 E050106 050102
Capua I. Marangon S. Control of avian influenza in poultry Emerg. Infect. Dis. 12 2006 1319 1324 17073078
Chang P. Kuchipudi S.V. Mellits K.H. Sebastian S. James J. Liu J. Shelton H. Chang K.C. Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and pro-inflammatory dysregulation Sci. Rep. 5 2015 17999 26642934
Chen C.J. Chen G.W. Wang C.H. Huang C.H. Wang Y.C. Shih S.R. Differential localization and function of PB1-F2 derived from different strains of influenza A virus J. Virol. 84 2010 10051 10062 20660199
Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. A novel influenza A virus mitochondrial protein that induces cell death Nat. Med. 7 2001 1306 1312 11726970
Cheng Y.Y. Yang S.R. Wang Y.T. Lin Y.H. Chen C.J. Amino acid residues 68-71 contribute to influenza a virus PB1-F2 protein stability and functions Front. Microbiol. 8 2017 692 28484439
Choi J.W. Shin E.K. Ha S.H. Kim H.A. Kim Y.H. Kim J.S. Hahn T.W. Optimal conditions for cryopreservation of primary chicken embryo kidney cells with dimethyl sulfoxide Mol. Biotechnol. 35 2007 237 241 17652787
Conenello G.M. Tisoncik J.R. Rosenzweig E. Varga Z.T. Palese P. Katze M.G. A single N66S mutation in the PB1-F2 protein of influenza A virus increases virulence by inhibiting the early interferon response in vivo J. Virol. 85 2011 652 662 21084483
de Bruin A.C.M. Funk M. Spronken M.I. Gultyaev A.P. Fouchier R.A.M. Richard M. Hemagglutinin subtype specificity and mechanisms of highly pathogenic avian influenza virus genesis Viruses 14 2022
DeLuca D.S. Keskin D.B. Zhang G.L. Reinherz E.L. Brusic V. PB1-F2 Finder: scanning influenza sequences for PB1-F2 encoding RNA segments BMC Bioinform. 12 13 2011 S6 Suppl
Dietze K. Graaf A. Homeier-Bachmann T. Grund C. Forth L. Pohlmann A. Jeske C. Wintermann M. Beer M. Conraths F.J. Harder T. From low to high pathogenicity-characterization of H7N7 avian influenza viruses in two epidemiologically linked outbreaks Transbound. Emerg. Dis. 65 2018 1576 1587 29790657
Dudek S.E. Wixler L. Nordhoff C. Nordmann A. Anhlan D. Wixler V. Ludwig S. The influenza virus PB1-F2 protein has interferon antagonistic activity Biol. Chem. 392 2011 1135 1144 22050228
Fogarty C.E. Bergmann A. The sound of silence: signaling by apoptotic cells Curr. Top. Dev. Biol. 114 2015 241 265 26431570
Fouchier R.A. Munster V. Wallensten A. Bestebroer T.M. Herfst S. Smith D. Rimmelzwaan G.F. Olsen B. Osterhaus A.D. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls J. Virol. 79 2005 2814 2822 15709000
Haanen C. Vermes I. Apoptosis and inflammation Mediators Inflamm. 4 1995 5 15 18475609
Hai R. Schmolke M. Varga Z.T. Manicassamy B. Wang T.T. Belser J.A. Pearce M.B. Garcia-Sastre A. Tumpey T.M. Palese P. PB1-F2 expression by the 2009 pandemic H1N1 influenza virus has minimal impact on virulence in animal models J. Virol. 84 2010 4442 4450 20181699
Hao W., Wang L., Li S., 2020. Roles of the non-structural proteins of influenza a virus. Pathogens 9.
Hayashi T. MacDonald L.A. Takimoto T. Influenza a virus protein PA-X contributes to viral growth and suppression of the host antiviral and immune responses J. Virol. 89 2015 6442 6452 25855745
Hohensee L. Scheibner D. Schafer A. Shelton H. Mettenleiter T.C. Breithaupt A. Dorhoi A. Abdelwhab E.M. Blohm U. The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens Vet. Res. 55 2024 5 38173025
Iverson A.R. Boyd K.L. McAuley J.L. Plano L.R. Hart M.E. McCullers J.A. Influenza virus primes mice for pneumonia from Staphylococcus aureus J. Infect. Dis. 203 2011 880 888 21278211
James J. Howard W. Iqbal M. Nair V.K. Barclay W.S. Shelton H. Influenza a virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window J. Gen. Virol. 97 2016 2516 2527 27558742
James J. Smith N. Ross C. Iqbal M. Goodbourn S. Digard P. Barclay W.S. Shelton H. The cellular localization of avian influenza virus PB1-F2 protein alters the magnitude of IFN2 promoter and NFkappaB-dependent promoter antagonism in chicken cells J. Gen. Virol. 100 2019 414 430 30672726
Jaworska J. Coulombe F. Downey J. Tzelepis F. Shalaby K. Tattoli I. Berube J. Rousseau S. Martin J.G. Girardin S.E. McCullers J.A. Divangahi M. NLRX1 prevents mitochondrial induced apoptosis and enhances macrophage antiviral immunity by interacting with influenza virus PB1-F2 protein Proc. Natl. Acad. Sci. U. S. A 111 2014 E2110 E2119 24799673
Kamal R.P. Alymova I.V. York I.A. Evolution and Virulence of Influenza A Virus Protein PB1-F2 Int. J. Mol. Sci. 19 2017
Keskinen P. Ronni T. Matikainen S. Lehtonen A. Julkunen I. Regulation of HLA class I and II expression by interferons and influenza A virus in human peripheral blood mononuclear cells Immunology 91 1997 421 429 9301532
Knobler, S.L., Mack, A., Mahmoud, A., Lemon, S.M. (Eds.), 2005. The threat of pandemic influenza: are we ready? Workshop Summary. Washington (DC).
Knowlton J.J. Dermody T.S. Holm G.H. Apoptosis induced by mammalian reovirus is beta interferon (IFN) independent and enhanced by IFN regulatory factor 3- and NF-kappaB-dependent expression of Noxa J. Virol. 86 2012 1650 1660 22090144
Kotredes K.P. Gamero A.M. Interferons as inducers of apoptosis in malignant cells J. Interferon. Cytokine Res. 33 2013 162 170 23570382
Koutsakos M. McWilliam H.E.G. Aktepe T.E. Fritzlar S. Illing P.T. Mifsud N.A. Purcell A.W. Rockman S. Reading P.C. Vivian J.P. Rossjohn J. Brooks A.G. Mackenzie J.M. Mintern J.D. Villadangos J.A. Nguyen T.H.O. Kedzierska K. Downregulation of MHC class I expression by influenza A and B viruses Front. Immunol. 10 2019 1158 31191533
Krammer F. Smith G.J.D. Fouchier R.A.M. Peiris M. Kedzierska K. Doherty P.C. Palese P. Shaw M.L. Treanor J. Webster R.G. Garcia-Sastre A. Influenza Nat. Rev. Dis. Prim. 4 2018 3 29955068
Krumbholz A. Philipps A. Oehring H. Schwarzer K. Eitner A. Wutzler P. Zell R. Current knowledge on PB1-F2 of influenza A viruses Med. Microbiol. Immunol. 200 2011 69 75 20953627
Le Goffic R. Bouguyon E. Chevalier C. Vidic J. Da Costa B. Leymarie O. Bourdieu C. Decamps L. Dhorne-Pollet S. Delmas B. Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells J. Immunol. 185 2010 4812 4823 20844191
Le Goffic R. Leymarie O. Chevalier C. Rebours E. Da Costa B. Vidic J. Descamps D. Sallenave J.M. Rauch M. Samson M. Delmas B. Transcriptomic analysis of host immune and cell death responses associated with the influenza A virus PB1-F2 protein PLoS Pathog. 7 2011 e1002202
Leymarie O. Jouvion G. Herve P.L. Chevalier C. Lorin V. Lecardonnel J. Da Costa B. Delmas B. Escriou N. Le Goffic R. Kinetic characterization of PB1-F2-mediated immunopathology during highly pathogenic avian H5N1 influenza virus infection PLoS One 8 2013 e57894 23469251
Leymarie O. Meyer L. Tafforeau L. Lotteau V. Costa B.D. Delmas B. Chevalier C. Le Goffic R. Influenza virus protein PB1-F2 interacts with CALCOCO2 (NDP52) to modulate innate immune response J. Gen. Virol. 98 2017 1196 1208 28613140
Liang C.C. Park A.Y. Guan J.L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro Nat. Protoc. 2 2007 329 333 17406593
Liu S. Zhuang Q. Wang S. Jiang W. Jin J. Peng C. Hou G. Li J. Yu J. Yu X. Liu H. Sun S. Yuan L. Chen J. Control of avian influenza in China: strategies and lessons Transbound. Emerg. Dis. 67 2020 1463 1471 32065513
Ma W. Brenner D. Wang Z. Dauber B. Ehrhardt C. Hogner K. Herold S. Ludwig S. Wolff T. Yu K. Richt J.A. Planz O. Pleschka S. The NS segment of an H5N1 highly pathogenic avian influenza virus (HPAIV) is sufficient to alter replication efficiency, cell tropism, and host range of an H7N1 HPAIV J. Virol. 84 2010 2122 2133 20007264
Mazur I. Anhlan D. Mitzner D. Wixler L. Schubert U. Ludwig S. The proapoptotic influenza A virus protein PB1-F2 regulates viral polymerase activity by interaction with the PB1 protein Cell Microbiol. 10 2008 1140 1152 18182088
McAuley J.L. Chipuk J.E. Boyd K.L. Van De Velde N. Green D.R. McCullers J.A. PB1-F2 proteins from H5N1 and 20 century pandemic influenza viruses cause immunopathology PLoS Pathog. 6 2010 e1001014
McAuley J.L. Hornung F. Boyd K.L. Smith A.M. McKeon R. Bennink J. Yewdell J.W. McCullers J.A. Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia Cell Host. Microbe 2 2007 240 249 18005742
McAuley J.L. Zhang K. McCullers J.A. The effects of influenza A virus PB1-F2 protein on polymerase activity are strain specific and do not impact pathogenesis J. Virol. 84 2010 558 564 19828614
Mitzner D. Dudek S.E. Studtrucker N. Anhlan D. Mazur I. Wissing J. Jansch L. Wixler L. Bruns K. Sharma A. Wray V. Henklein P. Ludwig S. Schubert U. Phosphorylation of the influenza A virus protein PB1-F2 by PKC is crucial for apoptosis promoting functions in monocytes Cell Microbiol. 11 2009 1502 1516 19523156
Pasick J. Berhane Y. Hooper-McGrevy K. Avian influenza: the Canadian experience Rev. Sci. Tech. 28 2009 349 358 19618638
Pasricha G. Mukherjee S. Chakrabarti A.K. Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells Adv. Virol. 2018 2018 5057184
Sagong M. Lee K.N. Lee E.K. Kang H. Choi Y.K. Lee Y.J. Current situation and control strategies of H9N2 avian influenza in South Korea J. Vet. Sci. 24 2023 e5 36560837
Scheibner D. Ulrich R. Fatola O.I. Graaf A. Gischke M. Salaheldin A.H. Harder T.C. Veits J. Mettenleiter T.C. Abdelwhab E.M. Variable impact of the hemagglutinin polybasic cleavage site on virulence and pathogenesis of avian influenza H7N7 virus in chickens, turkeys and ducks Sci. Rep. 9 2019 11556 31399610
Tran A.T. Cortens J.P. Du Q. Wilkins J.A. Coombs K.M. Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation J. Virol. 87 2013 1049 1060 23135712
van de Sandt C.E. Bodewes R. Rimmelzwaan G.F. de Vries R.D. Influenza B viruses: not to be discounted Future Microbiol. 10 2015 1447 1465 26357957
Varga Z.T. Grant A. Manicassamy B. Palese P. Influenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential J. Virol. 86 2012 8359 8366 22674996
Villarreal C. Avian influenza in Mexico Rev. Sci. Tech. 28 2009 261 265 19618630
Webster R.G. Bean W.J. Gorman O.T. Chambers T.M. Kawaoka Y. Evolution and ecology of influenza A viruses Microbiol. Rev. 56 1992 152 179 1579108
WOAH, 2023. Statement On Avian Influenza and Mammals.
Yoshizumi T. Ichinohe T. Sasaki O. Otera H. Kawabata S. Mihara K. Koshiba T. Influenza A virus protein PB1-F2 translocates into mitochondria via Tom40 channels and impairs innate immunity Nat. Commun. 5 2014 4713 25140902
Zamarin D. Garcia-Sastre A. Xiao X. Wang R. Palese P. Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1 PLoS Pathog. 1 2005 e4 16201016
Zamarin D. Ortigoza M.B. Palese P. Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice J. Virol. 80 2006 7976 7983 16873254
