Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective

Animals

Rainbow trout (Oncorhynchus mykiss) individuals of approximately 10 g were obtained from a commercial fish farm (PISZOLLA S.L., CIMBALLA FISH FARM, Zaragoza, Spain). Fish were maintained at the University Miguel Hernandez (UMH) facilities with a re-circulating dechlorinated-water system, at a stocking density of 1 fish/3L, at 14°C, and fed daily with a commercial diet (SKRETTING, Burgos, Spain). Fish were acclimatized to laboratory conditions over 2 weeks before experimentation. The number of fish used is indicated for each experiment/figure.

RBCs purification

Rainbow trout were sacrificed by overexposure to tricaine methanesulfonate (Sigma-Aldrich, Madrid, Spain) at 0.2 g/L. Peripheral blood was sampled from the caudal vein using insulin syringes (NIPRO Bridgewater, NJ). Approximately 100 µL of blood was diluted in RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fischer Scientific Inc., Carlsbad, CA) supplemented with 10% FBS (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 µg/mL gentamicin (Gibco), 2 µg/mL fungizone (Gibco) and 100 U/mL penicillin/streptomycin (Sigma-Aldrich). Then, RBCs were purified by two consecutive density gradient centrifugations with Histopaque 1077 (7206g, Ficoll 1.007; Sigma-Aldrich). Finally, RBCs were washed twice with RPMI 2% FBS. Purity of RBCs of 99.9% was estimated by optical microscopy evaluation. Then, purified RBCs were cultured in the above indicated medium at a density of 10⁷ cells/mL, in cell culture flasks, at 14°C, overnight.

Viral infection assays

Ex vivo rainbow trout RBCs along with CHSE-214 cell line (Chinook Salmon Embryo, ATCC CRL-1681) were infected using IPNV Sp strain¹⁷. IPNV was grown as previously described¹⁸. Ex vivo RBCs exposure to IPNV was performed by incubating RBCs with diluted IPNV at the indicated MOI (multiplicity of infection) in RPMI 2% FBS. After three hours of incubation at 14°C, RBCs were centrifuged at 1600 rpm for 5 minutes and then washed with medium to completely eliminate the non-adsorbed excess of virus. Finally, RBCs were placed in 24 well plates (Corning Costar, Sigma-Aldrich, Madrid, Spain) with 500 µl of RPMI 2% FBS. The whole process was done at 14°C. Infection of the CHSE-214 cell line was done by incubating IPNV diluted in RPMI 2% FBS at the desired MOI for 1 hour at 14°C. After that, medium was removed and RPMI 2% FBS was added to each well. Infected CHSE-214 cells were maintained at 14°C¹⁸.

In time course experiments, the initial supernatant with IPNV was not removed. When each of the time points was reached, RBCs were washed with cell culture medium and CHSE-214 cells with PBS supplemented with calcium.

Viral titration assay

The virus titer in IPNV-exposed RBCs supernatants was quantified by TCID₅₀ and by RT-qPCR. Briefly, different dilutions of the supernatants (from 10^-1 to 10^-4) were added to CHSE-214 cell monolayers, and incubated at 14°C for 90 minutes. Then, the virus was removed and infected CHSE-214 cell monolayers covered with a solution of RPMI 2% FBS. Cell plates were incubated at 14°C for 7 days. For RT-qPCR titration, 30 µL of IPNV with known titer (10⁹ TCID₅₀/mL) and 30 µL of IPNV-exposed RBCs supernatants were used to extract RNA and synthetize cDNA, as explained hereafter. Ten-fold serial dilutions from 10⁸ to 10² TCID₅₀/mL were done to obtain IPNV cDNA and create a standard line.

RNA isolation and DNAse treatment

The E.Z.N.A.® Total RNA Kit (Omega Bio-Tek Inc., Norcross, GA) was used for total RNA extraction, in accordance with manufacturer’s instructions. DNAse treatment was done in order to eliminate residual genomic DNA using TURBO™ DNase (Ambion, Thermo Fischer Scientific Inc.), following the manufacturer’s instructions. RNA was quantified with a NanoDrop® 377 Spectrophotometer (Nanodrop Technologies, Wilmington, DE).

Gene expression by RT-qPCR

cDNA was synthesized from RNA using M-MLV reverse transcriptase (Invitrogen, Thermo Fischer Scientific Inc.), as previously described¹⁹. Final concentration of cDNA was 6 ng/µL. RT-qPCR reactions were performed in a total volume of 20 μl using 12 ng of cDNA, 10 μl of TaqMan universal PCR master mix (Thermo Fischer Scientific), 900 nM final concentration of each primer (300 nM for IPNV segment A) and 300 nM of probe (150 nM for IPNV segment A). RT-qPCR was performed using the ABI PRISM 7300 System (Thermo Fischer Scientific). Cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.

Gene expression was analyzed by the 2^-ΔΔCt method²⁰. The eukaryotic 18S rRNA gene (Cat#4310893E, Thermo Fischer Scientific) was used as endogenous control. Primers and probes are listed in Table 1.

Antibodies

Several antibodies were used to stain cells for cytokines and to measure polypeptides in RBCs extracts by western blotting. They are briefly described below and their Research Resource Identifiers (RRIDs) given. For intracellular staining, mouse polyclonal antibodies against rainbow trout IL1β (RRID: AB_2716269)^21,22, IL8 (RRID: AB_2716272)²³ and TNF-α (RRID: AB_2716270)²⁴ were produced at the laboratory of Dr. Luis Mercado. Rabbit polyclonal antibody against rainbow trout Mx3 (RRID: ABA_2716267)^25,26 was produced at the laboratory of Dr. Amparo Estepa. Anti-IPNV-VP3 monoclonal antibody 2F12 (RRID: AB_2716296) was used for IPNV labelling²⁷. Anti-rabbit IgG (H+L) CF™ 488 antibody produced in goat and anti-mouse IgG (H+L) CF™ 488 antibody produced in goat were used as secondary antibodies for proteins and anti-mouse IgG (H+L) CF™ 647 produced in goat to detect 2F12 antibody.

For western blotting, rabbit polyclonal antibody against human eIF2α-P (Cat# E2152, RRID:AB_259283) and rabbit polyclonal antibody against human α-Actin (Cat#2066, RRID:AB_476693) were purchased from Sigma-Aldrich.

Western blot

Control and IPNV-exposed RBCs pellets (≈10⁷ cells) were used for protein extraction. Cell pellets were washed 3 times with PBS and then resuspended in 30 µl of PBS with a cocktail of protease inhibitors (Sigma-Aldrich). Then, cells were frozen/thawed 3 times and lysed using an eppendorf micropistile (Eppendorf, Hamburg, Germany). Samples were loaded in Tris–Glycine sodium dodecyl sulfate 12% polyacrylamide gels under reducing conditions. Electrophoresis was performed at 200 V for 60 min. For blotting, the proteins in the gel were transferred for 80 min at 100 V in transfer buffer (2.5 mM Tris, 9 mM glycine, 20% methanol) to nitrocellulose membranes (BioRad, Madrid, Spain). Then, membranes were blocked with 8% dry milk and 1% Tween-20 in PBS and incubated with eIF2α-P or α-Actin antibodies, at the recommended dilutions in PBS containing 0.5% dry milk and 0.5% Tween-20 at 4°C and overnight. Incubation with secondary antibody GAR-Po (Sigma-Aldrich) was done in 0.5% milk 0.5% Tween-20 in PBS for 45 min. Membranes were washed 3 times with PBS containing 1% dry milk 0.5% Tween-20 for 15 min after every antibody incubation. Finally, the membrane was washed 3 times with PBS before analysis of the peroxidase activity. Peroxidase activity was detected using ECL chemiluminescence reagents (Amersham Biosciences, Buckinghamshire, UK) and revealed by exposure to X-ray. Protein bands from western blotting were analysed by densitometry using the Scion Image 4.0.2 Software (RRID: SCR_008673) (www.scionorg.com).

Intracellular immunofluorescence stain and flow cytometry

RBCs were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) and 0.08% glutaraldehyde (Sigma-Aldrich) diluted in RPMI medium for 20 minutes. Then, RBCs were incubated with permeabilization buffer containing 0.05% saponin (Sigma-Aldrich) in RPMI, for 15 minutes. Primary antibodies were used at 1/50 dilution for IL-1β, IL-8 and TNF-α, 1/300 for Mx and 1/500 for 2F12 in permeabilization buffer and incubated for 60 minutes at room temperature. Secondary antibodies were incubated for 30 minutes at 1/200 dilution. RBCs were washed with permeabilization buffer after antibody incubations. Finally, RBCs were kept in PFA 1% in PBS. For nuclear staining, RBCs were stained with 1 μg/mL of 4′-6-408 Diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 minutes. Flow cytometry (FC) analysis was done in a BD FACSCanto™ II (BD Biosciences) flow cytometer. Immunofluorescence (IF) images were performed in the INCell Analyzer 6000 Cell imaging system (GE Healthcare, Little Chalfont, UK).

Antiviral activity of conditioned medium

Conditioned medium (CM) was obtained from control and IPNV-exposed RBCs at MOI 0.5, during 3 days. The CMs were clarified at 1600 rpm for 5 min. IPNV titer in the supernatants of IPNV-exposed RBCs resulted in 10 TCID₅₀/mL or less, therefore viral presence in the supernatants was obviated. To test the antiviral activity of the CM, confluent CHSE-214 cells (7.8×10⁴ cells/well), seeded in 96 well plates, were pre-treated with 100 µL of each supernatant at the indicated dilutions for 24 hours. After that, CHSE-214 cells were infected, as described previously, with IPNV at MOI 0.05, for 24 hours. Finally, intracellular staining of IPNV foci was carried out.

Intracellular staining of IPNV foci

CHSE-214 cells were fixed with PFA diluted at 4% in PBS followed by a second fixation with cold methanol. Each fixation step lasted 15 minutes. Cells were washed with PBS after each fixation step. Blocking buffer containing 5% goat serum (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) was added to each well with the cells for 1 hour. Then, anti-VP3 2F12 antibody was diluted 1/500 in antibody dilution buffer (1% BSA (Sigma-Aldrich), 0.3% Triton X-100) and was incubated for 1 hour. FITC-labelled goat anti-rabbit was used as secondary antibody at 1/300 dilution. Cells were washed three times after each antibody incubation with PBS. IF images were taken INCell Analyzer 6000 imaging system. IN Cell Developer Toolbox 1.9.2 (RRID: SCR_015790; GE Healthcare, Little Chalfont, UK) was used to count number of IPNV foci (positive areas after image segmentation were selected when >21000 fluorescence units and >2500 µm² criteria was reached).

MTT assays

Cell viability was tested using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay³². Briefly, 25 µl of MTT at a final concentration of 1.9 mg/mL were added to previously treated CHSE-214 cells monolayers, seeded in 96 well plates. Cells were incubated for 3 hours at 21°C with the reagent. Then, the medium was removed from the wells. Formazan crystals were dissolved in 100 µl of 100% DMSO, incubated for 30 minutes. Absorbance was read at 570 nm in the EON™ microplate spectrophotometer (Biotek, Winooski, VT). Percentage of viable cells was calculated as follows: absorbance treated cells/absorbance non-treated cells) x100.

Software and statistics

All the figures and graphics show the mean and standard deviation of the data. P-values associated with each graphic are represented by the legends: *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001, ****, p-value < 0.0001. Graphpad Prism 6 (RRID: SCR_002798, www.graphpad.com) (Graphpad Software Inc., San Diego, A) was used for preparing graphs and preforming statistical calculations. FC data were analyzed using Flowing Software 2.5.1 (RRID: SCR_015781)(www.flowingsoftware.com) to obtain Mean Fluorescence Intensity (MFI) and Mean Relative Fluorescence Intensity (MRFI) (relative to control cells) values.

Ethics approval

Methodology was carried out in accordance with the Spanish Royal Decree RD 53/2013 and EU Directive 2010/63/EU for animals used in research experimentation. All experimental protocols involving animal handling were also reviewed and approved by the Animal Welfare Body and the Research Ethics Committee at the Miguel Hernandez University (approval number 2014.205.E.OEP; 2016.221.E.OEP) and performed by qualified research personnel.

IPNV did not infect rainbow trout RBCs

To evaluate the infectivity of IPNV in rainbow trout RBCs, RBCs were exposed to IPNV at MOI 0.5 and the viral RNA was evaluated by RT-qPCR in the cell pellet at different times post-exposure. IPNV infectivity was also evaluated in parallel in the CHSE-214 cell line, used as a positive control of infection. IPNV segment A (IPNV-A) RNA levels inside RBCs and CHSE-214 cell line were similar at 1 and 3 hours post-exposure (hpe) (Figure 1A). After 6 hpe, IPNV-A RNA level was 3 logarithms lower in RBCs in comparison with CHSE-214 cells. On the other hand, the titer of IPNV in the supernatants from IPNV-exposed RBCs at a MOI of 0.5 and 5, was evaluated by TCID₅₀, at 3 days post-exposure (dpe), and showed a recovered titer of 5 and 4 logarithms lower, respectively (Figure 1B). Furthermore, the supernatants titrated by RT-qPCR, were below the lowest limit of detection 10² TCID₅₀ (Table 2). Moreover, FC analysis of control and IPNV-exposed RBCs for IPNV VP3 protein did not show significant differences (Figure 1C and D). Therefore, IPNV did not infect rainbow trout RBCs.

Figure 1. Infectivity of IPNV in RBCs.

(A) Time-course experiment of the expression of IPNV segment A (IPNV-A) in RBCs (●) (n = 6) and CHSE-214 cells (○) (n = 2) at MOI 0.5. Data is represented as mean±SD. Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed among all time-points post-exposure in comparison with control time point (0 hpi) (*, p-value < 0.05). (B) Recovered virus titer in supernatants from IPNV-exposed RBCs with an inoculum titer of 10⁶ (MOI 0.5) and 10⁷ (MOI 5) TCID₅₀/mL obtained after 72 hpe (n = 5). Data is represented as mean±SD. Mann-Whitney test was performed among both conditions (*, p-value < 0.05). (C) MFI (mean fluorescence intensity) of viral protein VP3 in control and IPNV-exposed RBCs at MOI 0.5 and 3 dpe (n = 6) Mann-Whitney test was performed among both conditions. (D) Representative flow cytometry histograms of IPNV VP3 protein detection in control and IPNV-exposed RBCs at MOI 0.5 and 3 dpi.

IPNV exposure increased the expression of interferon-related antiviral genes and proteins in rainbow trout RBCs

To determine if IPNV would induce an antiviral response in RBCs, RT-qPCR analysis of IFN-related antiviral genes was performed for IPNV-exposed RBCs. The results showed that mx1–3 and pkr genes were significantly expressed at 72 hpe. On the other hand, ifn1 gene presented a tendency to increase its expression after 6 hpe, having a peak at 24 hpe. Also, tlr3 gene expression tended to be upregulated at 24 hpe, whereas irf7 expression was upregulated at 72 hpe (Figure 2A). Three and six dpe with IPNV, RBCs were stained intracellularly with an anti-Mx antibody and analyzed by FC and immunofluorescence imaging (IF). The results showed a significant increment in the expression of Mx protein at 6 dpe by both FC an IF (Figure 2B and D). FC histograms showed, at 6 dpe, that RBCs depicted distinct peaks of Mx expression, showing that the expression of Mx in RBCs was heterogeneous in the total RBCs population (Figure 2C).

Figure 2. RBCs IFN-related antiviral response against IPNV.

(A) Gene expression of tlr3, irf7, inf1, mx1–3 and pkr in IPNV-exposed RBCs at the indicated times post-infection and MOI 0.5, measured by RT-qPCR. Data represent mean±SD (n = 6). Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed among all time-points post-exposure in comparison with control time point (0 hpi) (*, p-value < 0.05). (B) Mx protein MRFI (mean relative fluorescent intensity, relative to control cells) in IPNV-exposed RBCs at MOI 0.5 (n = 5). (C) Flow cytometry histograms of Mx protein expression from control (grey) and IPNV-exposed (red) RBCs at MOI 0.5 and the indicated days post-exposure (dpe). (D) Representative immunofluorescence images of Mx protein expression in control and IPNV-exposed RBCs at MOI 0.5 (FITC – Mx protein expression, DAPI - Nuclei) (IF representative of 40 images).

Conditioned medium from IPNV-exposed RBCs protected CHSE-214 cells against IPNV infection

To analyze if IPNV-exposed RBCs could secrete factors that were capable to protect other fish cells against IPNV infection, conditioned medium (CM) from control and IPNV-exposed RBCs (with IPNV titer <10 TCID₅₀/mL) were added to CHSE-214 cells prior to infection. Figure 3A shows a significant decrease in the number of IPNV infective focus forming units (FFU/mL) when pre-treating with 1/5 diluted CM from IPNV-exposed RBCs. CHSE-214 cells viability, by means of an MTT colorimetric assay, was not affected by the exposure to CM (Figure 3B).

Figure 3. Antiviral activity of the conditioned media from IPNV-exposed RBCs.

(A) Viral titers (FFU/mL) in CHSE-214 cells infected with IPNV at MOI 0.05 previously non-treated (black) or treated with either supernatants from control RBCs (white) or IPNV-exposed RBCs (grey), during 24 hours, at the indicated dilutions (n = 4, performing triplicates from each individual). Two-way ANOVA, with Sidak´s multiple comparison test, was performed among the different dilutions and conditions to test statistical differences. (B) Percentage of viable CHSE-214 cells pre-treated with conditioned medium from control and IPNV-exposed RBCs, during 24 hours, and relative to non-treated CHSE-214 cells. Percentage of viable cells was calculated as follows: absorbance treated cells/absorbance non-treated cells) x100.

IPNV exposure decreased the expression of cytokines in rainbow trout RBCs

To evaluate whether ex vivo rainbow trout RBCs could produce cytokines in response to IPNV exposure, RBCs were exposed to IPNV and IL-1 β, IL-8 and TNF-α protein levels were evaluated by means of FC and IF in control and IPNV-exposed cell cultures. The results showed a decrease in the protein expression of IL-1β, IL-8 and TNFα in IPNV-exposed RBCs (Figure 4A).

Figure 4. IPNV-exposure decreased cytokine levels in rainbow trout RBCs.

(A) Intracellular MFI (mean fluorescent intensity) values of IL-1β, IL-8 and TNFα from control and IPNV-exposed RBCs at MOI 0.5 and 3 dpe measured by FC (flow cytometry)(n = 6). Mann-Whitney test was performed among both conditions. (B) Phosphorylation of translation initiation factor eIF2α in IPNV-exposed RBCs. Representative western blot of eIF2α-P in control and IPNV-exposed RBCs from two individuals at MOI 0.5, 3 dpe. Densitometry ratios were done relativizing to α-actin. Mann-Whitney test was performed among both conditions.

IPNV exposure did not induce phosphorylation of the α-subunit of the eukaryotic translational initiation factor 2 (eIF2α) in rainbow trout RBCs

The phosphorylation of the translation initiation factor eIF2α is a key mechanism of global inhibition of translational initiation³³ and it has been described to happen after IPNV infection in the permissive cell line CHSE-214 cells³⁴. In this sense, since IPNV-exposed RBCs depicted a small downregulation of the evaluated cytokines protein levels, we further investigated whether IPNV exposure could reduce protein translation in RBCs by triggering the phosphorylation of eIF2α. However, the results revealed no changes in the phosphorylation of eIF2α (Figure 4B).