Generation of porcine PK-15 cells lacking the Ifnar1 or Stat2 gene to optimize the efficiency of viral isolation

Because pigs are intermediate or amplifying hosts for several zoonotic viruses, the pig-derived PK-15 cell line is an indispensable tool for studying viral pathogenicity and developing treatments, vaccines, and preventive measures to mitigate the risk of disease outbreaks. However, we must consider the possibility of contamination by type I interferons (IFNs), such as IFNα and IFNβ, or IFN-inducing substances, such as virus-derived double-stranded RNA or bacterial lipopolysaccharides, in clinical samples, leading to lower rates of viral isolation. In this study, we aimed to generate a PK-15 cell line that can be used to isolate viruses from clinical samples carrying a risk of contamination by IFN-inducing substances. To this end, we depleted the IFN alpha and beta receptor subunit 1 (Ifnar1) gene or signal transducer and activator of transcription 2 (Stat2) gene in PK-15 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 method. Treatment of PK-15 cells lacking Ifnar1 or Stat2 with IFNβ or poly (I:C) resulted in no inhibitory effects on viral infection by a lentiviral vector, influenza virus, and Akabane virus. These results demonstrate that PK-15 cells lacking Ifnar1 or Stat2 could represent a valuable and promising tool for viral isolation, vaccine production, and virological investigations.

Despite the importance of the IFN cascade as the first line of defense against viral infections, its induction can impair the isolation of infectious viruses from clinical samples if there is

Design of single guide RNAs (sgRNAs)
Specific sgRNAs targeting pig Ifnar1 and Stat2 were designed using the online CRISPR Design Tool (https://crispr.dbcls.jp).The sequence of sgRNAs used in this study is summarized in Table 1.Two partially complementary oligos were used to generate the sgRNA scaffold.The complementary oligos were mixed and heated at 95˚C for 5 min, followed by incubation at room temperature for 1 h for oligo annealing.The annealed oligos were diluted 250-fold with water and used for ligation with the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector (Addgene, Cat# 62988), which was predigested with BbsI-HF (NEB, Cat# R3539L) using a DNA Ligation Kit (Mighty Mix, TaKaRa, Cat# 6023).The ligated constructs were then transformed in NEB 5-alpha F 0 Iq Competent E. coli (High Efficiency).The plasmids were analyzed using a primer (5 0 -ACTATCATATGCTTACCGTAAC-3 0 ) to verify the sequence.

Generation of PK-15 cells lacking Ifnar1 or Stat2
To generate k/o cells, PK-15 cells were transfected with PX459-Ifnar1-k/o or PX459-Stat2-k/o plasmids using the TransIT-X2 Dynamic Delivery System (TaKaRa, Cat# V6100) according to the manufacturer's instructions.At 48 h after transfection, cells were treated with 5 μg/mL puromycin (InvivoGen, San Diego, CA, USA, Cat# ant-pr-1).After 1 week of incubation, single-cell clones were sorted in a 96-well plate using a Cell Sorter SH800S (Sony Biotechnology, Inc., San Diego, CA, USA).The single-cell clones were characterized by infection with an HIV-based lentiviral vector.

Measurement of ISG expression
Normal PK-15 cells, PK-15 Ifnar1 k/o cells, or PK-15 Stat2 k/o cells were plated in a 96-well plate in sextuplicate as previously described and treated with 0 or 100 ng/mL IFNβ.After overnight incubation, total RNA was collected using a CellAmp Direct RNA Prep Kit for RT-PCR (Real Time) (TaKaRa, Cat# 3732) according to the manufacturer's instructions.Porcine myxovirus resistance 1 (Mx1), interferon-stimulated gene 15 (ISG15), and viperin messenger RNA (mRNA) levels were measured by a qRT-PCR assay using the One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (TaKaRa, Cat# RR096A).The PCR protocol was 42˚C for 5 min, 95˚C for 10 s, and 40 cycles of 95˚C for 5 s and 60˚C for 34 s.Porcine Mx1, ISG15, and viperin mRNA levels were normalized to those of porcine β-actin, which was used as an endogenous control (ΔΔCt method).The specific sequence primers for ISG mRNA levels were described previously [8].The primer sets are listed in Table 2.

Virus replication assay
Normal PK-15, PK-15 Ifnar1 k/o, or PK-15 Stat2 k/o cells were seeded into a 96-well plate at a density of 1 × 10 4 cells/well and treated with 0 or 100 ng/mL pig IFNβ.After overnight culture, the cells were infected with IAV or AKAV.After incubation at 37˚C for 2 h, the cells were washed, and fresh DMEM supplemented with 2% FBS and 1× Pe/St was added to each well with or without 100 ng/mL pig IFNβ.For experiments using IAV, the cells were maintained in DMEM supplemented with 10% FBS, 1× Pe/St, and 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketonetreated trypsin (Sigma-Aldrich, St. Louis, MO, USA, Cat# 4352157) [30,31].The supernatant was harvested 2 days after infection and mixed with 2× RNA lysis buffer (2% Triton X-100, 50 mM KCl, 100 mM Tris-HCl [pH 7.4], 40% glycerol, and 0.4 U/μL Recombinant RNase Inhibitor [TaKaRa, Cat# 2313A]), as described previously [32].The replication levels of IAV and AKAV were determined via qRT-PCR using the corresponding primer pairs (Table 3), as described previously [33].The analysis was performed using the QuantStudio 5 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) via One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect Real Time).The relative levels of viral RNA were calculated using the ΔΔCt method.
In addition, normal PK-15, PK-15 Ifnar1 k/o, or PK-15 Stat2 k/o cells were seeded as described previously and then transfected with 0 or 100 ng/mL poly (I:C) (Sigma-Aldrich, St. Louis, MO, USA, Cat# P1530) using the TransIT-X2 Dynamic Delivery System, according to the manufacturer's instructions.After overnight culture, the cells were infected with IAV or AKAV as described previously.
To reconstitute IFNAR1, PK-15 cells lacking Ifnar1, or Stat2 were infected with retroviral vectors expressing pig IFNAR1 (WT), pig IFNAR1 (W70C), or ZsGreen protein.After 48 h of W70C), or ZsGreen protein were plated at 1 × 10 4 cells/well.After 24 h, total RNA was extracted using a CellAmp Direct RNA Prep Kit for RT-PCR (Real Time) to measure the Ifnar1 expression.We used a forward primer (5 0 -GCTGAGGACAAGGCGATTAT-3 0 ) and a reverse primer (5 0 -GGAGTACACGAATGAGGATGAG-3 0 ) based on the codon-optimized sequence of pig Ifnar1 (S1 Text).Expression was measured as previously described.To assess antiviral activity in reconstituted cells, cells were treated with 0 or 100 ng/mL IFNβ for 24 h, and the viral infection experiment was performed as previously described.The mRNA expression of IGSs in reconstituted cells was determined as previously described.

Statistical analysis
Differences between treated and untreated cells were evaluated by an unpaired, two-tailed Student's t-test.Multiple comparisons were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's test.p � 0.05 indicated statistical significance.The test was performed using GraphPad Prism 9 software v9.1.1 (GraphPad, San Diego, CA, USA).

PK-15 cells lacking Ifnar1 or Stat2 lost the ability to induce ISGs upon IFNβ treatment
We determined that the activation of the type I IFN signaling pathway triggers a complex cascade of events that culminate in the expression of a broad array of ISGs, contributing to an antiviral state in infected cells.

Reconstitution of Ifnar1 in PK-15 Ifnar1 k/o cells restored the response to IFNβ
Off-target effects are an important concern associated with the CRISPR/Cas9 method [38].To elucidate the specificity of Ifnar1 k/o, we reconstituted IFNAR1 by infecting PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells with retroviral vectors expressing pig IFNAR1.In this experiment, we used both WT IFNAR1 and mutant IFNAR1 carrying the W70C mutation (W73C in human IFNAR1)-IFNAR1 (W70C)-which was previously reported to be associated with the impaired activity of human IFNAR1 [39].Although we attempted to detect reconstituted IFNAR1 via western blotting, we could not detect pig IFNAR1 using three commercial antihuman IFNAR1 antibodies.Therefore, we used qRT-PCR for detection.IFNs play major role in innate immunity.IFNs are essential molecules for inducing an antiviral state, thereby protecting virus-exposed cells, and neighboring cells.IFNs bind to IFNAR1 on the cell surface, initiating a downstream cascade of immune responses.The stimulation of this cascade leads to the induction of ISGs such as Mx1, ISG15, and viperin gene [40].These ISGs block several stages of viral replication, including viral entry, viral genome replication, and progeny virion release [41][42][43].Mx protein has shown antiviral effects against the influenza virus [2,44,45] and bunyavirus [43,46].In this study, robust induction of ISGs and We examined the potential risk of off-target effects of gene editing in PK-15 Ifnar1 k/o cells.The reconstitution of PK-15 Ifnar1 k/o cells with WT IFNAR1, but not with IFNAR1 carrying the W70C mutation, restored the cells' resistance to viral infection and ISG induction.Our results are consistent with a previous report demonstrating that W70C mutation in IFNAR1 (W73C in humans) resulted in increased severity of COVID-19 infection because of the loss of IFN-mediated immune responses [39].
In conclusion, our study demonstrated that PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells can be used for viral isolation from samples with possible contamination by IFNs or IFN-inducing substances.We believe that PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells will contribute to the isolation of several zoonotic diseases such as influenza virus, Japanese encephalitis virus, and Nipah virus.Furthermore, considering the importance of maintaining the health of pigs for sustainable agriculture, the method developed in this study can be beneficial for isolating viruses from clinical samples to understand the pathogenicity of life-threatening viral diseases in pigs, including African swine fever, and foot-and-mouth disease.Furthermore, isolated viruses can serve as a vaccine seed for preparing an inactivated or live-attenuated vaccine.

Supporting information
Following infection with an HIV-1-based reporter virus, single clones of PK-15 Ifnar1 k/o or PK-15 Stat2 k/o cells were assessed.Normal PK-15 cells efficiently prevented infection by the HIV-1-based reporter virus (Fig 1).Conversely, PK-15 Ifnar1 and PK-15 Stat2 k/o cells did not resist this viral infection (Fig 1), suggesting that cells lacking Ifnar1 or Stat2 cannot respond to IFNβ treatment.
To characterize PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells, we evaluated the mRNA expression of three ISGs (Mx1, ISG15, and viperin gene) upon IFNβ treatment.Treatment of normal PK-15 cells with 100 ng/mL pig IFNβ robustly induced ISG expression (over 100-fold) compared to that in untreated cells (Fig 2A and 2B).Conversely, both PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells displayed no or marginal induction of these ISGs upon IFNβ treatment (Fig 2A and 2B).Furthermore, we tested the induction of ISG15 protein upon IFNβ treatment by western blotting.In normal PK-15 cells, we observed a significant induction of ISG15 upon IFNβ treatment (Fig 2C), whereas ISG15 expression was not induced by IFNβ treatment in PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells (Fig 2C).These results demonstrated that PK-15 cells lacking Ifnar1 or Stat2 were unresponsive to IFNβ treatment.PK-15 cells lacking Ifnar1 or Stat2 are susceptible to viral infection in the presence of IFNβ or poly (I:C) Next, we examined whether Ifnar1 k/o or Stat2 k/o augments the susceptibility of PK-15 cells to viral infection in the presence of IFNβ.We used PK-15 Ifnar1 k/o (Clone #4-11) and PK-15 Stat2 k/o cells (Clone #1-2) for further analyses.IAV replication was significantly inhibited in normal PK-15 cells treated with IFNβ (Fig 3A).By contrast, IAV replication was not inhibited in PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells upon IFNβ treatment (Fig 3A).Previous research demonstrated the inter-species transmission of AKAV from cows to pigs [37].Therefore, the prevalence, and pathogenicity of AKAV in the pig population should be investigated.To test the usefulness of PK-15 Ifnar1 k/o cells for this purpose, we tested the replication of AKAV in PK-15 cells treated with pig IFNβ (Fig 3B).Whereas AKAV replication was blocked in normal PK-15 cells treated with IFNβ, there was no inhibition of AKAV replication in PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells treated with IFNβ (Fig 3B).These results demonstrated that both PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells supported viral replication in the presence of IFNβ.Furthermore, we employed a synthetic double-stranded RNA analog poly (I:C) to investigate whether Ifnar1 k/o and Stat2 k/o enhance the susceptibility of PK-15 cells to viral replication in the presence of interferon-inducible substances.In normal PK-15 cells treated with poly (I:C), IAV replication was significantly inhibited (Fig 4A).However, after poly (I:C) treatment, IAV replication was not inhibited or potentially enhanced in PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells (Fig 4A).Furthermore, although AKAV replication was suppressed in normal PK-15 cells treated with poly (I:C), no inhibition of AKAV replication was observed in PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells upon poly (I:C) treatment (Fig 4B).These results demonstrate that both PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells support viral replication in the presence of poly (I:C).

Fig 1 .
Fig 1.Generation and screening of PK-15 cells lacking Ifnar1 or Stat2.Normal PK-15 cells, PK-15 Ifnar1 k/o cells, or PK-15 Stat2 k/o cells treated with various concentrations of pig IFNβ were infected with HIV-1-based reporter virus.Infectivity was calculated as relative light units (RLU) 2 days after infection.Relative infectivity was calculated in comparison that in untreated cells.The results are presented as the mean and standard deviation of quadruplicate measurements from one assay, and they are representative of at least three independent experiments.Differences were examined by one-way ANOVA followed by Tukey's test.****p < 0.0001.https://doi.org/10.1371/journal.pone.0289863.g001

Fig 2 .
Fig 2. PK-15 cells lacking Ifnar1 or Stat2 exhibited a loss of ISG induction upon IFNβ treatment.(a) Induction of ISG mRNAs in normal PK-15 cells, PK-15 Ifnar1 k/o cells, and PK-15 Stat2 k/o cells was measured by qRT-PCR 1 day after IFNβ treatment.The results are presented as the mean and standard deviation of sextuplicate measurements from one assay, and they are representative of at least three independent experiments.Differences between 100 ng/mL IFNβ-treated cells and untreated cells were examined by a two-tailed, unpaired Student's t-test.****p < 0.0001, ns (not significant).(b) The presented in Fig 2a were used to compare ISG induction among normal PK-15 cells, PK-15 Ifnar1 k/o cells, and PK-15 Stat2 k/o cells.Multiple comparisons were examined by one-way ANOVA followed by Tukey's test.****p < 0.0001.(c) Expression of ISG15 protein (15 kDa) in normal PK-15, PK-15 Ifnar1 k/o, and PK-15 Stat2 k/o cells was determined via western blotting, as depicted in the lower panel.The quantity of input protein was visualized using the Total Protein Detection Module, as depicted in the upper panel.https://doi.org/10.1371/journal.pone.0289863.g002 Ifnar1 k/o cells reconstituted with IFNAR1 (WT) or IFNAR1 (W70C) displayed significantly lower Ct values than control cells (Fig 5A), suggesting that IFNAR1 molecules were successfully reconstituted in these cells.We performed an infection assay in reconstituted cells using AKAV.The results demonstrated that PK-15 Ifnar1 k/o cells reconstituted with IFNAR1 (WT) showed resistance to viral infection, similar to that in normal PK-15 cells (Fig 5B).Conversely, PK-15 Ifnar1 k/o cells reconstituted with IFNAR1 (W70C) showed no resistance to viral infection, suggesting that the restoration of antiviral activity requires reconstitution with WT IFNAR1.As expected, neither IFNAR1 (WT) nor IFNAR1 (W70C) in PK-15 Stat2 k/o cells showed restoration of the resistance to viral infection (Fig 5B).Consistent with the results of infection activity, ISG

Fig 3 .
Fig 3. PK-15 cells lacking Ifnar1 or Stat2 are susceptible to viral infection in the presence of IFNβ.Replication of IAV (a) and AKAV (b) was examined in normal PK-15 cells, PK-15 Ifnar1 k/o cells, and PK-15 Stat2 k/o cells.Cells were pretreated with 0 or 100 ng/mL IFNβ before infection.Viral RNA levels in the culture supernatant were quantified by qRT-PCR 2 days after infection.The relative values were calculated in comparison to that in nonpretreated cells.The results are presented as the mean and standard deviation of sextuplicate measurements from one assay.Differences between 100 ng/mL IFNβ-treated cells and untreated cells were examined by a two-tailed, unpaired Student's t-test.***p < 0.001, **p < 0.05, ns (not significant).https://doi.org/10.1371/journal.pone.0289863.g003

Fig 4 .Fig 5 .
Fig 4. PK-15 cells lacking Ifnar1 or Stat2 exhibit susceptibility to viral infection in the presence of poly (I:C).The replication of IAV (a) and AKAV (b) was examined in normal PK-15, PK-15 Ifnar1 k/o, and PK-15 Stat2 k/o cells.Before infection, the cells were transfected with 0 or 100 ng/mL poly (I:C).Viral RNA levels in the culture supernatant were quantified via qRT-PCR 2 days after viral infection.The relative values were calculated with respect to nonpretreated cells.The results are presented as the mean and standard deviation of eight repeated measurements from one assay.Differences between 100 ng/mL poly (I:C)-treated cells and untreated cells were examined via a two-tailed, unpaired Student's t-test.****p < 0.0001, **p < 0.01, and *p < 0.05, ns (not significant).https://doi.org/10.1371/journal.pone.0289863.g004

S1
Dataset.Raw data for the luciferase assay.(XLSX) S1 Raw image.Raw images of western blotting.(PDF) S1 Text.Synthesized DNA for generating a plasmid expressing pig IFNAR1.The coding sequence of pig IFNAR1 was synthesized according to the amino acid sequence deposited in GenBank (Acc.Num.NM_213772.1)with codon optimization to pig cells.The start and stop codons are underlined.(TXT)