Pk 15

Viral stocks were propagated and titrated in the porcine kidney cell line (PK-15, ATCC^®:CCL-33, Manassas, VA, USA), cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum pestivirus-free at 37 °C in 5% CO₂. The highly virulent CSFV strain Margarita, which belongs to subgroups Genogroup 1.4 was employed for the challenge experiment and neutralization assay [17 (link),18 (link)]. Additionally, the Alfort/187 strain of genotype 1.1, was also employed. Virus titration was conducted by end-point dilution using a peroxidase-linked assay (PLA) and the titers calculated according to Reed and Muench [19 (link)].

Human embryonic kidney (HEK293T) cells and the porcine kidney cell line (PK-15) (ATCC) were cultivated in Dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum, 100 U penicillin/ml, and 100 μg streptomycin/ml in a humidified incubator with 5% CO₂ at 37 °C. Visa^–/– and wild-type mouse embryonic fibroblasts (MEFs) were kindly provided by Dr. Hong-bing Shu (Wuhan University). The antibodies used in this study were as follows: rabbit ployclonal antibodies against PCBP2 (Abcam), IgG (Ig) (Sigma), and VISA (CST); mouse monoclonal antibodies against flag, Myc, IgG (Ig), β-actin, GST (Sigma), HA (OriGene), and GFP (Thermo Fisher Scientific). Mouse anti-VP3 sera were prepared in our laboratory using a recombinant FMDV VP3 protein. Sendai virus (SeV) inducing the activation of interferon was previously described^{18 (link)}. The type O FMDV was propagated in PK-15 cells, and the supernatants of the infected cells were clarified and stored at –80 °C.

HEK293T (human) (CRL-11268; American Type Culture Collection [ATCC]), NIH3T3 (mouse) (CRL-1658; ATCC), MDTF (mouse), CRFK (cat) (CCL-94; ATCC), FEA (cat) (feline embryonic fibroblasts), Cf2Th (dog) (CRL-1430; ATCC), A72 (dog) (CRL-1542; ATCC), Mpf (ferret) (CRL-1656; ATCC), PK15 (pig) (CCL-33; ATCC), Vero 76 (CRL-1587; ATCC), and BHKT7/9 (a hamster kidney–derived BHK cell clone stably expressing T7 RNA polymerase) (31 (link)) cells were cultured in Dulbecco’s modified Eagle's medium (Sigma–Aldrich), supplemented with 10% heat-inactivated fetal calf serum and antibiotics (Thermo Fisher Scientific). The SFTSV YG1 strain, a field isolate from an SFTS patient in Japan, was kindly provided by Dr Ken Maeda, NIID. Virus stocks were prepared from the culture supernatants of Vero 76 cells.

A549, A549 PKR knockout (A549/PKRko), A549 RNase L knockout (A549/RNaseLko), and A549 PKR/RNase L double-knockout (A549/dko) cells were provided by B. Moss (NIH, USA) (26 (link)). A549 cells expressing human ACE2 and TMPRSS2 were purchased from InvivoGen (San Diego, CA, USA). PK15, BHK21, and HeLa cells were obtained from the ATCC. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), glutamine, and penicillin-streptomycin at 37°C with 5% CO₂. The vaccinia virus (VACV) Western Reserve (WR) strain and the E3L and K3L double-deletion mutant (VACVΔE3ΔK3) were described previously (18 (link)), and SARS-CoV-2 was an ancestral Canadian isolate (hCoV/Canada/ON-VIDO-01/2020) described previously (61 (link)). The VACV-related experiments were performed in our biosafety level 2 (BSL-2) laboratory, and SARS-CoV-2 infections were done in our BSL-3/4 containment laboratory.

Mouse fibroblast cell line L-929 (ATCC CCL-1) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM)/Nutrient Mixture F-12 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% non-essential amino acids. Pig kidney cell line PK-15 (ATCC CCL-33), mouse embryo fibroblast cell line NIH 3 T3 (ATCC CRL-1658), AAV-293 cells (Stratagene, USA) and African green monkey kidney cell line MARC-145 cells (ATCC CRL-12231) were grown in DMEM supplemented with 10% FBS and 1% non-essential amino acids. Primary PAMs were prepared from 6-week-old Lancrace pigs as described previously [7 (link)] and grown in DMEM/F-12 supplemented with 10% FBS, 1% non-essential amino acids and 20% L-929-conditioned medium. PRRSV strain VR-2332 (ATCC VR2332) is a prototype strain of North American genotype [28 (link)]. PRRSV strain CH-1R is an attenuated vaccine strain derived from the traditional Chinese strain CH-1a [29 ]. PRRSV strain JX-A1 is a highly pathogenic Chinese strain [30 (link)]. The three PRRSV strains were propagated and titrated on MARC-145 cells.

The porcine kidney cell line PK-15 was obtained from ATCC (CCL-33, Middlesex, England) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% foetal bovine serum (FBS), Pestivirus-free, at 37 °C in 5% CO₂.
The CSFV moderately virulent Catalonia 01 (Cat01) strain, which belongs to the 2.3 subgenotype and has been proven to induce postnatal persistent infection in newborn piglets, was used [15 (link),26 (link)]. The Alfort 187 strain, genogroup 1.1, was used in the neutralisation peroxidase-linked assay (NPLA). Viruses were grown in the PK-15 cell line that were infected with 0.1 TCID₅₀/cell in 2% FBS during 72 h of incubation. Peroxidase-linked assay (PLA) [27 (link)] was used for viral isolation and titration following the statistical methods previously described [28 (link)]. The attenuated vaccine (C-strain) belongs to the CSFV 1.1 genogroup and was used in Spain in the 1980s for CSF control. This vaccine has 100% homology with the Z46258 strain. This vaccine strain was employed to immunize animals in order to obtain IFN-γ producing PBMCs for the co-culture assays [29 (link),30 (link)].

HEK293T (ATCC CRL11268), mouse leukemic monocyte macrophage (RAW264.7; ATCC TIB-71), PK-15 (ATCC CCL-33), LF-BK (RRID:CVCL_RX26), BHK-21 (ATCC CCL-10), and Vero (ATCC CCL-81) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM-high glucose; Gibco, California, United States) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% antibiotic/antimycotic solution (Gibco). Cells were incubated at 37°C under a 5% CO₂ atmosphere.