Cell lines, viruses, antibodies, and magnetic beads

Human embryonic kidney (HEK) 293T cells, L929 cells, Vero cells, LLC-PK1 cells, Expi293F cells, and IPEC-J2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells, except Expi293F cells, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Cytiva, Shanghai, China) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 10 µg/mL streptomycin, and 100 U/mL penicillin (BasalMedia, Shanghai, China). The cultures were maintained at 37 °C in a 5% CO2 atmosphere. Expi293F cells were cultured in OPM-293-CD05 medium (81075, OPM, Shanghai, China) at 37 °C with 8% CO2 and shaken at 120 rpm. The PEDV strain CHFJFQ (GenBank: OP688373.1) was isolated in our laboratory in 2018 from intestinal samples of pigs with diarrhea in Fuqing, Fujian, China.

The anti-PEDV N protein monoclonal antibodies anti-PEDV NP-FITC (SD-2 F-1) and anti-PEDV NP (SD-2-1) were purchased from Medgene Labs (Brookings, SD, USA). GAPDH monoclonal antibody (60004-1-Ig), beta-actin monoclonal antibody (81115-1-RR), His-tag polyclonal antibody (10001-0-AP), His-tag monoclonal antibody (66005-1-Ig), HA-tag polyclonal antibody (51064-2-AP), DYKDDDDK-tag polyclonal antibody (20543-1-AP), and Siglec-15 polyclonal antibody (28594-1-AP) were obtained from Proteintech Biotechnology Co., Ltd. (Wuhan, China). Additionally, IRDye® 800CW donkey anti-mouse or anti-rabbit IgG secondary antibodies (LI-COR, Lincoln, NE, USA), as well as Alexa Fluor 555 donkey anti-rabbit IgG (H + L) and Alexa Fluor 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibodies (Beyotime, Shanghai, China), were utilized. For coimmunoprecipitation assays, Mouse IgG (A7028, Beyotime, Shanghai, China), BeyoMag™ Protein G magnetic beads (P2105-1 mL, Beyotime, Shanghai, China), BeyoMag™ Anti-Flag Magnetic Beads (P2115-0.5 mL, Beyotime, Shanghai, China), BeyoMag™ Anti-His Magnetic Beads (P2135-0.5 mL, Beyotime, Shanghai, China) and BeyoMag™ Anti-HA Magnetic Beads (P2121-0.5 mL, Beyotime, Shanghai, China) were used.

Plasmids and SgRNA design

The overexpression plasmids for CD62L, L1CAM, Siglec-16, Siglec-10, Siglec-2, CD24, Siglec-3, Siglec-15, and CD83 (PLVX-CD62L, PLVX-L1CAM, PLVX-Siglec-16, PLVX-Siglec-10, PLVX-Siglec-2, PLVX-CD24, PLVX-Siglec-3, PLVX-Siglec-15 and PLVX-CD83) were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). Plasmids for expressing human, porcine, and mouse Siglec-15 (pLVX-H-Siglec-15-His, pLVX-P-Siglec-15-His, pLVX-M-Siglec-15-His), as well as vectors for expressing truncated forms of human and porcine Siglec-15 (pCDNA3.1-H-Siglec-15 (20–263 aa)-Fc, pLVX-P-S15∆NRC-V-His, pLVX-P-S15∆NRC-C2-His, pLVX-P-S15∆V-C2-His), and vectors for the light and heavy chains of human Siglec-15 monoclonal antibodies (pFUSE2-CLIg-rk1-IL2SP-26H4, pFUSE2-CHIg-rG1-IL2SP-26H4) were synthesized by Beijing Tsingke Biotech Co., Ltd. (Beijing, China). The plasmid vectors used to overexpress the PEDV S1, S2, E, M, and N proteins (pLVX-S1-HA, pLVX-S2-HA, pLVX-E-Flag, pLVX-M-Flag, pLVX-N-Flag) were synthesized by Fuzhou Shangya Biotechnology Co., Ltd. (Fuzhou, China). The helper plasmids pCMV-VSV-G (#8454) and pSPAX2 for packaging lentivirus (#12260), the plasmid pX459 (#48139) for Siglec-15 knockout and the plasmid expressing TEV protease (pET28-MBP-super TEV protease) (#171782) were obtained from Addgene (Boston, MA, USA).

The sgRNA used for gene knockout was designed via the website http://www.e-crisp.org/ (sgRNA sequences are provided in Supplementary Table 1). Following annealing, the sgRNA was ligated with the BpiI-digested pX459 plasmid via T4 DNA ligase (2011 A, Takara, Tokyo, Japan). All plasmids were transformed into Trans1-T1 phage-resistant chemically competent cells (CD501-02, TransGen, Beijing, China) and cultured in Luria-Bertani (LB) media (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl). A FastPure EndoFree Plasmid Mini Plus Kit (DC204-01, Vazyme, Nanjing, China) was utilized to extract the plasmids.

PEDV amplification and titer assays

PEDV was propagated in Vero cells, and the culture supernatant and cells were harvested when the cytopathic effect exceeded 80%. The supernatant was centrifuged at 3000 rpm for 5 minutes for titer determination. To investigate the effects of sialic acid and sialidase on PEDV proliferation, 1 × 106 cells (per well) and PEDV (1 × 106 TCID50 or 2 × 105 TCID50), along with various concentrations of sialic acid (1 mM, 0.5 mM, 0.25 mM, 0.125 mM) (S20140-5 g, Yuanye, Shanghai, China) or sialidase (0.01 U/mL, 0.02 U/mL, 0.03 U/mL, 0.04 U/mL) (S10170-25U, Yuanye, Shanghai, China), were added to each well of a 6-well plate. The cells were harvested at 36 h post-infection (hpi) for RNA or protein extraction, and PEDV proliferation was assessed via RT-qPCR or Western blot analysis.

293T cells were seeded at 1 × 106 cells per well in 6-well plates and cultured for 12 h. Cells were then transfected with overexpression plasmids using Lipofectamine™ 3000 (L3000-008, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The following plasmids were used for transfection: pLVX-CD62L, pLVX-L1CAM, pLVX-Siglec-16, pLVX-Siglec-10, pLVX-Siglec-2, pLVX-CD24, pLVX-Siglec-3, pLVX-Siglec-15, and pLVX-CD83. Control cells were transfected with the empty vector pLVX-E. At 12 h post-transfection, 1 × 106 cells per well were seeded in 6-well plates and infected with PEDV (MOI = 0.1) for 36 h. Subsequently, Western blot analyses were performed to assess the expression of the PEDV N protein and determine the impact of CD62L, L1CAM, Siglec-16, Siglec-10, Siglec-2, CD24, Siglec-3, Siglec-15, and CD83 overexpression on PEDV infection.

PEDV was propagated in LLC-PK1, LLC-PK1Siglec − 15−/−, and LLC-PK1Siglec − 15−/−-P-S15 cells. A total of 1 × 106 cells (per well) were added to a 6-well plate for coculture with 1 × 106 TCID50 or 1 × 105 TCID50 of PEDV (per well). The culture supernatant was collected every 12 h for titer determination. The virus titer was assessed via the Spearman-Kärber method [78, 79].

PEDV adsorption and internalization assays

To investigate the effects of sialic acid and sialidase on PEDV cell adsorption, 293T cells were cultured in confocal dishes (2 × 105 cells/dish) or 6-well plates (1 × 106 cells/well) for 12 h. PEDV (1 × 107 TCID50 in 1 mL) was treated with sialic acid (1 mM) at 37 °C for 2 h, added to [79] were incubated with sialidase (0.04 U/mL) at 37 °C for 2 h. The culture media were then discarded, and the cells were incubated with 1 × 107 TCID50 of PEDV at 4 °C for 2 h. Afterward, the cells were washed twice with PBS (4 °C), and fixed with 4% paraformaldehyde (1 mL) at room temperature for 1 h, and PEDV cell adsorption was assessed by immunofluorescence. RNA was extracted from the cells in the 6-well plates via TRIzol™, and the cell adsorption of PEDV was subsequently assessed via RT-qPCR.

To explore the effects of Siglec-15 on PEDV adsorption and internalization, 1 × 106 LLC-PK1, LLC-PK1Siglec − 15−/−, or LLC-PK1Siglec − 15−/−-P-S15 cells were added to each well of a 6-well plate and cultured for 12 h. After the culture medium was discarded, 1 × 107 TCID50 of PEDV was added to each well, and the samples were incubated at 4 °C for 2 h. The cells were then washed three times with PBS. For internalization analysis, after PEDV adsorption, 2 mL of DMEM was added, and the cells were incubated at 37 °C for 2 h. A citric acid solution (pH = 3) consisting of 23.93 g/L sodium citrate dihydrate and 3.52 g citric acid was used to wash the cells three times. Following PEDV adsorption or internalization, the efficiency of adsorption and internalization was evaluated via RT-qPCR or immunofluorescence.

RNA sequencing and data processing

RNA was extracted from 1 × 10⁷ 293T cells per sample, with three biological replicates, for RNA sequencing. Total RNA was extracted via TRIzol™ reagent (Sigma-Aldrich) following the manufacturer’s instructions. A total of 2 µg of RNA was used as the starting material for RNA sample preparation. The sequencing libraries were constructed via the NEBNext Ultra™ RNA Library Preparation Kit (New England BioLabs, USA) according to the manufacturer’s guidelines. Index-labeled samples were clustered on the cBot Cluster Generation System via the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, Inc.). After cluster generation, the libraries were sequenced on the Illumina NovaSeq platform, which produced paired-end reads. Library construction and Illumina sequencing were performed by WUHAN IGENEBOOK BIOTECHNOLOGY CO., LTD. (Wuhan, China). Clean reads were obtained by removing adapter sequences, poly-N stretches, and low-quality reads from the raw data. The quality of the clean data was assessed by calculating the Q20, Q30, and GC contents, and sequence duplication levels. Clean reads were aligned to the reference genome (GRCh38.p14) via HISAT2 (v.2.2.1), and gene expression levels for each sample were quantified with featureCounts (v.2.0.6).

Construction of stable Siglec-15-overexpressing and Siglec-15-knockout cell lines

The pLVX-H-Siglec-15, pCMV-VSV-G, and pSPAX2 plasmids (8:4:6) µg were transfected into 293T cells (100 mm petri dish, approximately 85% confluence) via Lipofectamine™ 3000 to package the lentivirus. 5 × 105 293TSiglec− 15−/− cells were added to each well of a 6-well plate and cocultured with 1 mL of lentiviruses for 36 h. The Siglec-15 stable transgenic 293T cells were selected with puromycin (A1113803, Gibco, Grand Island, NY, USA) at a final concentration of 2 µg/mL.

For knockout experiments, 5 × 105 cells (293T, L929, or LLC-PK1) were plated in each well of a 6-well plate for 12 h. Each well was transfected with 4 µg of the knockout plasmid via Lipofectamine™ 3000, cultured for 36 h and selected with puromycin for 3 days (293T: 2 µg/mL, L929: 7 µg/mL, LLC-PK1: 4 µg/mL). The surviving cells were used to isolate monoclonal cells.

Genomic DNA was extracted via the FastPure Cell/Tissue DNA Isolation Mini Kit (DC102-01, Vazyme, Nanjing, China). One hundred nanograms of DNA were used for PCR to amplify the target fragment of Siglec-15 (the PCR primers are listed in Supplementary Table 2). Siglec-15 gene knockout was confirmed by sequencing analyses conducted by Beijing Tsingke Biotech Co., Ltd. (Beijing, China), and the sequences were analyzed via BioEdit software (v.7.0.5.3).

Western blotting

Proteins were extracted via RIPA buffer (P0013B, Beyotime, Shanghai, China), and their concentrations were determined via the Enhanced BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China). Sixty micrograms of protein were loaded for SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (IPFL00010, Millipore, Massachusetts, USA). The PVDF membrane was blocked with a 5% bovine serum albumin (BSA) solution (V900933, Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 2 h. Primary antibodies, including GAPDH (1:5000), β-actin (1:5000), anti-PEDV NP (1:1000), Siglec-15 (1:1000), His (1:500), Flag (1:500), and HA (1:500), were incubated with the samples at 4 °C for 12 h. Next, the sections were incubated with IRDye® 800CW donkey anti-mouse IgG secondary antibody (1:10,000) and IRDye® 800CW donkey anti-rabbit IgG secondary antibody (1:10,000) at room temperature for 2 h. The PVDF membrane was then visualized via a LI-COR Odyssey infrared fluorescence scanner (LI-COR) after washing with TBST. The primary antibodies were diluted in 5% BSA, while the secondary antibodies were diluted in TBST.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from cells and tissues was extracted via TRIzol™ (15596018; Thermo Fisher, Waltham, MA, USA). cDNA was synthesized from 1 µg of RNA via HiScript® III RT SuperMix for qPCR (+ gDNA wiper) (R323-01, Vazyme, Nanjing, China). The qPCR mixture consisted of 100 ng of cDNA (2 µL), forward and reverse primers (0.5 µL each at 100 µM), 10 µL of ChamQ Universal SYBR qPCR Master Mix (Q711-02/03, Vazyme, Nanjing, China), and 7 µL of ddH2O. The reactions were incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. GAPDH was used as the normalization control. The mRNA levels are presented as fold changes relative to those in the control group and were calculated via the 2−ΔΔCt method. The expression stability of each gene was verified by averaging triplicate Ct values (RT-qPCR primers are listed in Supplementary Table 3).

Flow cytometry

One million 293T, 293TSiglec− 15−/−, or 293TSiglec− 15−/−-H-S15 cells were added to each well of a 6-well plate and cocultured with 5 × 105 TCID50 of PEDV per well. The cells were digested with 0.25% trypsin and harvested every 12 h. For fixation and permeabilization, the cells were treated with 1 mL of fixation/permeabilization solution (554714, BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 15 min. Following centrifugation at 400 × g for 5 minutes, the supernatant was discarded, and 1 mL of ddH2O-diluted Perm/Wash™ Buffer (554714, BD Biosciences, Franklin Lakes, NJ, USA) was added and incubated for 10 min at room temperature. Next, 1 × Perm/Wash™ Buffer and 1 µL of anti-PEDV NP-FITC (SD-2 F-1, Medgene Labs, Brookings, SD, USA) were added and incubated at room temperature for 2 h. After another centrifugation at 400 × g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS containing 1% FBS. The samples were analyzed via a FACSymphony™ A5 cell analyzer (BD Biosciences, Franklin Lakes, NJ, USA), and the data were processed via FlowJo software (v.10.8).

Immunofluorescence and coimmunoprecipitation

HEK293T cells (5 × 106 cells per dish) were cultured in a 100 mm diameter dish for 12 h. The plasmids pLVX-H-Siglec-15-His, pLVX-P-Siglec-15-His, and pLVX-M-Siglec-15-His, which are designed to overexpress human, pig, and mouse Siglec-15, or the plasmids pLVX-P-S15∆NRC-V-His, pLVX-P-S15∆NRC-C2-His, and pLVX-P-S15∆V-C2-His for overexpressing truncated forms of porcine Siglec-15, were cotransfected into the 293T cells via Lipofectamine™ 3000 along with the plasmids pLVX-S1-HA and pLVX-M-Flag, which overexpress the PEDV S1 and M proteins, respectively. At 36 h posttransfection, 2 × 105 cells were cultured in confocal dishes for an additional 12 h, followed by washing with phosphate-buffered saline (PBS). The cells were then fixed at room temperature for 30 min with 1 mL of immunostaining fix solution (P0098-500 mL, Beyotime, Shanghai, China). The cells were subsequently permeabilized with 1 mL of immunostaining permeabilization buffer containing Triton X-100 (P0096-500 mL; Beyotime, Shanghai, China) for 15 min at room temperature and blocked with QuickBlock™ Blocking Buffer for immunostaining (P0260, Beyotime, Shanghai, China) for 15 min at room temperature.

Primary antibodies (His (1:200) with Flag (1:200) or His (1:200) with HA (1:200)) were added and incubated at 4 °C for 12 h. The cells were then washed twice with PBS, and Alexa Fluor 555 Donkey anti-rabbit IgG (H + L) and Alexa Fluor 488 Donkey anti-mouse IgG (H + L), both highly cross-adsorbed secondary antibodies (1:500), were added and incubated at room temperature for 2 h. After two additional washes with PBS, 200 µL of 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (C1005, Beyotime, Shanghai, China) was added, and the mixture was incubated at room temperature for 10 min. Imaging was performed via an LSM 780 laser confocal system (Zeiss, Oberkochen, Germany).

At 36 h posttransfection, the cells were harvested for protein extraction via RIPA buffer. Magnetic beads (20 µL) were added and incubated for 12 h at 4 °C. The magnetic beads were then separated via a DynaMag-2 magnet (Thermo Fisher Scientific, Waltham, MA, USA), and the beads were washed twice with TBST. Subsequently, 40 µL of RIPA buffer and 10 µL of SDS-PAGE loading buffer (5×) (P0015L, Beyotime, Shanghai, China) were added, and the mixture was heated at 95 °C for 5 min. The supernatant was collected for Western blot analysis.

For the coimmunoprecipitation experiment involving PEDV virus particles and Siglec-15, magnetic beads were coated with Siglec-15 and then incubated with PEDV (1 × 108 TCID50) at 4 °C for 12 h. The binding of Siglec-15 to PEDV was subsequently detected by Western blot analysis.

Hematoxylin-eosin (H&E) staining and immunohistochemistry

The mice were infected with PEDV, and tissues (heart, liver, spleen, lung, kidney, brain, and small intestine) were fixed in 4% paraformaldehyde at room temperature for 12 h. For hematoxylin-eosin (H&E) staining, the tissues were dehydrated in ethanol, embedded in paraffin, and sectioned at a thickness of 4 μm. These sections were dewaxed, made transparent, and stained with hematoxylin and eosin to evaluate histopathological changes. For immunohistochemistry, paraffin sections were dewaxed and rehydrated, and tissue antigens were retrieved at high temperatures and pressure. The samples were treated with 3% H2O2, followed by blocking with 10% BSA at room temperature for 2 h. The primary antibody (anti-PEDV NP at a dilution of 1:400) was added, and the samples were incubated at 4 °C for 12 h. Subsequently, 100 µL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000) (A0216, Beyotime, Shanghai, China) was added, and the mixture was incubated at 37 °C for 2 h. Finally, PEDV infection was observed via DAB staining with hematoxylin counterstaining.

Expression and purification of the TEV protease

A plasmid (pET28-MBP-super TEV protease) expressing TEV protease was transfected into competent BL21 (DE3) cells (Thermo Fisher Scientific, Waltham, MA, USA). Positive clones were selected by screening with kanamycin sulfate (K8020; SOLARBIO, Beijing, China). These positive clones were cultured in an LB medium and induced with 1 mol/L isopropyl-β-D-thiogalactopyranoside (IPTG) (I1020, SOLARBIO, Beijing, China) to express TEV protease. The TEV protease was purified via a nickel column and eluted with 200 mM imidazole.

H-Siglec-15 (20–263 aa) purification and immunization

Five hundred micrograms of plasmid (pCDNA3.1-H-Siglec-15 (20-263aa)-Fc) were transfected into Expi293F cells (cell density of 3 × 106 cells/mL in 200 mL) via polyethylenimine. Following transfection, the culture was maintained for 7 days, after which the supernatant was collected. The protein was enriched via Protein A Resin FF (L00464, GenScript, Nanjing, China) and eluted with a glycine solution (0.1 M). The protein was then concentrated in a 30 kDa ultrafiltration tube. H-Siglec-15 (20-263aa)-Fc was digested with TEV protease, and the Fc fragment, along with the TEV protease, was removed via a Protein A Resin FF and a nickel column. A total of 1.5 mL of H-Siglec-15 (20-263aa) (1 mg/mL) was mixed with 1.5 mL of Freund’s complete adjuvant (F5881, Sigma-Aldrich, St. Louis, MO, USA) and emulsified for immunization of a 3-month-old New Zealand rabbit. Immunization was performed every two weeks for a total of three immunizations.

H-Siglec-15 monoclonal antibody Preparation

The rabbits were immunized three times with H-Siglec-15 (20–263 aa), and venous blood was collected to extract B cells. The light and heavy chains (VL and VH) of the Siglec-15 antibodies were obtained through nested PCR and ligated to the expression plasmid (nested PCR primers are listed in Supplementary Table 4). Both the light and heavy chains (VL and VH) were double-digested with EcoRI and Eco91I and then linked via T4 DNA ligase. Plasmids expressing the human Siglec-15 monoclonal antibody light chain (pFUSE2-CLIg-rk1-IL2SP-26H4) and heavy chain (pFUSE2-CHIg-rG1-IL2SP-26H4) were cotransfected into Expi293F cells. After 6 days of culture, the antibodies were enriched with Protein A Resin FF (L00464; GenScript, Nanjing, China). Finally, the antibodies were eluted with a glycine solution (0.1 M) and concentrated in a 30 kDa ultrafiltration tube.

PEDV infection in mice

Six-week-old C57BL/6J wild-type mice and Siglec-15 knockout mice were divided into a control group and a PEDV inoculation group, with 5 mice in each group. The mice in the inoculation group received 1 × 106 TCID50 of PEDV (100 µL) via gavage, while those in the control group were administered DMEM. The experiment was terminated at 72 h post-infection (hpi). RT-qPCR and immunohistochemistry were used to detect PEDV infection in the organs of the mice, and H&E staining was used to assess tissue damage caused by PEDV infection. Specific pathogen-free C57BL/6J male mice (6 weeks old) were randomly allocated to the experimental groups and maintained at the Animal Experimental Center of Fujian Normal University under controlled conditions of 23–25 °C, 40–60% humidity, and a 12 h light/dark cycle, with free access to food. C57BL/6J wild-type mice were obtained from Wu’s Animal Center (Fuzhou, China), while C57BL/6J-Siglec15 knockout mice were obtained from Cyagen Biosciences Inc. (Guangzhou, China).

Siglec-15 antibody blockage of PEDV infection in cells and mice

One million 293T, 293TSiglec − 15−/−, or 293TSiglec − 15−/−-H-S15 cells were added to each well of a 6-well plate. After 12 h of culture, 10 µg (20 µL) of the Siglec-15 monoclonal antibody was added (the control group received an equal volume of PBS) and incubated at 37 °C for 30 min. The culture medium was then replaced with fresh medium, and 5 × 105 TCID50 (per well) of PEDV was added, followed by continuing culture for 2 h. The culture media was then replaced with fresh media, followed by continued culture for 36 h. RT-qPCR was employed to assess the blocking effect of the Siglec-15 monoclonal antibody on PEDV infection.

Six-week-old wild-type mice, Siglec-15 knockout mice, and Siglec-15 humanized mice were divided into a control group, a PEDV inoculation group, and a Siglec-15 antibody blocking group, with 5 mice in each group. The PEDV inoculation group received an intranasal inoculation of 1 × 106 TCID50 (50 µL) of PEDV, whereas both the PEDV inoculation group and the Siglec-15 antibody blocking group were treated with 50 µL of DMEM and 50 µg of Siglec-15 antibody via nasal drip 30 min prior to PEDV inoculation. The experiment was terminated at 72 h post-infection (hpi), and the lungs of the mice were collected for detection of PEDV infection via RT-qPCR and immunohistochemistry. C57BL/6J-Siglec15 humanized mice were obtained from GemPharmatech Co., Ltd. (Jiangsu, China).

Prediction and analysis of protein interaction structure

The amino acid sequences of the S1 and M proteins of PEDV and porcine Siglec-15 were obtained from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/), with the following protein IDs: Siglec-15 (XP_003121465.3, https://www.ncbi.nlm.nih.gov/protein/1191806540), PEDV-CHFJFQ S (WFG70067.1, https://www.ncbi.nlm.nih.gov/protein/2476855545), and PEDV-CHFJFQ M (WFG70070.1, https://www.ncbi.nlm.nih.gov/protein/2476855548). The 3D complex of porcine Siglec-15 with the PEDV S1 or M proteins was simulated via molecular docking via AlphaFold3 (https://www.alphafoldserver.com) [80], with the molar ratios of Siglec-15 to PEDV S1 and M proteins set at 1:3 and 1:1, respectively. Protein-protein interactions were analyzed via PDBePISA (v.1.52) [81], which provides thermodynamic properties of the complexes, including binding free energy (ΔiG), entropy change after dissociation (TΔS), and the interface area (Ų). Amino acid residues involved in interactions between protein complexes were analyzed via the PDBsum (v.2.2) online tool (https://www.ebi.ac.uk/pdbsum) [82]. Finally, the complex structure was visualized via PyMOL (v.3.0) [83].

Statistical analyses

Image processing was conducted via Adobe Illustrator 2022, BioEdit (v.7.0.5.3), and ImageJ (v.1.8.0). Statistical analyses were performed with GraphPad Prism (v.8.0), employing unpaired t-tests and two-way analyses of variance (ANOVAs). The results are presented as the means ± standard deviations (SDs), with significance set at P < 0.05.