All ASFV strains used in this study are listed in Table 1. To generate the intact ASFV virus particle for mice immunization, ASFV Lisbon/61, adapted in African green monkey kidney cells (Vero-76), was used (16). Briefly, Vero-76 cells (ATCC) grown in T-75 cell culture flasks were infected with ASFV Lisbon/61 at a multiplicity of infection (MOI) of 0.1. The infected cultures were incubated at 37°C in a 5% CO₂ incubator for 5–7 days until 90%–100% of the cells showed cytopathic effects. The flasks were then frozen overnight at −20°C, thawed the next day, and the contents were harvested and then sonicated for 30 s three times on ice. The resulting cell suspension was centrifuged at 3,600 × g for 10 min at 4°C to remove cellular debris. The supernatant was then ultra centrifuged (JA20 Beckmann rotor) at 8500 rpm for 6 h at 4°C. The resulting pellet was resuspended in DMEM, layered onto a 40% sucrose cushion, and centrifuged at 20,000 rpm (49,400 × g) for 45 min at 4°C using a Beckman SW-41Ti rotor. The pellet was resuspended in PBS and aliquoted into 2 mL cryovials. The vials were then immersed in a 70°C water bath for 60 min and subsequently exposed to gamma irradiation (5 million rad) to inactivate the virus. The treated ASFV were used for mouse immunization. Other ASF viruses used in this study were propagated and stored as previously described (17).

Monoclonal antibodies against ASFV were produced as previously described (18, 19). Briefly, female BALB/c mice were inoculated with irradiated and concentrated ASFV Lisbon/61 (~20 μg/mouse) in an equal volume of TiterMax Gold (TiterMax Inc., United States) subcutaneously. A total of six mice were immunized and boosted four times after the initial immunization. Three to 4 days before to fusion, mice were boosted with the same antigen in phosphate-buffered saline (PBS) by intravenous injection. Immunized spleen cells were then fused with myeloma cells (P3X63 Ag8.653). After 2 weeks, hybridoma supernatants were screened using an ELISA. The positive clones were subcloned using a limiting dilution method. The mAbs were isotyped using a mouse monoclonal antibody isotyping kit (Roche, Indianapolis, IN, United States).

Anti-ASFV porcine serum was collected 48 days post-inoculation (dpi) with ASFV (Estonia 2014). Microtitre plates (Nunc-Immunoplate Maxisorp, Roskilde, Denmark) were coated with polyclonal porcine anti-ASFV (Estonia 2014) serum diluted 1:5000 in a carbonate/bicarbonate buffer, pH 9.6 overnight at 4°C. After blocking in casein blocking buffer (Sigma-Aldrich, United States), ASF viruses (Table 1) were added. Concentrated and irradiated ASFV Lisbon/61 was used as an antigen for hybridoma screening. Hybridoma culture supernatants were then added. After incubation, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:2000, Jackson ImmunoResearch Laboratories, West Grove, PA, United States) was added, followed by-3,3′,5,5′-Tetramethylbenzidine (TMB, Pierce Biotechnology, Inc. Rockford, Illinois, United States). After stopping, optical density (OD) was measured at 450 nm using an Emax microplate reader (Molecular Devices, San Jose, CA, United States). Each incubation step was 60 min at 37°C with gentle shaking, followed by five washes (PBS with 0.1% Tween 20).

Irradiated and concentrated ASFV (Lisbon/61) was separated on a 4%–12% NuPAGE Novex Bis-Tris gel (Invitrogen, Carlsbad, United States); separated protein bands were transferred to nitrocellulose (NC) membranes using the iBlot Gel Transfer Device (Invitrogen, Carlsbad, United States). Membranes were then blocked using Casein blocking buffer at room temperature for 1 h and probed with either positive pig serum or mAbs diluted in Casein blocking buffer. Following primary antibody incubation, blots were incubated with HRP-conjugated anti-swine or anti-mouse secondary antibody (1:2000; Jackson ImmunoResearch Laboratories, West Grove, United States) for 1 h at room temperature. Blots were developed using the substrate 3,3′-diaminobenzidine (Sigma-Aldrich, St. Lucia, United States).

SDS-PAGE gel slices were de-stained using a combination of alternating washes with 100 mM ammonium bicarbonate and acetonitrile (Thermo Fisher Scientific, MA, United States). Once the Coomassie blue stain was removed, and the proteins within the gel slices were reduced and alkylated by the addition of 10 mM dithiothreitol and 66 mM iodoacetamide (Sigma Aldrich, MO, United States), respectively. The proteins within the gel slices were then digested using trypsin (Thermo Fisher Scientific, MA, United States), and the peptides were extracted from the gel slices using a combination of alternating washes with 1% formic acid (Millipore Sigma, MA, United States) and acetonitrile. The solution containing the tryptic peptides was dried to near completeness, and the peptides were resuspended in 2% acetonitrile, 0.1% formic acid (buffer A).

Digested peptide samples were analyzed using a nano-flow Easy nLC 1200 connected in-line to an Orbitrap Fusion Lumos mass spectrometer with a nano-electrospray ion source (2.3 kV) and a FAIMS Pro interface (Thermo Fisher Scientific, MA, United States). The samples were loaded (2 μL) onto a C18-reversed-phase Easy Spray column (50 cm long, 75 μm inner diameter, 2 μm particles, (Thermo Fisher Scientific) with 100% buffer A. Peptides were then eluted using a linear gradient of 3%–27% buffer B (80% acetonitrile, 0.1% formic acid) over 50 min, 27%–39% buffer B for 15 min, 39%–100% buffer B for 5 min and a wash at 100% buffer B for 10 min at a constant flow rate of 250 nL/min). Total liquid chromatography-mass spectrometry (LC-MS/MS) run-time was 100 min. The data-dependent acquisition was used with three different CV settings on the FAIMS Pro, (−40, −60, −80) 1 s each over a total cycle time of 3 s. The most abundant precursor ions in one-second survey scans were selectively isolated in the quadrupole (1.6 m/z isolation width) and fragmented by higher-energy collisional dissociation (HCD) (35% normalized collision energy). The survey scans were acquired in the Orbitrap over a range of m/z 375–1,500 with a target resolution of 120,000 at m/z 200, and the fragment ion scans were acquired in the linear ion trap with the scan rate set to “Rapid.”

Raw mass spectrometry data were converted to.mgf files using msconvert (ProteoWizard release 3.0.19254). Data was searched using Mascot (v3.1, Matrix Science) against a FASTA database of African Swine Fever Virus protein sequences and a database of common contaminant proteins. Mascot.dat files were imported into Scaffold (v 5.1.2) to generate a list of protein identifications.

A total of 14 overlapping peptides [15 amino acids (aa) in length, overlapping each other by 10 aa] were synthesized based on the ASFV A137R-encoded protein (pA137R) sequence (GenBank accession #AYW34014.1, ABclonal technology, MA, United States). Peptide ELISAs were performed as previously described (18, 19). Briefly, microtitre plates (Nunc Maxisorb) were coated with peptides (1 μg/well). After blocking, a diluted hybridoma culture supernatant was added to the plate. After incubation, HRP-conjugated anti-mouse IgG (1:2000, Jackson ImmunoResearch Laboratories, West Grove, PA, United States) was added. Then TMB was added and incubated for 15 min. After stopping, OD was measured at 450 nm using an Emax microplate reader. Each incubation step was 60 min at 37°C with gentle shaking, followed by five washes with a washing buffer.

Archived formalin-fixed, paraffin-embedded tissues were obtained from pigs experimentally inoculated with ASFV at the National Center for Foreign Animal Disease (NCFAD). Lymph nodes from animals infected with Gasson (7 dpi), Lillie SI-85 (5 dpi), Georgia 2007/1 (7 dpi), and Estonia 2014 (10 dpi). Tissues were sectioned at 5 μm onto positively charged slides for further testing in immunohistochemistry (IHC) and in situ hybridization (ISH) assays.

Tissue sections were quenched for 10 min in aqueous 3% hydrogen peroxide. Epitopes were retrieved using an in-house glycan retrieval solution in a Biocare Medical Decloaking Chamber. Primary antibody F88ASF-55 was applied to sections at pre-titrated dilutions and incubated at room temperature for 30 min. Reactions were visualized using the HRP-conjugated anti-mouse polymer Envision^® + System (Agilent Technologies, CO, United States) and developed with the chromogenic reagent diaminobenzidine (DAB). Sections were then counterstained with Gill’s hematoxylin. Lymph nodes from uninfected animals were tested as negative controls in the IHC assay, and no immunostaining was observed.

For the in situ hybridization (ISH) assay, the RNAScope 2.5 HD Detection Reagent-Red kit (Advanced Cell Diagnostics, Inc., CA, United States) was used. The procedure was performed following the manufacturer’s instructions. Briefly, tissue sections were cleared and hydrated in xylene and 100% ethanol and then air-dried. The sections were quenched for 10 min in aqueous H₂O₂, boiled in target retrieval solution for 15 min, rinsed in 100% ethanol and air-dried again. Then a final pre-treatment of protease plus enzyme for 15 min at 40°C was applied. The V-AFSV-01 probe (Advanced Cell Diagnostics, Inc., CA, United States) was applied and incubated at 40°C for 2 h. Sections were then washed twice in 1× wash buffer. After each of the subsequent hybridization steps, the sections were washed twice again. The signal was visualized with the chromogen Fast Red. The sections were then counterstained with Gill’s 1 hematoxylin, dried, cover-slipped with EcoMount (BioCare Medical, CA, United States), and examined by the pathologist. A negative control lymph node from a non-infected animal was tested in an ISH assay, and no immunostaining was observed. In addition, a negative control probe on positive tissue (lymph node from an ASFV Georgia 2007/1 infected animal) was tested, and no staining was observed.

Following mouse immunization with concentrated ASFV Lisbon/61, splenocytes were harvested and fused with myeloma cells. Supernatants from the resulting hybridomas were screened using ASFV Lisbon/61 as the antigen in an ELISA. Positive clones were selected and then subcloned. To determine the specificity of the clones, subcloned hybridoma culture supernatants were further tested in an ELISA using foot-and-mouth disease virus and recombinant classical swine fever E2 protein as negative controls. Only one hybridoma clone, F88ASF-55, showed specificity for ASFV and no cross-reactivity with other antigens (data not shown). The F88ASF-55 mAb is an IgG1 isotype and contains a ƙ light chain.

To identify the mAb’s binding protein, western blot analysis was performed. Irradiated and concentrated ASFV Lisbon/61 was separated on NuPAGE Novex Bis-Tris gels and the proteins were transferred to nitrocellulose membranes. The ASFV proteins were detected with a positive pig serum (Figure 1A) and F88ASF-55 (Figure 1B). The ASFV-positive porcine serum detected ASFV proteins with molecular weights (MW) of approximately 30, 54, 72, and 17 kDa, corresponding to ASFV structural proteins p30, p54, p72, and k145Rp, respectively. The porcine polyclonal serum showed a strong reaction to p30, but was weak to p54, p72, and k145Rp (Figure 1A). Additionally, the polyclonal serum showed a weak reaction to a 14 kDa protein. While F88ASF-55 reacted strongly to a protein with a MW of 14 kDa and weakly to a protein with a MW of 33 kDa (Figure 1B). The epitope recognized by F88ASF-55 is linear because the mAb reacted with denatured ASFV protein in western blot analysis.

A proteomic approach was performed to determine the sequence identities of the protein band (14 kDa) recognized by F88ASF-55. The stained protein band was excised, digested, and analyzed by LC-MS/MS. A total of 10 exclusive unique peptides and 15 exclusive unique spectra were identified (data not shown). The amino acid coverage (88/137) of the structural protein encoded by A137R was 64.2% (Figure 2). The results confirmed that the mAb-binding epitope is located on the protein encoded by A137R (formerly known as p11.5).

To further locate binding epitopes, 14 overlapping peptides representing the whole polypeptide encoded by A137R were synthesized. The reactivity of the mAb to ASFV pA137R peptides was examined using a peptide ELISA. The mAb reacted with peptide #12, corresponding to amino acids 111–125 (VDFQANIENMDDLQK) (Figure 3). Sequences of this region (111–125 aa) were aligned with ASFV isolates from 1950–2008 (Table 2). The results showed that the mAb-binding epitope was 100% identical across the ASFV 12 genotypes examined.

Although numerous genotypes have no whole genome sequence information available, this epitope is 100% identical in 256 of 258 sequences in GenBank from encompassing the years 1950–2023. The epitope sequence from two strains, Uvira B53 (Genotype X; 2019; MT956648) and BUR/18/Rutana (Genotype X; 2018; MW856067) varied by one amino acid (Q124E; peptide Q14E). In addition, the epitope recognized by F88ASF-55 cannot be found in any other organism.

A DAS ELISA was used to determine whether the mAb could detect ASFV. In the ELISA, polyclonal porcine anti-ASFV was used as capture agent, and mAb F88ASF-55 was used as detection agent. The DAS ELISA identified 5 strains of ASFV genotypes I and II viruses in addition to Lisbon/61 (Figure 4). The ELISA is expected to be specific for ASFV, as cell culture supernatants and virus-free controls showed a very low background. Not all ASFV genotypes were tested due to limited sequence information available for other genotypes. F88ASF-55 has the potential to detect numerous ASFV genotypes in ELISA because this mAb targets a conserved epitope shared among ASFV genotypes with A137R sequences, as shown in Table 2. However, a full evaluation is required.

The mAb was used in the IHC assay to detect ASFV in infected porcine tissues. IHC results indicated that F88ASF-55 was able to specifically recognize the viral antigen with low non-specific background staining in submandibular lymph nodes from animals experimentally infected with ASFV (Figure 5). The immunostaining observed was intense and specific with an excellent signal-to-noise ratio. The mAb showed no cross-reactivity with negative control tissue (data not shown).

To confirm the specificity of the IHC assay results, an ISH was performed in parallel using the same tissues (Figure 5). ASFV RNA could be detected with a similar distribution to IHC assay results, indicating that the IHC assay using F88ASF-55 detected specific ASFV antigen. However, IHC detection showed stronger signals than ISH for the four ASFV strains tested. Viral antigen concentrations in tissues at 5–10 dpi may be higher than viral RNA detected by ISH.