The ASFV Armenia/07 strain was used in all experiments. The ASFV was obtained from the spleen of infected pigs. The virus (Armenia/07) was first isolated in 2007 from the spleen of an ASFV-infected swine. Virus titration was performed as described previously and expressed by hemadsorption unit (HADU) as lg10 HADU50/mL for non-adapted cells [9]. Measurements in HADU make it possible to estimate the number of infectious units of the virus when studying hemadsorbing strains of the ASF virus. Therefore, this technique complements quantitative measurements of viral genome copies well. The titer was expressed as hemadsorption units—an HADU50/mL.

The virus for experiments was received after 48 h infections of PAMs. The HADU technique was carried out on the primary culture of porcine alveolar macrophages [10]. The ASFV was inactivated by incubating in a water bath (65 °C at 10 min). After heat inactivation, the virus was tested for infectivity using in vitro cell culture.

All the information about software/equipment/drugs/reagents are included in Supplementary Material 1. Human acute myeloid leukemia cell line THP-1 obtained from ATCC (Manassas, VA, USA), validated by STR profiling and tested negative for mycoplasma, was maintained in RPMI 1640 (Life Technologies) medium and supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM Hepes, and 100 U/mL penicillin, 100 µg/mL streptomycin (Sigma) in a humidified incubator at 37 °C with 5% CO${}_2$. M0 macrophage-like THP-1 cells (M$\phi$0) were generated in a 24-well plate at a density of 2 $\times$10${}^5$ cells per milliliter by the treatment of THP-1 cells with phorbol 12-myristate 13-acetate (PMA) for 72 h, followed by a resting period in fresh RPMI medium for 24 h. The plates were washed with RPMI to remove non-adherent cells before other experimental viral treatments. Polarized M0 status was confirmed by flow cytometry and microscopy.

For in vitro experiments, ASFV was grown in a culture of THP-1-derived M0 macrophages (M$\phi$0) for 24 h and 48 h. M$\phi$0, seeded as described above, were inoculated with ASFV Arm07 at 10${}^4$ HADU50/mL from the 1st passage; the dose was chosen to prevent spontaneous apoptosis caused by viral particles (0,1 MOI). Following adsorption at 37 °C for 1 h, the infected cell monolayer was washed twice to remove any unbound viruses. A complete medium was then added and analyzed at the indicated time points. The ASFV infection was performed on M$\phi$0 in two stages—24 hours post-infection (hpi) and 48 hpi. After 1 hpi, cells were washed twice with medium and cultivated at 37 °C with 5% CO${}_2$ [9].

For comparison with human macrophages, we used a primary culture of PAMs, which were seeded according to the generally accepted standard method and inoculated with ASFV Arm07 at 10${}^4$ HADU50/mL. After adsorption at 37 °C for 1 h, the infected cell monolayers were washed twice to remove unbound viruses. Afterward, a complete medium was added and analyzed at the indicated time. All data were obtained at 24 and 48 hpi [9].

Macrophage apoptosis and viability during infection were assessed using Annexin V and propidium iodide (PI). The cells were harvested, trypsinized, and resuspended in Annexin binding buffer before adding 5 µL Annexin V-FITC (Biolegend) and incubated in the dark at 4 °C for 30 min. Immediately before cytometric analysis, the cells were labeled with PI. Data were acquired on a FACS Calibur (BD Biosciences) and analyzed using the FlowJo vX0.7 software (Tree Star, Inc., San Carlos, CA, USA).

To better characterize cellular morphology, investigated cells were cultured in 6-well chamber slides. For morphological analysis, cells were fixed in pure methanol and stained with Pappenheim (Cypress Diagnostics, Belgium), according to the manufacturer’s protocol. Additionally, Hematoxylin–Eosin staining was used (Sigma-Aldrich, Germany), according to the manufacturer’s protocols.

To detect viral factories, cytospectrophotometry and Feulgen–Naphthol Yellow S staining procedures were used as described previously [11].

Flow cytometry analysis was used to evaluate the effect of inactive and active ASFV (inASFV and aASFV) treatments on M$\phi$0 phenotype and inflammatory markers. At the end of each experiment, the cells were washed twice and incubated for 15 min with PBS supplemented with 1% bovine serum albumin (BSA) to prevent non-specific binding of antibodies and conjugated with the following antibodies: CD36-FITC, CD80-FITC, CD163-PE, CD11b-PerCP, HLA-DR-PerCP, CD11b-PerCP, CD282 (TLR2)-Pe-Cy7, CD182 (CXCR2)-PeCy7, CDD197 (CCR7)-APC, CD11c-APC-Cy7, and CD14-APC-Cy7. To analyze the intracellular expression of the TLRs, the cells were fixed and permeabilized after surface-staining and labeled with CD283 (TLR3)-PE and CD289 (TLR9)-APC (all Biolegend). Positive and negative thresholds for fluorescence signals were defined using isotype-specific negative controls. Data were acquired on a Becton Dickinson LSRII flow cytometer and analyzed using the FlowJo vX0.7 software. In all experiments, a minimum of 10,000 events were counted. Results are expressed as the percentage and mean fluorescence intensity (MFI).

For determining the phagocytic capacity of macrophages, pH-sensitive fluorochrome pHrodo-green-labeled Zymosan BioParticles (ThermoFisher) were used at a concentration of 50 µg/mL, according to the manufacturer’s recommendation. Briefly, particles were added to the attached cells and incubated at 37 °C and 5% CO${}_2$ for 2 h. All cell lines were validated by STR profiling and tested negative for mycoplasma. Cells were all cultured in a humidified incubator at 37 °C and 5% CO${}_2$. Cells were placed on ice to stop further phagocytosis and analyzed using a FACS Calibur (BD Biosciences). The frequency of pHrodo+ cells and/or the mean fluorescence intensity (MFI) was determined.

After the indicated stimulations, the cells were removed from the plate by trypsinization, washed, and stained with surface lysosome-associated membrane protein (LAMP-1-PE, CD107a). Then, the cells were fixed, permeabilized, and stained with LAMP-1-APC (Biolegend) for intracellular staining. Afterward, the cells were washed and analyzed by flow cytometry.

After the treatments described above, cell-free supernatants were quantified for the presence of interleukin (IL)-10, tumor necrosis factor (TNF), and interferon (IFN)-$\alpha$ using an enzyme-linked immunosorbent assay (Biolegend), according to the manufacturer’s protocols. Absorbance was read at 450 nm using a HiPo MPP-96 Plate Reader.

To determine ASFV expression in PAM cell lines, total viral RNA/DNA was isolated using the HiGene™ Viral RNA/DNA Prep kit (BIOFACT). RNA/DNA samples were then reverse transcribed using the FIREScript® RT cDNA synthesis kit (Solis Biodyne). Both methods were conducted following the manufacturer’s instructions. When measuring DNA/RNA concentrations with a NanoDrop® ND-1000, UV-Vis Spectrophotometer A260/280 values were acceptable. A ratio of ~1.8 is considered “pure” for DNA; a ratio of ~2.0 is considered “pure” for RNA. Quantitative real-time PCR was performed using the SYBR green methods previously described [12, 13] on an Eco Illumina Real-Time PCR system device (Illumina Inc). Each reaction mixture (20 µL) was composed of 4 µL of 5$\times$ HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX) (Solis BioDyne), 0.2 µL of each specific primer, 4 µL of template DNA/cDNA, and 11.6 µL of ddH${}_2$O. Positive and negative controls were used. DNA isolated from an ASFV-infected pig’s spleen was used as a positive control. For the negative control, ddH${}_2$O was added to the reaction mix instead of a sample. Reactions were carried out in the following conditions: polymerase activation: 95 °C for 12 min, 40 cycles: 95 °C for 15 s, 52 °C for 30 s, and 72 °C for 30 s. Standard curves were created using serial 10-fold dilutions of viral DNA. The fluorescence threshold value (Ct) was calculated using the ECO-Illumina system software. Primers used for amplification were designed and ordered from Integrated DNA Technology-IDT (https://www.idtdna.com/pagesasfollows).

Viral genes were measured at 48 hpi to determine the viral amount and after 24 hpi to analyze the transcriptional activity of separate genes. For alignment of the cDNA plots and infection titers of ASFV, Cq values were rescaled after comparing with viral genome copies and modified in absolute amounts along the y-axis for better visualization. To evaluate the ASFV replication effectivity profile, the genes with different temporal expression patterns were identified [8, 14]. All primers are listed in Table 1.

Porcine IFN-$\alpha$ (MBS162596), IL-10 (MBS2019681), and TNF-$\alpha$ (MBS2019932) were purchased from MyBioSource. ELISA was performed using the manufacturer’s description. The IFN levels and/or receptors were measured in duplicate using a colorimetric reader (Stat Fax 303 Plus) and calculated according to the standard curve supplied by the kit.

Statistical analyses were performed using GraphPad Prism software (version 6.01, https://www.graphpad.com/scientific-software/prism/www.graphpad.com/scientific-software/prism/). The results are expressed as the mean $\pm$ standard error (SEM). Statistical significance for the differences between groups measured in M$\phi$0 cells was calculated using one-way ANOVA with Tukey’s multiple comparisons test. *p 0.05, **p 0.01.

All in vitro experiments with virus analysis were conducted in triplicate. The significance has been evaluated using a two-tailed Student’s t-test. SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) has been applied for statistical analyses.

The virus was blindly passaged to determine the possible susceptibility of M$\phi$0 to ASFV infection. We found that the genome copies of ASFV decreased in M$\phi$0 starting from the first passage to the fourth (Fig. 1A). The K196R gene belongs to the group of late genes. Since the late genes in the ASF virus in an unusual target cell are not always transcribed or transcribed very late, we chose one of the late genes to better assess the virus’s ability to replicate fully. However, simultaneously with the K196R gene, we also studied other genes that showed similar results (data not shown). Similar data were obtained when the virus was titrated by HADU (Fig. 1B). The number of infectious virus particles at the third passage was 2.5–3 lg lower than the initial one and stock virus. However, the ASFV was detected in all examined samples in the third passage. In the fourth passage, only one of the six samples contained an infectious virus (Fig. 1B). Despite this, transcription of some viral genes continued (or persisted) in the third passage, although it had almost disappeared in the fourth passage (Fig. 1A).

Fig. 1.

African swine fever virus (ASFV) amounts in porcine alveolar and human macrophages. (A) Quantitative real-time polymerase chain reaction (PCR) results of genome amount ASFV K196R and complementary DNA (cDNA) in 1st, 3rd, and 4th passages. *significant compared with 1st passage (p 0.05); ** tendency (p 0.1) compared with 1st passage. (B) ASFV hemadsorption unit (HADU) titers in M$\phi$0 in 1st, 3rd, and 4th passages. *significant compared with porcine alveolar macrophage (PAM) (p 0.05); ** tendency (p 0.1) compared to PAM.

We studied thirteen genes involved in viral replication to identify and analyze the transcriptional activity of the ASFV genes in human M$\phi$0 cells. The level of the virus genes was compared with the viral transcript levels. All studied genes were divided into two groups: early genes (Fig. 2A) and ambivalent to replication time genes—late genes (Fig. 2B).

Fig. 2.

Quantitative real-time PCR results of ASFV K196R, R298L, A2410L, F778R, F334L, and E165R mRNA (cDNA) in ASFV-infected human M$\phi$0 and PAM lysates at 24 hpi. (A) Early genes and their transcriptional activity (cDNA) in ASFV-infected M$\phi$0. (B) Ambivalent and late genes and their transcriptional activity (cDNA) in ASFV-infected M$\phi$0. (C) Early genes and their transcriptional activity (cDNA) in ASFV-infected PAMs. (D) Early genes and their transcriptional activity (cDNA) in ASFV-infected PAMs. *significant compared to PAMs (p 0.05); ** tendency (p 0.1) compared to DNA levels. mRNA, messenger RNA; M$\phi$0, M0 macrophages; hpi, hours post infection.

The following ASFV early genes can be transcribed in M$\phi$0 cells: F778R ribonucleotide reductase (large subunit), F334L ribonucleotide reductase (small subunit), E165R deoxyuridine triphosphatase, G1211R DNA polymerase $\alpha$-like, P1192R DNA topoisomerase type II. At the same time, our data did not reveal the transcriptional activity of early genes, such as the F1055L helicase superfamily and A240L thymidylate kinase (Fig. 2A).

Ambivalent genes O174L DNA polymerase X-like and K196R thymidine kinase exhibited transcriptional activity, whereas EP1242L RNA polymerase subunit 2 and A859L helicase superfamily II did not display any detectable transcriptional activity. Both late genes R298L serine protein kinase and B646L major capsid protein demonstrate transcriptional activity (Fig. 2B). In conclusion, regardless of the timing of transcription in susceptible cells, certain ASFV genes show transcriptional activity, and others do not.

The same genes were examined for transcriptional activity in PAMs infected with a similar dose of ASF virus. As follows from Fig. 2, all genes, both early (Fig. 2C) and late (Fig. 2D), demonstrate high transcriptional activity.

A morphological examination revealed that intact macrophages generally have a small and round shape, with few vacuoles and small pseudopodia. Nonetheless, signs of macrophage activation can occasionally be detected within the intact population. Additionally, intact macrophages were also characterized by cytoplasmic vacuolization, which was observed only in a minority of cells (Fig. 3A).

Fig. 3.

Morphological characteristics of the ASFV-infected human macrophages. (A) Intact M$\phi$0 with vacuolized cytoplasm; Hematoxylin and Eosin (H–E) staining. Scale bar: 10 µm. (B) Infected (24 hpi) with active ASFV enlarged M$\phi$0 with massive vacuolization, which includes nucleus (arrowed), H–E staining. Scale bar: 10 µm. (C) M$\phi$0 incubated with inactive ASFV (48 hpi) severe vacuolization, Pappenheim staining. Scale bar: 10 µm. (D) M$\phi$0 incubated with inactive ASFV (48 hpi) severe vacuolization, cytoplasmic basophilia (arrowed), H–E staining. Scale bar: 10 µm. (E) Infection (48 hpi) with active ASFV M$\phi$0 with mild vacuolization and pronounced pseudopodia (arrowed), H–E staining. Scale bar: 10 µm. (F) Infection (48 hpi) with active ASFV M$\phi$0 with mild vacuolization and pronounced pseudopodia (arrowed), Pappenheim staining. Scale bar: 10 µm. (G) Infected (24 hpi) with active ASFV M$\phi$0 without viral factories, Feulgen staining. Scale bar: 10 µm. (H) Infected (24 hpi) with active ASFV porcine alveolar macrophages with typical viral factory (arrowed), Feulgen staining. Scale bar: 10 µm.

Analysis of the morphology of M$\phi$0 cells infected with the virus showed noteworthy variations at 24 and 48 hpi. At 24 hpi, the morphology of macrophages infected with the inactivated virus resembled that of macrophages infected with the active virus, possibly due to viral envelope components. Both groups of infected M$\phi$0 cells exhibited prominent cytoplasmic vacuolization, which occupied most of the volume of the cytoplasm and sometimes the nucleus (Fig. 3B). Cytoplasmic vacuolization and basophilia were typically absent in intact macrophages (Fig. 3A), they were observed in M$\phi$0 cells infected by both inactivated and active virus (Fig. 3C,D). By 48 hpi, significant differences were observed between the two groups with the inactivated virus, where macrophage activation continues in groups with the infectious virus (Fig. 3E,F), where many signs of inhibition of cell activity were observed. Pronounced pseudopodia were visible in some ASFV-infected M$\phi$0 cells (Fig. 3E).

Furthermore, it is worth mentioning that none of the samples analyzed for the presence of viral factories on Feulgen-stained preparations revealed DNA-positive structures in the cytoplasm of infected cells (Fig. 3G). In contrast, when ASFV infects susceptible cells, such as primary cultures of PAMs, factories are typically detected in significant numbers and are clearly visible (Fig. 3H). Hence, M$\phi$0 infected with ASFV (Arm07) do not exhibit the typical factories observed in infected susceptible cells, such as PAMs.

To investigate the viability of M$\phi$0 cells upon infection with ASFV, we analyzed the rates of dead cells and apoptosis. Flow cytometry analysis has shown that active and inactive viruses do not alter the viability of M$\phi$0 cells after infection for 48 hours. Despite the apoptotic rate being increased in both suited groups (aASFV and inASFV), their differences from the control group were not significant (Fig. 4A).

Fig. 4.

Functional tests of THP-1-derived M$\phi$0 cells infected with active virus (aASFV) and inactivated virus (inASFV) (n = 6). (A) Percentage of live, apoptotic, and dead cells. (B) Representative overlay of histograms of pH changes in M$\phi$0 labeled with pHrodo-green-labeled Zymosan BioParticles. (C) Expression levels of surface and intracellular lysosome-associated membrane protein (LAMP-1) quantified as mean fluorescent intensity (MFI). Each experiment was repeated three times, and the results were averaged. Ctl, control.

Next, we studied the phagocytic activity of ASFV-infected M$\phi$ with pH-sensitive fluorescent pHrodo dye. After engulfment, the intensity of pHrodo light emission was slightly elevated in M$\phi$0 infected with inASFV (p = 0.05) compared to non-infected M$\phi$0 (Fig. 4B).

The analysis revealed low lysosomal membrane glycoprotein LAMP-1 expression at the M$\phi$0 surface. Both M$\phi$0+aASFV and M$\phi$0+inASFV exhibited increased surface LAMP-1, indicative of lysosome exocytosis of the marker on the cell surface. In opposition to the surface marker expression, intracellular expression of LAMP-1 showed decreased expression in both studied groups (not significant) (Fig. 4C).

To elucidate the response of M$\phi$0 cells toward the presence of the ASFV, we characterized the phenotype of the cells. Histograms of a representative experiment and a summary of the surface marker expression profile for each studied group are shown in Fig. 5. Overall, flow cytometry analysis revealed that both aASFV and inASFV caused detectable changes in marker expression in M$\phi$0 cells. As shown in Fig. 5, the expression of several CD markers was upregulated by the cells infected with active virus (aASFV), namely CD36, CD163, CXCR2, and CD80. Interestingly, the expression of CD11c was downregulated in infected cells, which is indicative of cell activation, albeit not significantly. The process of downregulating the expression of CD11c is triggered by TLR4, TLR3, and TLR9 signaling [15].

Fig. 5.

Differential expression of surface CD markers and Toll-like receptors (TLRs) by human THP-1-derived M$\phi$0 cells infected with active virus (aASFV) and inactivated virus (inASFV). The graph shows the percentage of M$\phi$0 cells expressing TLR3 and TLR9 and mean fluorescent intensity (MFI) of CD11c, CD36, CD163, CD11b, CXCR2, TLR2, HLA-DR and CD80 on the surface of M$\phi$0 cells. Data are presented as the mean $\pm$ standard error of the mean. *p 0.05, **p 0.01, ns, not significant. Each experiment was repeated three times, and the results were averaged.

Intending to identify cellular pattern recognition receptors (PRRs) responsible for the viral activation in macrophages, we examined the expression levels of TLR3 and TLR9, which have been described as receptors that mediate the sensing of the ASFV (Ayanwale et al. [16] 2022). TLR2 was analyzed as a receptor able to recognize viral antigens and sense endogenous danger-associated molecular patterns (DAMPs) to trigger the process of self-healing and tissue repair [17]. We found that TLR2 expression was slightly upregulated in both the inASFV and aASFV. Interestingly, the percentage of M$\phi$0 cells positive for TLR3 (receptor mediating sensing of ASFV) was significantly increased only in a group of the cells treated with the aASFV (Fig. 5).

Next, we measured the accumulation of TNF-$\alpha$, IL-10, and IFN-$\alpha$ in culture supernatants of ASFV-infected M$\phi$0 cells (Fig. 6A). The induction of TNF-$\alpha$ resembled mainly immunophenotyping results. Production of IL-10 was significantly increased in the aASFV group compared with the inASFV group. Notably, the production of IFN-$\alpha$ was reduced by the active virus.

Fig. 6.

The effect of ASFV on production of cytokines TNF$\alpha$, IFN-$\alpha$, and IL-10. (A) In culture supernatants of THP-1-derived M$\phi$0 cells infected with active virus (aASFV) and inactivated virus (inASFV) (n = 7). (B) In culture supernatants of PAMs (n = 4). Data are presented as the mean $\pm$ standard error of the mean. *p 0.05, **p 0.01, ns, not significant. The statistical analysis of cytokine levels in the supernatants of THP-1-derived M$\phi$0 cells was conducted using one-way ANOVA, while the cytokine levels in the supernatants of PAMs were assessed using the Wilcoxon test. TNF$\alpha$, tumor necrosis factor $\alpha$; IFN-$\alpha$, interferon-$\alpha$; IL-10, interleukin-10.

Similar to THP-1-derived M$\phi$0 cells, the levels of measured TNF-$\alpha$ were increased in the PAMs infected with ASFV, while the supernatant levels of IL-10 remained unchanged (Fig. 6B). However, the production of IFN-$\alpha$ differed between the THP-1-derived M$\phi$0 and PAMs. Particularly, PAMs infected with ASFV secreted higher amounts of IFN-$\alpha$ than the control group.