PAMs were isolated from the lungs of 3–8-week-old ASFV-negative piglets (Guangxi State Farms, China) by lung lavage. PAMs were cultured in RPMI-1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, and 10% heat-inactivated fetal bovine serum (FBS; PAA, Pasching, Austria). Animals were euthanized and carcasses were treated innocuously. All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. ASFV isolate GD19 was propagated and titrated in PAMs and was used throughout the study. ClO₂ was provided by Guangzhou WellFar Co, Ltd., Guangzhou, China.

The cytotoxicity of ClO₂ was tested using the alamarBlue^® assay (Invitrogen, USA) according to the manufacturer's instructions. Briefly, PAMs at the density of 1 × 10⁵ per well were seeded in 96-well plates respectively, ClO₂ at different concentrations were added. Mock-treated cells were set up simultaneously. After incubation for 48 h, 10 μl of alamarBlue^® was added to each well and the cultures were incubated for another 3 h. Fluorescence intensity was measured at 570 nm excitation and 590 nm emission wavelengths using a microplate reader (Synergy2, BioTek, USA) and normalized to the control for each sample.

Total RNA was extracted from PAMs using TRIzol reagent (Magen, China) according to the manufacturer's instructions. Briefly, Reverse Transcription System (A3500, Promega, USA) was used for reverse transcription in 20 μL reaction volume. The reverse-transcription primers were Oligo (dT) 15 primer (C110A, Promega, USA) and Random primer (C118A, Promega, USA). SYBR Green (TaKaRa, Osaka, Japan) real-time PCR was performed using a Light-Cycler 480 PCR system (Roche, Basel, Switzerland). Relative quantities of mRNA accumulation were evaluated using the 2^−ΔCt method. The primers used for qRT-PCR are listed in Table 1.

PAMs were fixed with 4% paraformaldehyde at room temperature (RT) for 15 min and then permeabilized with 0.3% Triton X-100 at RT. After washed three times with PBS, PAMs were blocked with 1% Bovine serum albumin (BSA) in PBS for 30 min at RT, then incubated overnight with an anti-ASFV p30 protein mAb (diluted 1: 200 in PBS; MEDIAN, Republic of Korea) at 4°C and followed by Alexa Fluor^® 488-conjugated anti-mouse IgG secondary antibody for 2 h. The nuclei were stained with Hoechst dye 33342 (Sigma-Aldrich, St. Louis, MO, USA). Finally PAMs were examined using fluorescence microscopy (Carl Zeiss, Jena, Germany).

ASFV-infected or mock-infected PAMs treated with or without ClO₂ were lysed in RIPA lysis buffer (Beyotime, China), and then electrophoresed onto a 12% SDS-PAGE gel and transferred to a polyvinylidene-fluoride (PVDF) membranes (Roche, USA). The membranes were blocked and incubated with a monoclonal antibody against ASFV p72 protein or GAPDH (Cell Signaling Technology, USA). After washing, membranes were incubated with anti-mouse or anti-rabbit IgG, HRP-conjugated antibody (Cell Signaling Technology, USA). Signal detection was performed using a chemiluminescence reagent (Fdbio Science, China).

The inhibitory effect of treatment with ClO₂ on ASFV was analyzed with three different approaches. (I) Pre-treatment: PAMs were pre-treated with ClO₂ at a concentration of 0.2 μg/mL for 4 h, ASFV at a multiplicity of infection (MOI) of 1 was then added and cells were cultured for another 36 h. (II) Co-treatment: Cells were inoculated with ASFV (MOI = 1) in the presence or absence of ClO₂ (0.2 μg/mL) for 36 h. (III) Post-treatment: PAMs were infected with ASFV at an MOI of 1 for 4 h at 37°C, and then the viral inoculum was removed and fresh RPMI-1640 medium containing ClO₂ (0.2 μg/mL) was added and cells were further incubated for 36 h. In all above antiviral assays, the cells were collected for the IFA and qRT-PCR analysis.

PAMs were cooled at 4°C for 30 min, and then infected with ASFV (MOI = 1) in the presence or absence of ClO₂ for 3 h at 4°C. After washing three times with ice-cold PBS to remove unbound viral particles, cells were switched to 37°C for a further 4 h. Cells were harvested for IFA and qRT-PCR analysis.

Cells were initially infected with ASFV (MOI = 1) for 3 h at 4°C. After binding to cell surface, the viral inoculum was removed and then cells were washed with ice-cold PBS three times and incubated for 4 h at 37°C in the presence or absence of ClO₂. After washed with PBS three times, cells were harvested to detect ASFV infection by IFA and qRT-PCR.

ASFV was pre-incubated with various concentrations of ClO₂ for 2 h at 37°C or pre-incubated with ClO₂ at the indicated concentration for 1 or 4 h at 37°C. The mixtures were added to PAMs which were then incubated for 2 h at 37°C. The mixtures were finally removed and cells were cultured with RPMI-1640 medium containing 2% FBS for additional 24 h at 37°C, and harvested for the analysis of IFA and qRT-PCR.

All experiments were performed with at least three independent replicates. Student's t-test and one-way ANOVA were used to analyze the data. All data were presented as means ± standard errors (SE). Statistical analysis was performed using SPSS 17.0 and GraphPad Prism 6.0. Differences with p-values < 0.05 were considered significant.

To examine the inhibitory effect of ClO₂ on ASFV replication, the half maximal inhibitory concentration (IC₅₀) and half maximal cytotoxic concentration (CC₅₀) values of ClO₂ were assessed using a non-linear regression model in GraphPad Prism 9.0. As shown in Figure 1A, ASFV infection was inhibited by ClO₂ at an IC₅₀ of 0.08 μg/ml, as determined by analyzing the cell infection rate from IFA images, and the CC₅₀ value of ClO₂ measured using the alamarBlue^® assay was calculated to be 0.56 μg/ml in PAMs. We next performed IFA and qRT-PCR assays to determine the effect of ClO₂ on ASFV, the concentration of ClO₂ used in subsequent experiments was non-cytotoxic to PAMs. Upon ClO₂ treatment, ASFV p30-specific staining was remarkably decreased in a dose-dependent manner in PAMs compared to that in mock-treated cells following ASFV infection (Figure 1B), suggesting that the infection and the spread of the virions to the neighboring cells are blocked by ClO₂. The number of infected cells was notably diminished with ClO₂ treatment (Figure 1C). Consistently, the mRNA and protein levels of the viral p72 were significantly reduced by ClO₂ treatment compared with the mock-treated cells post ASFV infection, and the inhibition pattern is also in a dose-dependent manner (Figures 1D,E). To further test its antiviral activity on ASFV replication, PAMs were infected with ASFV at different MOIs (at MOIs of 1, 1.5 or 2) in the presence or absence of ClO₂ (0.2 μg/mL). As shown in Figure 2, consistent with these findings above, ClO₂ significantly suppressed the viral infection and replication in PAMs infected with ASFV at various MOIs.

Since the order in which the virus and the compound are added to the cells may affect the antiviral effect of the compound, we therefore assessed the effects of ClO₂ treatment on ASFV replication using three different approaches as described in the Materials and Methods. As shown in Figure 3, ClO₂ displayed a strong antiviral activity both at the protein level of viral p30 and at the transcription and protein levels of viral p72 when administered with either the pre-, co-, or post-treatment approach. Taken together, these data demonstrate that ClO₂ shows a powerful antiviral effect on ASFV infection in PAMs in vitro.

Previous studies have shown that ClO₂ has great ability to inactivate bacteria, virus, fungi, parasites and other cellular pathogens (23–25). To demonstrate that whether ClO₂ can kill the ASFV in vitro, a virucidal assay was performed on PAMs. As shown in Figure 4, a lot of fluorescence representing viral burden in visual field was observed following the treatment of ASFV only using a fluorescence microscope. However, the viral fluorescence was dramatically reduced in ASFV-infected cells upon ClO₂ treatment. The decline was even more pronounced when the virus was incubated with ClO₂ for 4 h at 37°C. Consistently, the mRNA expression of the viral p72 was notably repressed in the presence of ClO₂ treatment for 1 or 4 h (Figure 4C). Similar phenomenon was also observed when the virus was incubated with different concentrations of ClO₂ for 2 h (Figure 5). Overall, ClO₂ can effectively inactivate the ASFV virions in vitro.

Viral infection of target cells involves the following processes: adsorption, entry, and uncoating. To characterize the molecular mechanism of anti-ASFV activity of ClO₂ and to identify which stage of the viral life cycle is suppressed by ClO₂, a viral adsorption assay described in Materials and Methods was first performed to test whether ClO₂ is able to inhibit ASFV attachment to PAMs. As shown in Figures 6A,B compared with the mock-treated cells inoculated with ASFV (MOI = 1), the translation level of viral p30 protein and the transcription level of viral p72 were sharply decreased in infected cells in the presence of ClO₂ during the period of virus adsorption. The data indicate that ClO₂ is capable of diminishing the quantity of infectious virions attached to cell surface. We next investigated whether ClO₂ can restrain the entry phase of virus life cycle (the protocol was described in Materials and Methods). The ASFV p30-specific staining which represents the virus yield in virus-infected cells with the treatment of ClO₂ was comparable to that of cells following the treatment of ASFV only, during the entry of the virus (Figure 6C). The level of mRNA expression of the viral p72 protein showed a similar phenomenon (Figure 6D). Taken together, these data indicate that ClO₂ shows a potent inhibitory effect on the viral adsorption rather than the internalization process of ASFV life cycle in PAMs.

Previous studies have shown that ClO₂ produces its antiviral activity against hepatitis A virus by acting on viral nucleic acids and proteins (19, 26). Since ClO₂ has the ability to kill ASFV, we next investigated that whether ClO₂ can damage the nucleic acids of the virus. ClO₂ at the concentration of 0.1 μg/mL was incubated with ASFV for 30 or 60 min at 37°C, and then the mixtures were harvested for viral nucleic acids analysis by PCR. The starting amount of ASFV added to each reaction is 5.8 × 10⁶ copies. As shown in Figure 7A, there were no detectable fragments of viral p32 gene and partial p72 gene upon ClO₂ treatment, indicating that the ASFV nucleic acids can be destroyed by ClO₂ treatment. To determine the minimum concentration at which ClO₂ destroys the viral nucleic acids, quantitative PCR was performed (27). As shown in Figure 7B, ClO₂ at the concentration of 1.2 μg/mL completely destroyed the viral genome (no Ct values were detectable). The viral nucleic acids were almost degraded by ClO₂ treatment at the concentration of 0.2 μg/mL for 60 min at 37°C (Figure 7C). Since the effect of ClO₂ in killing ASFV virions is also affected by temperature, we next investigate the ability of viral nucleic acids damage by ClO₂ in different temperatures. As expected, the Ct values decreased with the drop of temperature post ClO₂ treatment (Figure 7D), indicating that ClO₂ degrades ASFV nucleic acids in a temperature-dependent manner. The effect of ClO₂ on the viral proteins was also tested. ClO₂ at different concentrations was incubated with ASFV for 30 min at 37°C. As expected, ASFV was degraded with ClO₂ treatment, and the minimum concentration of degradation is 0.2 μg/mL (Figure 7E). Taken together, ClO₂ is capable of damaging the ASFV nucleic acids and degrading the viral proteins, which may contribute to its inactivation of ASFV.

Since PAMs are immune cells and play an important role in the innate immune response, we next investigated the effects of ClO₂ on the expression of cytokines such as interleukin-6 (IL-6) and interferon-β (IFN-β), which are known to be related to the host antiviral and inflammatory reactions. As shown in Figure 8, the upregulation of IL-6 and IFN-β induced by ASFV infection was significantly diminished in the presence of ClO₂ in PAMs at 6 hpi. The expression level of IL-6 and IFN-β in cells treated with ClO₂ alone was comparable to that of mock-treated cells. These data suggest that ClO₂ inhibition of cytokines expression caused by ASFV may contribute to its capacity of suppressing virus replication.