The channel catfish ovary (CCO) cell line is a brown bovine head cell line known as CCO/BB (22). Cells used in this study were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 28°C. The CCV strain VR-665 (kindly provided by Prof. Jun-Fa Yuan, Huazhong Agricultural University, China (23, 24), was maintained in our laboratory and used in the present study. CCO cells were used for CCV propagation, and CCV titers were determined using median tissue culture infectious dose (TCID₅₀) assays on CCO cells, as previously described (23).

Heparan sodium and collagen were purchased from Sangon Biotech (Shanghai, China). Hyaluronic acid was purchased from Selleck Chemicals (Houston, TX, USA). Heparinase I was purchased from Sigma-Aldrich (St. Louis, MO, USA). All drugs except heparinase I were dissolved in water according to the manufacturer's instructions and then stored as stock solution at −80°C. Heparinase I was re-suspended in phosphate-buffered saline (PBS) at 100 U/mL and stored at −20°C.

Cells were infected with virus at a multiplicity of infection of 0.01 for 6 days. After three freeze-thaw cycles, the supernatant was collected by centrifugation at 6,000 × g for 40 min at 4°C. The pellet was re-suspended in PBS and stored at −80°C. The intracellular virus growth curve of CCV was determined as previously described (25).

Viral titers were determined using the TCID₅₀ assay. Briefly, CCO/BB cells were seeded in 96-well plates and incubated overnight at 28°C to form a confluent monolayer prior to infection. Ten-fold serial dilutions of virus were added to the wells of the plates and absorbed for 4 h at 28°C. Thereafter, unbound virus was removed and the cells were washed twice with DMEM. The cells were incubated with DMEM at 28°C and the cytopathic effect (CPE) was observed after 2 days. Drug-treated groups were incubated with CCO/BB cells for 1 h before CCV infection, and the TCID₅₀ was calculated using Reed and Munch mathematical analysis (26).

Cytotoxic effects of all drugs were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, CCO/BB cells were seeded in a 96-well plate overnight at 28°C before treatment. Each inhibitor was diluted in different concentration gradients according to its solubility. Then, medium was changed to one containing different concentrations of the inhibitors. After 24 h of incubation, 10 μL of MTT solution was added to each well and incubated for 4 hours. The cell viability analysis was performed using a MTT Cell Proliferation and Cytotoxicity Assay Kit (Sangon Biotech). Optical density was measured at 570 nm using a Spark microplate reader (TECAN, Männedorf, Switzerland). The optical density of the control group was set to 100%. Three parallel wells were used for each experiment.

To analyze CCV uptake, quantitative real-time PCR (qPCR) was used to analyze the gene copy number of the virus. CCO/BB cells were seeded in 24-well plates and incubated overnight at 28°C before infection. Then, CCO/BB cells were pretreated with heparin sodium salt, hyaluronic acid, and collagen for 1 h at 28°C, respectively. CCV was then added to the medium at a multiplicity of infection of 0.01 and incubated at 28°C for 4 h. The inoculum was removed, the cells were washed twice with DMEM medium, the DMEM was replaced and the cells were incubated for about 24 h at 28°C. Cells were harvested by centrifugation for viral DNA extraction and western blotting analysis. The ultrastructure of the virions in infected cells was examined using transmission electron microscopy (TEM) (Axio Vert A1, 10X; Carl Zeiss, Oberkochen, Germany). To confirm the effect of the drug on the CCV infection of CCO/BB, the drug was first incubated with cells or virus (pre or post), respectively.

To further investigate the role of adsorption factors in virus binding, cells were treated with heparinase I to remove HS from the cell surface. Heparinase I was dissolved in 1 × PBS at 100 U/mL before the assay and stored at −20°C. CCO/BB cells were seeded in 24-well plates and cultured at 28°C overnight, the medium was removed before the assay, cells were incubated with different concentrations of heparinase I for 1 h, and then washed twice with fresh medium. CCV was added to the medium at a multiplicity of infection of 0.01 and incubated for 4 h at 28°C. After 4 h of incubation, the cells were washed twice with fresh medium and then the medium replaced with DMEM medium. After 24 h of culture, the cells were collected for western blotting or qPCR analysis. The ultrastructure of the virions in infected cells was examined using TEM (Axio Vert A1, 10X).

Heparan-Sepharose beads 6FF (Sangon Biotech) or control Sepharose beads (Sigma) were washed three times with PBS at room temperature and equilibrated, then 100, 300, or 500 μL of CCV was added, and the mixture was shaken at low speed at 4°C for 2 to 3 h. Unbound virions were collected and the pellet was washed three times with PBS. Samples were re-suspended in 5 × protein sample buffer containing 5% 2-mercaptoethanol, subjected to electrophoresis, and detected using western blotting analysis. The eluted CCV virus particles were treated using the negative staining method and observed using TEM.

Total DNA was extracted from the collected CCO/BB cells using a viral RNA/DNA extraction kit (Takara, Dalian, China) according to the manufacturer's instructions. Briefly, an equal volume of Buffer VGB was added, and the Carrier RNA and Proteinase K were added to remove excess viral RNA and protein. Carrier RNA and Proteinase K must be stored at −20°C and operated on ice. The mixture was kept at 56°C for 10 min to fully lyse the virus. After adding absolute ethanol, the mixture was centrifuged at 14,000 × g for 2 min. Further processing was carried using Buffer RWA and Buffer RWB. Absolute ethanol should be added to Buffer RWB before first use to ensure elution efficiency. Finally, RNase-free ddH₂O was added and the viral DNA was eluted by centrifugation. The mixture was allowed to stand at room temperature for 5 minutes before centrifugation to ensure reaction efficiency. All reagents used, except Carrier RNA and proteinase K, were stored at room temperature. The extracted DNA was stored at −20°C for subsequent quantitative PCR analysis.

qPCR was performed on the LightCycler^® 96 System (Roche, Basel, Switzerland). The cycling conditions were: An initial denaturation at 98°C for 60 s, followed by 35 cycles of 98°C for 10 s, 60°C for 15 s, and 72°C for 30 s. The copy number of the viral genomic DNA was determined according to the copy number of the CCV ORF39 gene. A known amount of the pGBKT7-ORF39 plasmid containing the CCV ORF39 gene was serially diluted 10-fold as a standard. The standard curve of the cycle threshold values was determined, a standard curve of CCV ORF39 was established, and the viral gene copy number was calculated according to the standard curve. The primer sequences for CCV ORF39 are as follows, forward: 5′-GAAGATAGCCCGTCTCACCG-3′; reverse: 5′-ATCTCGATCAGCATCTGGCG-3′. The pGBKT7 plasmid is maintained in our laboratory. A ChamQ Universal SYBR qPCR Master Mix was purchased from Vazyme (Nanjing, China) and stored at −20°C. qPCR was performed in triplicate in each sample. All qPCR reactions were performed in triplicate. The resulting data were analyzed using the LightCycler^® 96 SW 1.1 software (Roche).

For western blotting analysis, cells were lysed with cell lysis buffer (Beyotime, Jiangsu, China) and the protein concentration was determined using the Bradford method. Proteins obtained after lysing the cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Pall, Shanghai, China) using a semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) at 120 V about 20 min. The membrane was then further incubated with anti-ORF39 (prepared in our laboratory) (27) or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies (ABclonal, 1:2,000). Horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Sigma, 1:4000) were used as secondary antibodies. Immunoreactive protein bands were detected using an ECL kit (Tiangen Biotechnology, Beijing, China).

Statistical analyses were expressed as means ± SD and performed using GraphPad PRISM software version 8 (GraphPad Software, San Diego, CA, USA). The probability of P < 0.05 was considered to be statistically significant.

To study the effect of various adsorption factors on CCV infection of CCO/BB cells, their safe concentrations on CCO/BB cells was tested first. To this end, different concentrations of drugs were added to the CCO/BB cell culture supernatant, and cytotoxicity was tested using the MTT assay after 24 h. The MTT method is a common method for activity detection of active factors, drug screening, and cytotoxicity assay. It is widely used for the in vitro detection of changes in cell proliferation activity caused by inducers and inhibitors. Succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT solution to blue-violet crystalline formazan and deposit it in the cells, thereby detecting cell viability. In the presence of specific solvents, formazan can be completely dissolved. Figure 1 shows that after the cells were treated with hyaluronic acid at a concentration of <1 mg/mL, there was no significant change in the morphology of the cells under the microscope, and the cell viability was above 95%. After the cells were treated with collagen at a concentration of <1 mg/mL, there was no significant change in the morphology of the cells under the microscope, and the cell viability was above 95%. After the cells were treated with heparin sodium salt at a concentration of <10 mg/mL, the morphology of the cells did not change significantly under the microscope, and the cell viability was above 95%. The cell viability was higher after cells were treated with heparinase I at a concentration of <5 U/mL. The results showed that CCO/BB cells had good tolerance to ECM components, and the cells could be treated with higher concentrations of these drugs without significant cytotoxicity.

To determine whether the main adsorption factors are involved in CCV attachment, CCO/BB cells were incubated with hyaluronic acid, heparin sodium salt or collagen for 1 h and then infected with CCV for approximately 4 h, CCO/BB cells were harvested 24 h later and the amount of bound virus was detected using western blotting and qPCR analysis. As shown in Figure 2, the viral genome DNA copy number and protein expression levels were not significantly different after treatment with 0–500 μg/mL hyaluronic acid or collagen. The results suggested that hyaluronic acid and collagen are not associated with CCV attachment. heparin sodium salt reduced CCV adhesion at 5–10 mg/mL, and caused about 90% inhibition at 10mg/mL. The qPCR and western blot results showed the same trend. The copy number and protein expression levels were decreased, suggesting that heparan might play an important role in CCV attachment. To confirm that the exogenous adsorption factor plays an inhibitory role, different concentrations of collagen, hyaluronic acid, and heparin sodium salt were added after CCV infection for 4 h. Heparan exerted a competitive inhibitory effect on CCV adsorption to CCO/BB cells.

Changes in ECM composition and structure can be mediated by proteases and have an impact on the overall structure and biomechanical properties of the formed network and cell signaling (28). If the interaction between heparin-like GAGs and the virus is required for viral infection, digestion of GAGs on the cell surface should inhibit infection. Cell surface heparan was removed using heparinase I, and CCV attachment was detected by western blotting and qPCR analysis. As shown in Figure 3, the viral gene copy number was significantly reduced in CCO/BB cells pretreated with heparinase I. At a heparinase concentration of 5 U/mL, the reduction was more than 90%, and the level of viral protein expression was also reduced. Thus, the ability of heparinase treatment to reduce viral gene copy numbers supports the notion that cell surface HS is a receptor for the virus. These results suggested that CCV attachment to the ECM is highly dependent on HS, which might be an important attachment factor for CCV infection.

To further determine the effect of heparan on CCV infection of CCO/BB cells, viral particle release was measured after ~48 h of treatment using the TCID₅₀ method. The CCV was serially diluted 10-fold, and the cells grown overnight in 96-well plates were incubated with different concentrations of heparin sodium salt or heparinase I for 1 h, the CCO/BB cells were then infected with different dilutions of CCV for about 4 h, and the culture medium was replaced. The cells were washed and treated for about 48 hours. The effect of heparin on CCV-infected CCO/BB cells was studied by observing the CPE produced by CCV-infected CCO/BB cells and calculating TCID₅₀. The results showed that with the increase of heparin sodium salt or heparinase I concentration, the TCID₅₀ value of CCV decreased significantly (Figures 4A, B), which further verified the important role of HS in CCV infection of CCO/BB cells. TEM images showed that the number of CCV particles in cells treated with 10 mg/mL heparin sodium salt and 5 U/mL heparinase I was lower than that in the control cells (Figure 4C and Supplementary Figure S1). Compared with the control group, the number of viruses in the treatment group decreased significantly after 4 h of infection. These results suggest that heparan and heparinase I can inhibit the production of offspring viruses.

To further confirm that CCV can bind heparan into CCO/BB cells, heparan affinity chromatography was used to detect the interaction between CCV virions and heparin (29, 30). Western blotting results showed that CCV virions could bind well to heparan (Figure 5A), and with the increase in the number of CCV virions, the binding increased, while the control group had no obvious binding, indicating that CCV could bind to heparan in a dose-dependent manner, which was consistent with previous results. The virus solution eluted was negatively stained, and TEM showed that the virus particles were round or oval, with capsule and typical herpesvirus characteristics (Figure 5B).