The EHV-1 specific primer pair referred to the EHV-1 sequence. The DNA standards constructed had a size of 302 bases (nt. 1,207 to 1,509 EHV-1 sequences). The amplified product was purified and cloned into the pMD19-T vector (Takara Biotechnology, Japan) for sequencing. Conversion to genome equivalents was done using the following equations: optical density at 260 nm (OD₂₆₀) of 1 = 50 μg/mL and 1 bp = 660 g/mol. This resulted in a molecular mass for the plasmids (2,994 bp) of 9 × 10¹² g/mol. For the standard curve, a 10-fold dilution series of the plasmid pEHV-1 was included in each test; based on the temperature of 57°C, the plasmid was diluted to 10⁴, 10⁵,10⁶, 10⁷, 10⁸, 10⁹, and 10¹⁰ copies/μL, which has an excellent linear relationship with cycle threshold (CT) values. All experiments were conducted using three replicates with the templates, and the fluorescence intensity of the corresponding CT value was used to obtain the amplification curve (17).

BBM was dissolved in dimethyl sulfoxide (DMSO; Sigma, United States) and diluted to 100 mM. Next, 96-well plates containing 5 × 10⁴ RK-13 cells cultured in Dulbecco’s modified eagle medium (DMEM; Biological Industries, Israel) with 10% fetal bovine serum (FBS; Biological Industries) were treated with different doses of BBM (1, 5, 8, 10, 15, and 20 μM) (18) for 8 h at 37°C; 0 μM BBM was used as a negative control. Subsequently, 3–2,5-diphenyl tetrazolium bromide solution (MTT; Sigma) at 5 mg/mL was added to each well and the cells were incubated for another 4 h at 37°C. The culture medium was then aspirated, the wells were washed with phosphate-buffered saline (PBS) and allowed to dry, and 200 μL DMSO was added to each well. The OD was read with a multi-well microplate reader at 570 nm (19). Cell viability was expressed as a percentage of the negative control group. For this part, we operated in the shadows to avoid light falling on the experiment. The experiments were performed in triplicate and expressed as mean ± SD (*p < 0.05).

In 6-well plates (1 × 10⁵ RK-13 cells/well), cell suspension was collected which treated with DMSO or 10 μM of BBM for 8 h at 37°C, infected with 0.7 MOI of EHV-1, and observed at 0, 12, 24, 36, and 48 h using an inverted microscope. Virus underwent freezing and thawing three times and was reinfected in 96-well plates (5 × 10⁴ RK-13 cells/well) using the Reed–Muench method to calculate the 50% tissue culture infective dose (TCID₅₀) for 7 d (20).

The specific primer pair for qPCR was designed using Oligo 6.0 and the EHV-1 glycoprotein B (gB) gene sequence (GenBank accession number: M36298). The primer pair (forward, 5′-GCCATACGTCCCTGTCCGACAA-3; reverse, 5′-CCTCCACCTCCTCGACGATGC-3′) yielded a product of 302 bases (nt. 1,207 to 1,509 EHV-1 sequences) under PCR cycle conditions of 95°C for 2 min, 95°C for 5 s, 60°C for 30 s, 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. RK-13 cells were seeded in 6-well plates (1 × 10⁵ cells/well), treated with 10 μM BBM for 8 h at 37°C, and infected with a neuropathogenic strain of EHV-1 (YM2019 kindly provided by Prof. Duoliang Ran (21), GenBank: MT063054.1) at an MOI of 0.7. After 4 h, the media was changed for fresh DMEM. At 0, 12, 24, 36, and 48 h later, suspension DNA was extracted for cells provided with the E.Z.N.A^® Viral DNA Kit (D3892-01, Omega Bio-Tek, the United States), and quantified to 6.07 × 10⁵ copies/mL using a QuantiNova SYBR Green PCR Kit (No. 208054) and 7,500 Fast Real-Time PCR software (22).

Western blotting was performed, as previously described in 3.3, on RK-13 cells treated with BBM and infected with EHV-1 at an MOI of 0.7. Harvested RK-13 cells were lysed with RIPA lysis buffer (containing 1 mM PMSF). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% skim milk for 2 h and then incubated with primary antibodies against EHV-1 (1:1,000 EHV-1 positive serum) (23) and GAPDH (1,1,000; No. 10494-1-AP, Proteintech Group, United States) at 4°C overnight, followed by incubation with secondary antibodies at room temperature. Blots were exposed using an enhanced chemiluminescence detection kit (P0018S, Beyotime, China) and a chemiluminescence immunoassay analyzer. Relative blot intensity (ratio to GAPDH) was quantified using ImageJ software.

Binding was studied by adding EHV-1 at an MOI of 0.7 to chilled RK-13 cells. After 0, 2, 4, 6, and 8 h of incubation at 4°C, the cells were washed extensively with chilled PBS, and then lysed. For internalization, the cells were washed extensively with chilled PBS following incubation with EHV-1 for 3 h, and the temperature was changed to 37°C to allow internalization of the attached virus. At 3, 4, 5, and 6 h post-internalization, the cells were washed extensively with acidic PBS to remove cell surface-attached viruses. Total DNA of each lysate sample was collected with the E.Z.N.A^® Viral DNA Kit (24).

To evaluate the role of BBM in EHV-1 infection of Syrian hamsters, 48 specific-pathogen-free (SPF) male Syrian hamsters (age 3 weeks, purchased from Charles River, Beijing, China) were randomly divided into three concentration groups and one no-infection group (received 0 mg/kg BBM only). The BBM concentrations were configured as 0 mg/kg (DMSO 660 μL, PEG300 3,960 μL, Tween80 1,320 μL, H₂O 7,260 μL), 50 mg/kg (BBM 66 mg, DMSO 660 μL, PEG300 3,960 μL, Tween80 1,320 μL, H₂O 7,260 μL), and 100 mg/kg, (BBM 132 mg, DMSO 660 μL, PEG300 3,960 μL, Tween80 1,320 μL, H₂O 7,260 μL). The hamsters were given the indicated concentration of BBM by oral administration every 24 h for 3, 7, and 14 d, and infection with 0.1 mL DMEM containing10⁹ TCID₅₀ of EHV-1 by nasal drip (23); 0 mg/kg BBM with and without EHV-1 infection were the positive and negative controls, respectively. Brain, lung, intestine, spleen, liver, and kidney tissue samples (25) were collected and 0.2 g of each tissue was ground and cracked, then the DNA was extracted, quantified to 6.07 × 10⁵ copies/mL, analyzed detected using qPCR. Animals were cared for according to the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

To detect the effects of BBM on existing EHV-1 infection, 48 Syrian hamsters were randomly divided into two BBM concentration groups (0 or 100 mg/kg) and one no-infection group (received 0 mg/kg BBM only, as negative control). Hamsters were infected with 0.1 mL DMEM containing 10⁹ TCID₅₀ of EHV-1 by nasal drip 3 d. After 48 h, BBM was orally administered to each hamster every 24 h for 0 (positive control) 3, 7, and 14 d. Brain, lung, liver, kidney, intestine, and spleen samples were collected. Brain and lung histopathologic changes were detected by microscope. DNA was quantified in the collected tissue samples to measure attenuation of EHV-1 infection. All procedures were approved by the Animal Welfare and Ethics Committee of Xinjiang Agricultural University.

Tissues were fixed in 10% neutral buffered formalin for 48 h and processed using routine methods for histology. Next, 5-mm sections were stained with hematoxylin and eosin (H&E). Central nervous system sections were taken from the olfactory bulb (OB); piriform cortex; septostriatal, rostral, and caudal diencephalon; rostral mesencephalon; and cerebellum with cerebellar peduncles. Histological lesions were classified as mild, moderate, or severe (26). The severity of the histopathological changes in EHV-1 infected hamsters was investigated. All evaluations were performed by investigators blinded to the treatment and were conducted separately by two pathologists. Histopathological severity was scored such that higher numbers reflected a higher degree of tissue inflammation, injury, and necrosis. Severity was determined by the extent of necrotic cells and the number and relative area of inflammatory cell infiltration.

All statistical analyses were conducted using SPSS 17.0. A t-test was used to evaluate distribution type. Normally distributed data are expressed as mean ± standard deviation (SD), and an independent sample t-test was used for comparisons between the two cohorts.

The bands in agarose gel electrophoresis following PCR amplification of EHV-1 DNA extracts indicated that the PCR product was 302 bp (Figure 1A), which was consistent with the expected product size. The coincidence rate of the sequence of positive plasmids was 99%, similar to the sequences documented in the GenBank database. The regression equation of the standard curve was Y = −3.0523X + 36.138, Y(CT), X(Copies), R² = 0.9991 (Figure 1B). Amplification efficiency was 113.066%. The melting curve was a single peak (Figure 1C), and the amplification curve was obtained using T_m values between 84.5 and 86 (Figure 1D).

An MTT assay was used to reveal the effects of BBM on the viability of RK-13 cells. There was a significant reduction in the viability of the RK-13 cells following treatment with 15 μM and 20 μM BBM for 24 h (p < 0.01) (Figure 2A). BBM decreased the viability of the RK-13 cells in a concentration-dependent manner; the IC₅₀ of BBM was 17.99 μM (Figure 2B).

To explore the effect of BBM on EHV-1 DNA replication, the anti-EHV-1 activity of BBM was investigated in RK-13 cells. BBM significantly inhibited viral generation from 24 h to 48 h post-infection (pi) (p < 0.01) in virus titration assays (Figure 2C) and as quantified in qPCR assays (Figure 2D). From 24 h to 48 h, viral generation decreased about 50 times compared with DMSO treatment.

To determine whether BBM could inhibit increased levels of EHV-1 protein in cells infected with EHV-1, RK-13 cells were treated with 10 μM BBM and infected with EHV-1 for 0, 12, 24, 36, and 48 h. Western blot results showed that BBM significantly inhibited EHV-1 compared with DMSO treatment (Figure 2E).

Next, the cytopathic effect (CPE) of EHV-1 infection was evaluated in the RK-13 cells that were treated with BBM compared with those that were not treated. A CPE was exhibited from 12 h pi in the cells that were not treated with BBM, while BBM treatment reduced cell death and syncytia formation (Figure 2F), and delayed cell death until at least 24 h pi. These data suggest that EHV-1 infection results in the development of a syncytial plaque-forming phenotype in RK-13 cells, but BBM could delay this change, particularly at 24, 36, and 48 h pi.

To illustrate the mechanism of BBM inhibition of EHV-1, a binding assay was used. RK-13 cells were infected with EHV-1 at 4°C for different durations. Attached viral DNA copies were determined by qPCR, and percentage changes in DNA copies were derived by comparing the number of DNA copies in the BBM samples with that of the untreated cells. In parallel, EHV-1 was pre-blocked or controlled with 10 μM BBM for 1 h at 37°C before inoculating onto chilled RK-13. BBM inhibited EHV-1 infection by repressing viral binding. This effect was related to time, and the qPCR results demonstrated that the number of bound EHV-1 DNA copies was significantly reduced by BBM at 4 h and 8 h pi (p < 0.01) compared with controls, and was also reduced at 6 h (p < 0.05). As shown in Figure 3A, there was no significant difference in bound EHV-1 DNA copies between BBM and the controls at 0 h and 2 h (p > 0.05); however, at 4 h pi, there was an approximate 100-fold decrease in EHV-1 DNA copies in BBM-treated cells compared with control. This indicated that BBM repressed viral binding.

Viral internalization was also explored to explain BBM inhibition of EHV-1 replication. For the internalization assays, we pre-treated RK-13 cell with BBM and used different incubation times. The number of internalized viral DNA copies was determined by qPCR. BBM inhibited EHV-1 infection by repressing viral internalization at all times examined in the assay (3, 4, 5, and 6 h). Figure 3B shows that, compared with controls, the number of internalized EHV-1 DNA copies treated with BBM significantly decreased at different intervals pi (p < 0.01), particularly at 4 h incubation where a decrease of approximately 100 times was detected. This indicated that BBM had antiviral activity that inhibited viral internalization from EHV-1 infection.

Following pre-treatment with different concentrations of BBM 3 d pi and infection with EHV-1 YM2019 (Figure 4A), the hamsters began to show ruffled fur, dyspnea, crouching and a hunched posture in corners at 2 d pi with EHV-1. The hamsters also exhibited increased reactivity to external stimulation, and sialorrhea. On day 2 pi with EHV-1, the animals showed decreased survival (Figure 4B) and a significant increase in the level of EHV-1 DNA (p < 0.01). However, these changes could be reduced by pre-treatment of the hamsters with 50 mg/kg BBM (p < 0.05) and significantly reduced by pre-treatment with 100 mg/kg BBM (p < 0.01). Furthermore, the changes were dose- and time-dependent, especially in brain and lung tissues, and there was little difference in the other tissues (Figures 4C–E). EHV-1 replication predominantly induced changes in the brain and lungs (Table 1). Analysis of the pathological tissue sections showed that EHV-1 infection led to infiltration of neutrophils and plasma cells in the meninges (meningitis), severe lymphocytic meningoencephalitis, the OB were expanded by macrophages and lymphocytes, the alveolar septa exhibited diffuse thickening with macrophages and a few neutrophils and lymphocytes, and there was multifocally amorphous eosinophilic fluid (edema) with erythrocytes (hemorrhage) in the alveolar lumen. The 50 mg/kg BBM group experienced a reduction in these histopathological characteristics; occasionally, there was an increase in erythrocytes in the alveolar lumen with contained eosinophilic, Multifocally, within there was degeneration, necrosis cells to the lung. The hamsters that received 100 mg/kg BBM had no histopathological symptoms (Figures 4F–H). Other tissues did not exhibit any significant pathological changes expect a little hemorrhage.

Hamsters were infected with EHV-1 YM2019 for 3 d and then treated with different concentrations of BBM (Figure 5A). The hamsters began to show severe neurological symptoms with difficulty breathing and increased reactivity to external stimulation. In addition, the animals showed decreased survival after EHV-1 infection (Figure 5B). The level of EHV-1 DNA was significantly increased in EHV-1-infected hamsters (p < 0.01), but this was reduced in hamsters that received 50 mg/kg BBM (p < 0.05) and significantly reduced in those that received 100 mg/kg BBM (p < 0.01). All reductions in the level of EHV-1 DNA by BBM were time- and dose-dependent, especially in the brain and lung; there was a lower level of EHV-1 DNA in the spleen and liver, but not in the kidneys and intestine (Figures 5C–F). EHV-1 infection leads to marked tissue damage in the brain, lung, spleen, liver, and a little in the intestine, with hardly any tissue damage in the kidneys (Table 2). Histopathological section analysis showed that EHV-1 infection could increase connective tissue in the liver and red pulp in the spleen, the OB were expanded by macrophages and lymphocytes, the alveolar septa exhibited diffuse thickening with macrophages and a few neutrophils and lymphocytes, and there was multifocally amorphous eosinophilic fluid (edema) with erythrocytes (hemorrhage) in the alveolar lumen. Occasionally, erythrocyte increased in the alveolar lumen with contained eosinophilic, Multifocally, within there was degeneration, necrosis cells to the lung, while the hamsters that received 100 mg/kg BBM had no histopathological symptoms (Figures 5G–J). There was no significant pathological changes expect a little hemorrhage in the intestine and kidneys. These results demonstrated that BBM significantly inhibited EHV-1 infection in Syrian hamsters. Inflammatory cells increased in the brain, and neutrophils, giant cells, and erythrocytes increased transference cure. BBM significantly inhibited EHV-1 and histopathological changes, indicating that BBM significantly affected EHV-1 pre-infection in Syrian hamsters.