Antiviral activity of diallyl trisulfide against H9N2 avian influenza virus infection in vitro and in vivo

The H9N2 avian influenza virus (A/mallard/Jiangxi/39/2011) stored in our laboratory was isolated from wild duck in Jiangxi Province, China in 2011. The complete sequences of all 8 genomic segments (JQ901621, JQ901632, JQ901643, JQ901654, JQ901665, JQ901676, JQ901687, and JQ901698) of the virus are available from the GenBank. The H9N2 AIV used in this study has been shown to infect and induce acute lung injury in mice [23]. The virus was propagated in 10-day old pathogen free chicken embryos (Ingelheim Vital Biotechnology Co, Ltd, Beijing, China) at 37 °C for 72 h (h). The 50% tissue culture infection dose (TCID50) was determined in human alveolar epithelial cell line A549 cells, and the titer of viral stock was 10^6.9 TCID50/100 μl.

A549 cells were purchased from the Cell Bank Academy of Science (China) and grown in Dulbecco's Minimum Essential Medium (DMEM) (Gibco, USA) supplemented with 10% FBS at 37 °C with 5% CO₂. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (purity > 98%), amantadine hydrochloride (AMT) (purity > 99%), and DATS (purity > 97%) were purchased from Sigma-Aldrich. DATS was dissolved in 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich) at 3 mM as stock solution and stored at − 20 °C before use. AMT was dissolved in 0.1% DMSO at 4 mM and used as positive anti-virus control drug. MTT (5 mg/ml) was dissolved in phosphate buffered saline (PBS) and filtered through a 0.2-µm microporous membrane (Millipore).

The cytotoxicity of DATS and AMT for A549 cells was determined using the MTT assay. A549 cells were seeded at a concentration of 1 × 10⁵ cells/well in a 96-well plate (Corning) and incubated at 37 °C in a 5% CO₂ incubator for 24 h. When the cells reached 75% confluency, they were replenished with 0.1 ml of maintenance medium (with 2% FBS) containing various concentrations of DATS (75–3000 µM) or AMT (125–4000 µM). Five wells of cell were used for each concentration and incubated at 37 °C in a 5% CO₂ incubator for 48 h. Cells in wells with 0.1 mL maintenance medium without drugs were used as negative controls. After removal of culture medium in each well, MTT (5 mg/ml, 20 µl) was added and incubated for 4 h at 37 °C. The reaction was stopped by addition of 100 µl of DMSO, and the absorbance (Abs) of the purple formazan formed due to MTT reduction by NAD(P)H-dependent cellular oxidoreductase in live cells was read at 570 nm using a microplate reader (Thermo Fisher Scientific,USA). The percentage of cell viability after drug treatment was calculated as follows: % Cell viability = [Abs of treatment group/Abs of control group] × 100%.

The effect of DATS on H9N2 AIV infection was investigated with two different conditions, pre-treatment and post-treatment. A549 cells were seeded in 96-well plates or T-25 culture flasks and grown to 75–90% confluency. For pre-treatment experiments, A549 cells were treated with three different concentrations (375 µM, 187.5 µM and 93.75 µM) of DATS for 24 h, washed twice with PBS, and then infected with H9N2 AIV. For post-treatment experiments, the cells were infected with H9N2 AIV for 1 h, washed twice with PBS, and then incubated in maintenance medium containing three different concentrations (375 µM, 187.5 µM, and 93.75 µM) of DATS. Cells treated with 500 µM AMT were used as positive drug control. To test the effect of DATS on virus adsorption, the cells were inoculated with H9N2 AIV at a multiplicity of infection (MOI) of 0.1 After 1 h of incubation at 37 °C, the cells in wells of a 96-well plate were washed twice with PBS and then incubated in maintenance medium for 48 h. The inhibitory activity of DATS on cytopathic effect (CPE) induced by H9N2 AIV was determined by MTT assay. The cells in T-25 culture flasks were incubated at 37 °C under 5% CO2 with a small amount of maintenance medium, and the supernatants were harvested at 24 h and 48 h after drug treatment and assayed for TCID50 on MDCK cells.

Quantitative real-time RT-PCR (qRT-PCR) was performed to determine the expression levels of IL-6, TNF-α, RIG-I, IRF-3, IFN-β, and H9N2 AIV M gene. All qRT-PCR primers (Table 1) were designed using the software Primer Premier 5.0 (Premier, Canada). Total RNA was extracted from culture supernatants and cells using the RNeasy Mini Kit (QIAGEN). Synthesis of the first-strand complementary DNA was conducted using the Invitrogen Transcription SuperScript™III RT Kit (Invitrogen, US). Each qRT-PCR reaction contained 10 μL SYBR Premix Ex Taq (2 ×), 0.4 μL forward primer, 0.4 μL reverse primer, 0.4 μL ROX reference dye (50 ×), 2 μL cDNA, 6.8 μL H₂O (total volume 20 μL), and SYBR Premix Ex Taq RR420A-Tli RNase H Plus (Takara Clontech, Dalian). PCR was performed as follows: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 31 s. Expression levels of various genes were normalized to that of the housekeeping gene GAPDH as its expression level was stable in A549 cells. Four independent PCRs were performed for each sample. All data were analyzed using the Sequence Detector Systems software (Applied Biosystems, USA). Fold change in gene expression was calculated using the 2^−ΔΔCt method.

Pathogen free BALB/c female mice (aged 6–8 weeks) were purchased from Shanghai Slake Co, Ltd. (China). To evaluate the effect of DATS on H9N2 AIV-induced lung injury, the mice were randomly divided into infected group, DATS-treated infected group, DATS control group, and uninfected control group (n = 26 mice/group). Mice in infected group and DATS-treated infected group were lightly anaesthetized with diethyl ether and inoculated intranasally with 80 μL of H9N2 AIV in allantois fluid (1 × 10⁶ 50% egg infection dose per 0.1 ml, EID50). Mice in uninfected control group were inoculated with the same dilution and volume of sterile allantois fluid intranasally. One day after virus inoculation, mice were intraperitoneally injected with DATS (30 mg/kg body weight) or 0.9% saline daily for 2 weeks. Five mice from each group were euthanized at days 2, 4, and 6 post-infection to obtain whole lungs, kidneys, spleens, and intestines. The pulmonary index was calculated according to the following formula: Pulmonary index = [Lung weight (g)/Body weight (g)] × 100. A portion of each lung was fixed in formalin and processed for histological examinations as described previously [24]. The other part of each lung was used for viral titration on MDCK cells and expression assessments of inflammatory cytokine genes and H9N2 AIV M gene by qRT-PCR. The viral loads in other organs of infected mice were also determined on MDCK cells at days 2, 4, and 6 post-infection. The remaining mice were monitored daily for clinical signs and body weight for 14 days after infection.

All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS for Windows, version 19.0 (SPSS Inc, USA) and GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). The one-way analysis of variance (ANOVA) followed by post-hoc Tukey test or unpaired two-tailed t-test was used to determine the significance of difference between two groups. In all statistical analyses, p < 0.05 or p < 0.01 was considered significant.

The cytotoxicity of DATS and AMT on A549 cells was examined using the MTT-based cell viability assay. Results showed that 375 µM of DATS and 500 µM of AMT had little or no cytotoxicity to A549 cells (Fig. 1). Therefore, 93.75–375 µM of DATS and 500 µM of AMT were used for subsequent experiments.

To investigate anti-H9N2 AIV activity of DATS, its effect on different stages of infection was examined. DATS was added to A549 cell cultures to a final concentration of 93.75, 187.5, or 375 µM 24 h before (pre-treatment), or 1 h after (post-treatment) H9N2 AIV infection. H9N2 AIV-infected A549 cells were treated with 500 µM AMT as positive anti-viral control in the same manner. As AMT was dissolved in 0.1% DMSO, cells in untreated control groups were treated with 0.1% DMSO before or after infection to serve as negative controls.

In the pre-treatment experiment, the viability of untreated H9N2 AIV infected- cells was 33.6%, and that of AMT-treated infected group was 47.8%. The viability of H9N2-infected cells treated with 93.75, 187.5, or 375 µM of DATS was 36.7%, 44.8%, and 44.9%, respectively (Fig. 2a), suggesting that pre-treatment of A549 cells with DATS for 24 h rendered them less susceptible to H9N2 AIV infection. In the post-treatment experiment, the viability of untreated H9N2 AIV infected-cells was 53.5%, and that of AMT-treated infected cells was 82.2%. The viability of infected cells treated with 93.75, 187.5, or 375 µM of DATS 1 h after H9N2 AIV infection was 67.7%, 72.7%, and 79.3%, respectively. The anti-viral activity of DATS was dose dependent and was more effective in the post-treatment experiment.

Culture supernatants of H9N2 AIV-infected cells were assayed at 24 h and 48 h after infection for TCID50 on MDCK cells. Results showed that DATS effectively inhibited virus proliferation in both pre-treatment and post-treatment experiments at 24 h and 48 h (Fig. 2b). To further assess the effect of DATS on H9N2 AIV replication, the expression levels of the M gene of H9N2 AIV in both pre-treatment and post-treatment experiments were examined by qRT-PCR 48 h after infection. Results showed that M gene expression level in AMT pre-treatment experiment was 0.71 fold and that in AMT post-treatment experiment was 0.54 fold of the level of untreated H9N2 AIV-infected cells (Fig. 2c). H9N2 AIV M gene expression in A549 cells in DATS pre-treatment experiment was 0.6 fold (40% decrease) that of untreated H9N2-infected cells for all 3 DATS doses. The inhibitory effect of DATS on H9N2 AIV replication was more profound in the post-treatment experiment as M gene expression in A549 cells treated with 93.75, 187.5, or 375 µM of DATS after infection was 0.27, 0.18, and 0.16 fold that of untreated cells, indicating that DATS at 93.75, 187.5, and 375 µM inhibited H9N2 AIV replication by 73, 82, and 84% when it was added to the cells 1 h after infection. Taken together, these results indicated that DATS was inhibitory to H9N2 AIV replication and was more effective when it was added 1 h after infection. Accordingly, 375 µM of DATS added 1 h post-infection was used for subsequent experiments.

To investigate the effect of DATS on H9N2 AIV-induced inflammation, the expression levels of inflammatory cytokines TNF-α and IL-6 in cells of post-treatment experiment were measured by qRT-PCR at 24 h and 48 h after infection. The level of TNF-α and IL-6 mRNAs thus determined was then compared to that of uninfected control, which was set as 1.0. For IL-6, H9N2 AIV infection increased its expression by 15.8 fold at 24 h and 9.6 fold at 48 h (Fig. 3a). Treatment of H9N2 infected cells with 500 µM AMT decreased IL-6 expression by 5.6 fold at 24 h and 2.5 fold at 48 h (Fig. 3a). Treatment of H9N2 infected cells with 375 µM of DATS reduced IL-6 expression by 9.0 fold at 24 h and 2.3 fold at 48 h. For TNF-α, H9N2 AIV infection increased its expression by 11.3 fold at 24 h and 23.7 fold at 48 h (Fig. 3b). Treatment of H9N2 infected cells with 500 µM AMT decreased TNF-α expression by 2.9 fold at 24 h and 2.3 fold at 48 h. Treatment of H9N2 infected cells with DATS decreased TNF-α expression by 11.3 fold at 24 h and 4.8 fold at 48 h. These results showed that DATS decreased proinflammatory response induced by H9N2 AIV infection.

The effect of DATS on the expression of anti-viral factors RIG-I, IRF-3, and IFN-β in A549 cells were also investigated by qRT-PCR. The level of RIG-I, IRF-3, and IFN-β mRNAs thus determined was then compared to that of uninfected control, which was set as 1.0. H9N2 AIV infection was found to increase RIG-1 expression by 14.8 fold at 24 h and 12.9 fold at 48 h post-infection (Fig. 3c). Treatment of A549 cells with 500 µM AMT 1 h after H9N2 AIV infection resulted in a further increase in RIG-1 expression, 29.8 fold at 24 h and 17.9 fold at 48 h. Treatment of cells 1 h after infection with 375 µM of DATS increased RIG-1 expression 26.9 fold at 24 h and 15.3 fold at 48 h (Fig. 3c). For IRF3, H9N2 AIV infection increased its expression by 19.8 fold at 24 h and 3.3 fold at 48 h (Fig. 3d). Treatment of A549 cells 1 h after H9N2 AIV infection with 500 µM AMT increased IRF3 expression 62.8 fold at 24 h and 24.4 fold at 48 h. Treatment of cells with 375 µM of DATS 1 h after infection increased its expression 51.8 fold at 24 h and 34.2 fold at 48 h (Fig. 3d). For IFN-β, H9N2 AIV infection increased its expression by 6.1 fold at 24 h and 4.7 fold at 48 h (Fig. 3e). Treatment of A549 cells 1 h after H9N2 AIV infection with 500 µM AMT increased its expression 13.6 fold at 24 h and 9.7 fold at 48 h. Treatment of cells with DATS 1 h after H9N2 AIV infection increased its expression 9.2 fold at 24 h and 8.2 fold at 48 h (Fig. 3e). These results suggest that DATS exerts its anti-H9N2 AIV by enhancing innate immune responses.

Based on results of in vitro experiments, we hypothesized that DATS can attenuate the symptoms of H9N2 AIV infection in mice. It has been shown that H9N2 AIV-infected mice exhibit marked inactivity, emaciation, ruffled fur, lack of appetite, labored breathing, respiratory distress, and decreased body weight [24]. In this study, the decrease in body weight caused by H9N2 AIV infection was most profound (12.8% loss) 3 days after infection. Treatment of infected mice with DATS (30 mg/kg/day), starting 1 day after infection, diminished such loss (8.2% loss) 3 days after infection. The body weight of mice in DATS-treated group and uninfected control group was not significantly changed (Fig. 4).

To investigate pulmonary edema caused by H9N2 virus infection, lung index (weight lung to body weight ratio) was determined at different time points during infection. Results showed that lung index was increased in mice of infected groups at days 2–6 after H9N2 AIV infection, but it was lower in mice of DATS-treated group (Fig. 5a). At day 6, DATS effect was not significant. These results indicated that H9N2 AIV infection caused edema in the lungs, thus increasing lung index, and DATS treatment reduced the severity of lung edema. Similar patterns of DATS effect were observed in viral loads in the lungs. A significant decrease in viral loads was observed in DATS treatment for 2 and 4 days (Fig. 5b). Since the levels of influenza M protein reflect the levels of virus replication, mRNA levels of the M gene were determined. Results showed significantly reduced levels of M gene mRNA after treatment with DATS for 2 or 4 days (Fig. 5c).To examine lung lesion, HE staining of lung sections was carried out. Results showed that the lungs of H9N2 AIV-infected mice exhibited collapsed alveolar spaces, infiltration of inflammatory cells, interstitial and alveolar edema, and hemorrhage (Fig. 6). These pathological changes were relatively mild in mice with DATS treatment. These results suggest that DATS treatment reduced the severity of edema and viral loads in the lungs.

The expression levels of inflammatory cytokines (IL-6 and TNF-α) and anti-viral cytokines (RIG-1 and IFN-β) induce by H9N2 AIV infection in lungs were then determined. The mRNA level of these cytokines was then compared to that of uninfected control, which was set as 1.0. Results showed that the effects of H9N2 AIV infection and DATS treatment on the expression of TNF-α and IL-6 were most profound at 6 days post infection (Fig. 7). At that time, H9N2 AIV infection increased TNF-α expression by 6.5 fold, and DATS treatment reduced it to 3.9 fold. For IL-6, H9N2 AIV infection increased its expression by 20 fold, and DATS treatment reduced it to 18 fold. The effect on RIG-I expression was most profound (16.9 fold increase) at day 6 post-infection. DATS treatment further increased it to 29 fold (Fig. 7). The effect on IFN-β expression was most profound (tenfold increase) at 2 days post-infection. DATS treatment increased IFN-β expression 12 fold. These results showed that H9N2 AIV infection caused a profound increase in the expression of inflammatory and anti-viral cytokines. DATS treatment reduced the expression of TNF-α and IL-6, but increased the expression of RIG-1 and IFN-β.