Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo

HeLa and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. CVB3M strain was amplified in HeLa cells by infection. This strain was a kind gift from Dr S.A. Huber, University of Vermont, U.S.A. [18]. To assess the validity of shRNA-2B, HeLa cells were seeded into 12-well plates (1 × 10⁵ cells/well) overnight. When reached 50-60% confluency, cells were washed with phosphate buffered saline (PBS) and transfected with plasmid or transduced with lentivirus. At 24 h post transfection or 48 h post transduction, cells were washed and infected with CVB3 (MOI = 0.01) for 1 h. The cells were then overlaid with complete medium and incubated at 37°C with 5% CO₂. At different time points post infection, supernatants were collected to determine the virus titers by viral plaque assay. Experiments were carried out 3 times.

pHelper 1.0, pHelper 2.0 and pGCSIL-GFP plasmids were purchased from Shanghai GeneChem Co. Ltd. (Shanghai, China). To construct the recombinant vector, RNAi stem-loop DNA oligos containing the target sequences (GGACTATGTGGAACAGCTT) in the region of CVB3 2B were chemically synthesized, annealed and cloned into the AgeI/EcoRI-digested pGCSIL-GFP, thus generating the plasmid pGCL-2B. The lentiviral vector pGCSIL-GFP contained a U6 promoter to express continuously the shRNA and CMV promoter to express the green fluorescent protein (GFP), which could be used to detect the transfection efficiency of viral packaging and infection. Recombinant lentiviruses were generated by co-transfection of 293T cells with 20 μg pGCL-2B and packaging vectors (15 μg pHelper1.0, 10 μg pHelper2.0) using 100 μl of Lipofectamine 2000™ reagent according to the manufacturer’s instructions (Invitrogen, USA). The recombinant lentivirus was designated Lenti-2B. The viral supernatant was harvested 48 h after transfection, passed through 0.45 μm filters and concentrated. The viral titer was determined by infecting 293T cells with serial dilutions of concentrated lentivirus, and then determining the GFP expression of infected cells by fluorescence microscopy 96 h after infection. Therefore, the titer is expressed as “transduction unit (TU)/ml”. A scramble siRNA sequence (5’-UUCUCCGAACGUGUCACGU-3’) was used to generate the non-silence control plasmid and lentivirus that were designated pGCL-NC and Lenti-NC.

6-week-old BALB/c male mice were injected via caudal vein with plasmid or lentivirus. Entranster™-in vivo transfection reagent (Engreen Biosystem Co, Ltd. China) was used to deliver the plasmids. Control mice were injected with pGCL-NC, Lenti-NC and minimum essential medium (MEM), respectively. After 24 h, all mice were inoculated intraperitoneally with a dose of 8 × 10³ plaque-forming units (pfu)/mouse of CVB3 virus. Some mice were observed for survival time. Other mice were euthanized on days 3, 5 and 7 after infected with CVB3 (n = 10 per group). Experiments were carried out 3 times. The animal experiment has been performed with the approval of the ethics committee of Capital Institute of Pediatrics and followed internationally recognized guidelines.

Hearts and pancreases from mice were weighted, homogenized in 0.5 ml MEM, and centrifuged for 10 minutes at 1000 rpm. In the supernatants, infectious virus was analyzed by viral plaque assay, and the viral titer in infected tissue was expressed as pfu/g. To assess severity of myocarditis and pancreatitis, paraffin-embedded sections of tissues were stained with hematoxylin-eosin and examined histopathologically for evidence of inflammation and necrosis. The immunohistochemistry staining for CVB3 and GFP were performed using anti-coxsackievirus B3 monoclonal antibody (Chemicon International, USA) and anti-GFP monoclonal antibody (Chemicon International, USA) as the primary antibody following the procedure described previously [19].

HeLa cells were seeded into six-well plates (4 × 10⁵ cells/well) and incubated at 37°C with 5% CO₂ for 48 h. When cell confluency reached approximately 90%, cells were washed with PBS to remove the fetal bovine serum and then overlaid with 200 μl of 1:10 diluted supernatant. The cells were incubated at 37°C for 60 min and the supernatants were removed, and washed with PBS. Finally, cells were overlaid with 2 ml of sterilized soft Bacto-agar/MEM (2 × MEM:1.5% Bacto-agar = 1:1). The cells were incubated at 37°C for 72 h, fixed with Carnoy’s fixative for 30 min and then stained with 1% crystal violet. The plaques were counted and the viral pfu/ml was calculated [20]. Each sample was tested in triplicate.

In this study, recombinant plasmid expressing the shRNA targeting 2B sequence was cloned into pGCSIL-GFP and named pGCL-2B. This plasmid was used to pack the infectious lentivirus particles by co-transfection of 293T cells with other packaging plasmids. The lentivirus was named Lenti-2B and its titer was determined by fluorescent microscopy. The titer of the recombinant lentiviruses was approximately 2 × 10⁹ TU/ml.

To evaluate the transfection efficacy of pGCL-2B, HeLa cells were transfected with pGCL-2B at a series of doses and the GFP-expressing cells were visualized through fluorescence microscopy after 24 h. The transfection rate of pGCL-2B was determined by the percent of the area of GFP-expressing cells versus that of whole HeLa cells in one field calculated using Anymicro DSS™ digital shoot system (Beijing Yutianshijiweiye Technology Development Co. Ltd., China). Figure 1A shows that 1.25 μg/well was the lowest dose of pGCL-2B to reach about 60% of cells expressing GFP and was less toxic to the cells. Higher the dose of plasmid used, more vacuolar change occurred in the cells. When HeLa cells were transfected with pGCL-2B of 6.25 μg/well, the cells died after 24 h. Subsequently, pGCL-2B of 1.25 μg/well was used to transfect HeLa cells, and GFP expressions detected at different time. After 48 h, GFP expression rate reached to the peak (Figure 1B). The transfection rate of control plasmid was similar to that of pGCL-2B. We also studied its antiviral efficacy as a function of time and dose in HeLa cells. Compared to the viral titer of pGCL-NC, an approximate 96% decrease in viral titer was detected in these cultures treated with pGCL-2B (1.25 μg/well) at 48 h post challenge. This protective effect was reduced after 60 h and 72 h (Figure 1C). Determination of CVB3 titers 48 h after infection showed a strong antiviral effect of the pGCL-2B transfection with titers reduced 95.83% at the plasmid dose of 1.25 μg/well and 96.46% at the plasmid dose of 1.75 μg/well (Figure 1D). Expectedly, HeLa cells pre-treated with a dose of 1.25 μg/well of pGCL-2B were efficiently protected against CVB3-induced cell lysis at 48 h after infection, while strong CPE occurred in other two groups (Figure 1E).

To evaluate the efficacy of Lenti-2B on coxsackieviral replication, Lenti-2B (1, 10, 100 TU/cell) or Lenti-NC were used to transduce HeLa cells respectively. After 48 h, cells were infected with CVB3 (MOI = 0.01), and then the culture supernatants were collected to determine the CVB3 titers by viral plaque analysis at 48 h post challenge. Lenti-2B at a dose of 10 TU/cell or 100 TU/cell achieved an approximately 84% and 90%, respectively, reduction of CVB3 titer compared with that of control cells treated with Lenti-NC. The antiviral effect was almost absent at the dose of 1 TU/cell because of the low transduction efficacy of the lentivirus (Figure 2A). Lenti-2B also exhibited the protective effect against CPE (Figure 2B). These results indicate that recombinant plasmid and lentivirus expressing shRNA-2B are highly efficient antiviral agents in vitro.

To reveal the effect of shRNA-2B on survival, virus replication, and tissue damage, pGCL-2B (20 μg/mouse, 30 μg/mouse or 40 μg/mouse) or Lenti-2B (1 × 10⁷ TU/mouse, 5 × 10⁷ TU/mouse or 1 × 10⁸ TU/mouse) was injected into mice before infection with CVB3 virus. The control vector pGCL-NC or Lenti-NC was applied in the same way. Transfection of pGCL-NC or Lenti-NC had no effect on survival compared with the infected but non-treated animals. All mice died in 9 days after infected with CVB3. In contrast, in the group infected with 1 × 10⁸ TU/mouse Lenti-2B, 50% mice were still alive at day 9 and 40% mice lived at 30 days after challenge with CVB3 (Figure 3A). The pGCL-2B group (40 μg/mouse) also showed a higher survival rate than that of the pGCL-NC control group (30% vs. 0 survival rate, P < 0.01) at day 9 post CVB3 infection (Figure 3B). However, transfection of pGCL-2B or Lenti-2B with lower doses had no difference on survival between treated group and control group. Both pGCL-2B and Lenti-2B improved significantly the life span of mice, suggesting that shRNA-2B could be the potential agent to reduce the mortality caused by viral myocarditis.

To further determine the potency of Lenti-2B, mice were euthanized to directly compare the tissue virus titers by plaque-forming assay at days 3, 5 and 7 post CVB3 infection. The virus titers in the hearts and pancreases of mice infected with Lenti-2B were approximately 4-log₁₀ lower than those in the control group (P < 0.01 versus Lenti-NC group for both organs, n = 5 in each group) (Figure 4). Virus titers were 1-log₁₀ lower in the heart and 2-log₁₀ lower in the pancreas of mice that received pGCL-2B than that in the respective control mice (Figure 5). Furthermore, the virus titer was 10³-fold lower in the heart of Lenti-2B group than that of pGCL-2B group, suggesting that lentivirus vector expressed shRNA-2B more effectively than plasmid in vivo (P < 0.01).

To confirm the protect effect of Lenti-2B on the hearts and pancreases of CVB3 infected mice, hematoxylin-eosin staining was used to detect inflammation and immunohistochemistry staining was used to detect the distribution of CVB3 viruses or GFP expression in the organs. No, or only slight, myocardial inflammation and CVB3 infection were revealed in the Lenti-2B group; however, there were large extent of necrosis, calcification and serious virus infection in the Lenti-NC group and non-shRNA-treated group (Figure 6).