Specific interference shRNA-expressing plasmids inhibit Hantaan virus infection in vitro and in vivo

Abstract

Aim: To investigate the antiviral effects of vectors expressing specific short hairpin RNAs (shRNAs) against Hantaan virus (HTNV) infection in vitro and in vivo.

Methods: Based on the effects of 4 shRNAs targeting different regions of HTNV genomic RNA on viral replication, the most effective RNA interference fragments of the S and M genes were constructed in pSilencer-3.0-H1 vectors, and designated pSilencer-S and pSilencer-M, respectively. The antiviral effect of pSilencer-S/M against HTNV was evaluated in both HTNV-infected Vero-E6 cells and mice.

Results: In HTNV-infected Vero-E6 cells, pSilencer-S and pSilencer-M targeted the viral nucleocapsid proteins and envelope glycoproteins, respectively, as revealed in the immunofluorescence assay. Transfection with pSilencer-S or pSilencer-M (1, 2, 4 μg) markedly inhibited the viral antigen expression in dose- and time-dependent manners. Transfection with either plasmid (2 μg) significantly decreased HTNV-RNA level at 3 day postinfectin (dpi) and the progeny virus titer at 5 dpi. In mice infected with lethal doses of HTNV, intraperitoneal injection of pSilencer-S or pSilencer-M (30 μg) considerably increased the survival rates and mean time to death, and significantly reduced the mean virus yields and viral RNA level, and alleviated virus-induced pathological lesions in lungs, brains and kidneys.

Conclusion: Plasmid-based shRNAs potently inhibit HTNV replication in vitro and in vivo. Our results provide a basis for development of shRNA as therapeutics for HTNV infections in humans.

Figure 1

ShRNA interference with HTNV production in Vero-E6 cells. Vero-E6 cells were transfected with 60 nmol/L shRNA-S1, -S2 (A), -M1, -M2 (B), or shCRK as a control and were then were infected 24 h later with HTNV. The cells were harvested for RNA purification and real-time PCR at 24 and 48 hpi. The viral titers of the frozen-thawed culture samples collected at 96 hpi were measured (C). The data are expressed as the log₁₀ values of the vial titers. All experiments were repeated three times, the replications produced similar results. ^bP<0.05.

Figure 2

Viral antigen detection in the shRNA expressing plasmid-treated Vero-E6 cells with HTNV infections. The Vero-E6 cells were transfected with 0.25, 0.5, 1.0, 2.0 and 4.0 μg of pSilencer-S (A–E), pSilence-M (data not shown) or pSilence-blank (F) followed by infection with HTNV 76–118. The samples were fixed at 72 hpi and stained with the monoclonal antibody A35 targeting the nucleocapsid (A–G, green color). Evans blue was applied to indicate the cytoplasm (red). Normal cells are shown in (G). The percentages of positive cells were assessed by counting the green-labeled cells and red-labeled cells in 4 quadrants of each slide, and the results are plotted in (H) (pSilencer-S) and (I) (pSilence-M). Representative fluorescence images are shown. The experiments were repeated three times, and the replications produced similar results. ^bP<0.05. (J) presents the dynamic inhibitory curves of pSilencer-S and pSilencer-M in terms of the viral antigen expressions in the HTNV-infected Vero-E6 cells. The cells were transfected with 2.0 μg shRNA expression plasmid followed by infection with HTNV 76–118. Slides were prepared at 1, 3, 5, 7, and 9 dpi to detect the viral antigens as described above. The inhibitory rates were calculated using the following formula: inhibitory rate (%)=(the percentage of positive cells_{viral control group}–the percentage of positive cells_{treatment group})/the percentage of positive cells_{viral control group}×100%. All presented data are the results of experiments performed in triplicate.

Figure 3

ShRNA expression plasmid interference with HTNV production in Vero-E6 cells. Vero-E6 cells were transfected with 2.0 μg of pSilence-S, pSilence-M or pSilence-blank and then infected 24 h later with HTNV. The cells were harvested for RNA purification and real-time PCR at 48 hpi (A). The viral titers of the frozen-thawed culture samples were measured at 5 dpi and are shown in (B). All experiments were repeated three times, and the replicates produced similar results. ^bP<0.05.

Figure 4

Effects of the pSilence-S and -M treatments in the lethal HTNV challenge model. Suckling mice were infected with lethal doses of HTNV 76–118 and treated with pSilence-S or pSilence-M at 1 and 3 dpi, respectively. For each group, 8 mice were monitored for survival (A), 4 mice per group were sacrificed on the indicated days, and samples were collected for virological analyses (B–D). On 8 dpi, the viral loads in the lungs, kidneys and brains were determined by viral titration (B). The HTNV RNA profiles in the brains were determined with qRT-PCR on 2, 3, 4, and 5 dpi. ^bP<0.05.

Figure 5

Effects of the pSilence-S and -M treatments on the organ histopathologies in the suckling mice infected with HTNV. The suckling mice were infected with lethal doses of HTNV 76–118 and treated with pSilence-S or pSilence-M at 1 and 3 dpi, respectively. Hematoxylin and eosin (H&E)-stained sections of the lungs, kidneys and brains collected from each experimental group at 8 dpi are shown.