The oncolytic efficacy and safety of avian reovirus and its dynamic distribution in infected mice

Abstract

Primary liver cancer is a major public health challenge that ranks as the third most common cause of cancer worldwide despite therapeutic improvement. Reovirus has been emerging as a potential anti-cancer agent and is undergoing multiple clinical trials, and it is reported that reovirus can preferentially cause the cell death of a variety of cancers in a manner of apoptosis. As few studies have reported the efficacy of oncolytic activity and safety profile of avian reovirus, in our study, LDH assay, MTT assay, DAPI staining, and flow cytometry assay were performed to demonstrate the oncolytic effects of avian reovirus against the HepG2 cells, and quantitative real-time PCR (qRT-PCR) and animal experiments were conducted to investigate the dynamic distribution of avian reovirus in infected mice and then illustrate the safety and tissue tropism of avian reovirus. LDH assay, DAPI staining, and flow cytometry assay confirmed the efficacy of the oncotherapeutic effects of avian reovirus, and MTT assay has indicated that avian reovirus suppressed the proliferation of HepG2 cells and decreased their viability significantly. qRT-PCR revealed the dynamic distribution of avian reovirus in infected mice that avian reovirus might replicate better and have more powerful oncolytic activity in liver, kidney, and spleen tissues. Furthermore, histopathological examination clearly supported that avian reovirus appeared non-pathogenic to the normal host, so our study may provide the new insights and rationale for the new strategy of removing liver cancer.

Impact statement: We demonstrated the efficacy of oncolytic activity of avian reovirus (ARV) by LDH assay, MTT assay, DAPI staining, and flow cytometry assay, and also investigated the dynamic distribution of ARV in infected mice and then illustrated the safety and tissue tropism of ARV by quantitative real-time PCR (qRT-PCR) and animal experiments. Collectively, our study may provide the new insights and rationale for the new strategy of removing liver cancer.

Keywords: Hepatocellular carcinoma; avian reovirus; dynamic distribution; oncolytic virus; real-time PCR; safety.

Figure 1.

Histopathology in five organs stained with H&E from Kunming mice of three groups. 100×view of five organs from Kunming mice of the oral groups challenged with 3 × 106TCID50/0.2 mL of ARV S1133 (a) and 0.2 mL PBS (b) at 14 dpi, 100×view of five organs from Kunming mice of intramuscular injection groups challenged with 3 × 106TCID50/0.2 mL of ARV S1133 (c) and 0.2 mL PBS (d)at 14 dpi. (e) The body weight of the four groups, the control group in the graph represents the average weight of mice challenged with PBS orally and intramuscularly; error bars here and later indicate the standard deviations. (A color version of this figure is available in the online journal.)

Figure 2.

Dynamic distribution of the ARV S1133 in infected Kunming mice. The foldchange represents the relative expression of ARV S1133 in infected Kunming mice of oral group (a) and intramuscular injection group (b) at 1, 3, 5, 7 and 14 dpi.

Figure 3.

Comparison of relative expression of ARV S1133 of five organs in infected Kunming mice of two experimental groups. Fold change of ARV S1133 between the two groups at 3 dpi (a), 5 dpi (b) and 7 dpi (c). The peak foldchange of virus replication of five organs between the two groups (d) showed the viral load in liver and kidney was significantly higher than that in other tissues in oral groups and the highest viral load occurred in liver tissue of intramuscular injection groups.***P < 0.001.

Figure 4.

Syncytia formation of HepG2 cells infected with ARV S1133. Experimental groups infected at a MOI of 1 and control groups followed by DAPI staining under the fluorescent microscope (100×magnification). (A color version of this figure is available in the online journal.)

Figure 5.

Cytotoxic effects of ARV S1133 on growth and proliferation of HepG2 cells. Cells were treated with ARV S1133 at a MOI of 1, and the apoptosis was measured by LDH-cytotoxicity assay as the rise of absorbance (a). ARV S1133 on growth and proliferation of HepG2 cells was measured by MTT assay as the decline of absorbance (b). The increase of viral load in HepG2 cells (c).*P < 0.05,**P < 0.01,***P < 0.001. (A color version of this figure is available in the online journal.)

Figure 6.

Apoptosis of HepG2 cells was analyzed by flow cytometry with double staining of Annexin V and PI. The apoptosis was detected after the HepG2 cells infected with ARV S1133 at 24 hpi (a), 48 hpi (b), 72 hpi (c) and the corresponding control groups (d–f). The percentage of early apoptosis, late apoptosis, total apoptosis and live cells was compared among 24 hpi, 48 hpi and 72 hpi (g).The percentage of total apoptosis was compared between ARV S1133 groups and control groups at 24, 48 and 72 hpi (h). ***P < 0.001. (A color version of this figure is available in the online journal.)