目的: 使用不同种牛、猪源病毒感染人间充质干细胞（HMSC）,并通过对比牛、猪源病毒感染人源细胞系293T/17和MRC-5后的感染和扩增情况,综合分析HMSC对常见猪、牛源病毒的易感性。方法: 将HMSC、人胚肺成纤维细胞（MRC-5）、人胚肾细胞（293T/17细胞）、非洲绿猴肾细胞（Vero细胞）以及猪肾细胞（PK-15细胞）分别按照7×103个 · cm-1接种24孔板和25 cm2培养瓶,分别以感染复数（MOI）为0.02接种猪细小病毒（PPV）;将HMSC、MRC-5、293T/17细胞、Vero细胞以及牛鼻甲骨（BT）细胞分别按照7×103个 · cm-1接种24孔板和25 cm2培养瓶,分别以MOI为0.02接种牛细小病毒（BPV）、牛腹泻病毒（BVDV）、牛副流感病毒3型（PI3病毒）、牛腺病毒3型（BAV-3）以及呼肠孤病毒（REO-3）,其中24孔板细胞培养7 d,25 cm2培养瓶细胞盲传3代,第3代盲传至24孔板,各代次分别进行细胞病变观察、免疫荧光检测和病毒核酸qPCR检测。结果: 与PPV敏感细胞PK-15相比,接种PPV后的Vero、HMSC和MRC-5各代均未观察到细胞病变和荧光,PPV拷贝数随着传代次数持续下降,而293T/17细胞虽未观察到细胞病变,但在盲传3代后观察到绿色荧光,且PPV拷贝数持续维持在较高水平,为1.49× 107 copies · mL-1。与牛源病毒敏感细胞BT细胞相比HMSC对PI3、REO-3、BPV这3种牛源检测相关病毒也呈现出易感性,感染后均能产生不同程度的细胞病变和免疫荧光阳性,通过qPCR法均能检测到对应病毒核酸,其中PI3、REO-3可以在HMSC中扩增,但扩增水平低于293T/17。结论: HMSC和MRC-5相似,对BPV、REO-3和PI3病毒易感染,对PPV、BVDV和BAV-3病毒不易感染;293T/17细胞对PPV、REO-3和PI3病毒易感,对BPV、BVDV和BAV-3病毒不易感染。在HMSC的病毒安全风险控制中,如引入猪源或牛源动物源性材料,应更加关注BPV、REO-3和PI3病毒的感染风险。

Objective: To assess the risk of introducing bovine and porcine viruses in the production of human mesenchymal stem cells, which may involve the use of raw materials such as bovine serum and trypsin, this study infected human mesenchymal stem cells (HMSC) with various bovine and porcine viruses. The aim was to provide a foundation for risk assessment in the prevention and control of these viruses in HMSC. Additionally, by comparing the infection and proliferation patterns of bovine and porcine viruses in human cell lines 293T/17 and MRC-5, this study offered a comprehensive analysis of the susceptibility of HMSC to common bovine and porcine viruses. Methods: HMSC, human diploid MRC-5 cells, 293T/17, Vero, and PK-15 cells were seeded in 24-well plates and 25 cm2 flasks at a density of 7×103 cm-1, respectively, and then inoculated with porcine parvovirus (PPV) at a multiplicity of infection (MOI) of 0.02. Similarly, HMSC, MRC-5, 293T/17, Vero, and BT cells were seeded in 24-well plates and 25 cm2 flasks at a density of 7×103 cm-1, respectively, and inoculated with bovine parvovirus (BPV), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (PI3), bovine adenovirus type 3 (BAV-3), and reovirus type 3 (REO-3) at an MOI of 0.02. The cells in the 24-well plates were cultured for 7 days, while those in the 25 cm2 flasks were blindly passaged for three generations. The third generation of blind-passaged cells were transferred to 24-well plates. For each passage, cytopathic effects (CPE) were observed, immunofluorescence assays were performed, and viral nucleic acids were detected by qPCR. Results: Compared to PPV-sensitive PK-15 cells, no CPE or fluorescence were observed in Vero, HMSC, or MRC-5 cells after PPV inoculation, and the PPV copy number continuously decreased with successive passages. In contrast, although no CPE was observed in 293T/17 cells, green fluorescence was detected after three blind passages, and the PPV copy number remained consistently high at 1.49×107 copies · mL-1. Compared to bovine virus-sensitive BT cells, HMSC also exhibited susceptibility to three bovine viruses (PI3, REO-3, and BPV), showing varying degrees of CPE and positive immunofluorescence upon infection. Viral nucleic acids were detected via qPCR for all three viruses. Among them, PI3 and REO-3 were able to replicate in HMSC, though at lower levels compared to 293T/17 cells. Conclusion: HMSC and MRC-5 cells exhibit similar viral susceptibility profiles, demonstrating permissiveness to BPV, REO-3, and PI3, but resistance to PPV, BVDV, and BAV-3. In contrast, 293T/17 cells show susceptibility to PPV, REO-3 and PI3, while remaining non-permissive to BPV, BVDV and BAV-3. In the control of viral safety risks in human mesenchymal stem cells, greater attention should be paid to the infection risks of BPV, REO-3, and PI3 viruses when introducing porcine or bovine-derived materials of animal origin.