PDF(Pubmed)
Abstract：
Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin-Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID50 assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC50) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC50) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.
摘要：
冠状病毒（CoV）属于有包膜的正义单链RNA病毒，是呼吸道，胃肠道,以及许多宿主物种的中枢神经系统疾病，即，鸟,哺乳动物,和人类。Beta-CoV通过在21世纪引起人类的三种流行病/大流行，揭示了跨越物种之间障碍的巨大潜力。考虑到迫切需要强大的抗病毒剂用于净化，预防,和治疗BCoV感染，我们将注意力转移到光敏剂与光照射相结合的光动力灭活的可能性上。在本研究中,我们评估,第一次,与亚甲蓝（MB）相比，甲苯胺蓝O（TBO）对β-冠状病毒1（BCoV）的抗病毒活性。首先,我们用ISO10993-5/附件C测定了MB和TBO对Madin-Darby牛肾（MDBK）细胞系的体外细胞毒性。BCoV在MDBK细胞中繁殖，用数字液滴PCR检测病毒滴度，TCID50测定和空斑测定。使用直接灭活方法估计无毒浓度的TBO的抗病毒活性。通过开发用于非线性建模和响应面分析的程序,在MAPLE15®数学软件中计算所有效应。在MDBK细胞中孵育72小时后，TBO的中位抑制浓度（IC50）为0.85μM。TBO直接灭活BCoV后的抗病毒活性(MOI=1)显著强于MB。TBO的中值有效浓度(EC50)为0.005μM。细胞病变效应以浓度依赖性方式降低，从0.0025到0.01µM，在0.02至0.3µM的TBO浓度下完全消失。病毒颗粒的数量也减少了，根据应用的浓度，通过ddPCR分析证明。总之,TBO在体外表现出直接灭活BCoV的巨大潜力，具有非常高的选择性指数，并应接受进一步调查，针对其在兽医和/或人类医疗实践中的应用。
