Leang Sook-Kwan, Hurt Aeron C
WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity.
WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity;
J Vis Exp. 2017 Apr 15(122):55570. doi: 10.3791/55570.
The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.
神经氨酸酶（NA）抑制剂是唯一一类被批准用于治疗和预防流感且对当前流行毒株有效的抗病毒药物。除了用于治疗季节性流感外，许多国家还储备了NA抑制剂以备大流行时使用。因此，监测流行流感病毒对这类抗病毒药物的敏感性很重要。有多种不同类型的检测方法可用于评估流感病毒对NA抑制剂的敏感性，但使用荧光底物或化学发光底物的酶抑制检测方法是应用最广泛且被推荐的。本方案描述了一种基于荧光的检测方法，用于评估流感病毒对NA抑制剂的敏感性。该检测方法基于NA酶切割2'-（4-甲基伞形酮基）-α-D-N-乙酰神经氨酸（MUNANA）底物以释放荧光产物4-甲基伞形酮（4-MU）。因此，根据将NA活性降低50%所需的NA抑制剂浓度（以IC50值表示）来确定NA抑制剂对流感病毒NA的抑制作用。
