Rabies is a zoonotic disease that can be prevented by vaccination. The confirmation of rabies virus inactivation is a critical step during the vaccine quality test; however, the current protocol conducted in Japan requires a large number of mice. The development and introduction of animal-free alternative assays are essential from the perspective of the 3Rs (reduction, refinement, and replacement) of animal testing. Here, we propose a novel inactivation assay for confirming the complete inactivation of the viable rabies virus using cultured Neuro-2a cells and an enzyme-linked immunosorbent assay (ELISA). The detection ability of ELISA was similar to that of a direct immunofluorescence assay, with the detection limit of ELISA being as low as 0.014 focus forming units/test. These results suggest that the assay could be used as a viral inactivation test. In comparison with a traditional in vivo assay, this assay has a higher detection ability, an objective interpretation, and would shorten the test duration from 25 days to 8 days.

狂犬病是一种人畜共患疾病，可以通过接种疫苗来预防。狂犬病病毒灭活的确认是疫苗质量检测的关键步骤；然而，目前在日本进行的协议需要大量的老鼠。从动物试验的 3R（减少、改进和替代）的角度来看，无动物替代检测的开发和引入是必不可少的。在这里，我们提出了一种新的灭活测定，用于使用培养的 Neuro-2a 细胞和酶联免疫吸附测定 (ELISA) 确认活狂犬病毒的完全灭活。ELISA的检测能力与直接免疫荧光法相似，ELISA的检测限低至0.014个焦点形成单位/测试。这些结果表明该测定可用作病毒灭活测试。与传统的相比体内试验，该试验具有更高的检测能力，客观的解释，并将测试时间从25天缩短到8天。