Department of Pathogenetic and Preventive Veterinary Science, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Transboundary Animal Diseases Research Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
Virus Res. 2021 Apr 2;295:198331. doi: 10.1016/j.virusres.2021.198331. Epub 2021 Feb 1.
Monitoring of the epidemic situation is imperative to control the risk of infection with avian influenza H5 and H7 subtype viruses. A microneutralization (MN) assay was employed to detect hemagglutinin (HA) subtype-specific antibodies. However, the conventional MN assay raises biosafety concerns and is labor-intensive and time-consuming. Therefore, a safer and more convenient assay that can be applied in a high-throughput format is warranted. In this study, PB2 knockout (PB2-KO) influenza viruses of H5 and H7 subtypes expressing different colored fluorescent proteins were generated using a reverse genetics system and applied to a novel MN assay for the detection of specific antibodies. The detection sensitivity of our PB2-KO virus-based MN assay was evaluated by observing fluorescent proteins under a fluorescence microscope and measuring fluorescence intensities using a plate reader. In addition, the PB2-KO virus-based MN assay was used for the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Expression of the reporter fluorescent protein from H5 and H7 PB2-KO viruses was restricted toin PB2 protein-expressing cells. The MN titer as determined using fluorescence microscopy and plate reader revealed that the detection sensitivity of our PB2-KO virus-based MN assay was comparable to that of the conventional MN assay. Moreover, H5 and H7 PB2-KO viruses could be usedapplied forto the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Our study demonstrates a safe and convenient assay for the detection of H5 and H7 subtype-specific antibodies.
监测疫情对于控制禽流感 H5 和 H7 亚型病毒的感染风险至关重要。采用微量中和（MN）试验来检测血凝素（HA）亚型特异性抗体。然而，传统的 MN 试验存在生物安全隐患，且操作繁琐、耗时。因此，有必要开发一种更安全、更便捷的高通量检测方法。本研究利用反向遗传学系统，生成了表达不同颜色荧光蛋白的 H5 和 H7 亚型 PB2 缺失（PB2-KO）流感病毒，并将其应用于新型 MN 试验，以检测特异性抗体。我们通过荧光显微镜观察荧光蛋白和酶标仪测量荧光强度，评估了基于 PB2-KO 病毒的 MN 试验的检测灵敏度。此外，该试验还可用于在单个试验中同时检测 H5 和 H7 亚型特异性抗体。H5 和 H7 PB2-KO 病毒的报告荧光蛋白表达仅限于表达 PB2 蛋白的细胞中。荧光显微镜和酶标仪检测的 MN 滴度表明，我们基于 PB2-KO 病毒的 MN 试验的检测灵敏度与传统 MN 试验相当。此外，H5 和 H7 PB2-KO 病毒可用于在单个试验中同时检测 H5 和 H7 亚型特异性抗体。本研究证明了一种用于检测 H5 和 H7 亚型特异性抗体的安全便捷的方法。
