Dataset.

Duck Tembusu virus (DTMUV) belongs to the family Flaviviridae and genus Orthoflavivirus. It causes disease in ducks, affecting the nervous system and significantly reducing egg production. The first outbreak of DTMUV in Thailand was reported in 2013, with widespread cases across various regions. However, serological diagnosis of DTMUV is challenging due to antibody cross-reactivity with other flaviviruses. To address this issue, we developed an ELISA based on subviral particles. The cassette encoding the membrane precursor and envelope genes of DTMUV (strain KPS54A61) were cloned into a pCAGGS vector with an OSF-tag and transfected into HEK-293T cells to generate subviral particles. The subviral particles were detected in the supernatant of the transfected cell via immunoblotting using anti-DTMUV E protein and anti-Strep-tag antibodies, which revealed a protein band of approximately 59 kDa. An electron microscopy confirmed the presence of particles approximately 35 nm in diameter. To optimize the SP-based ELISA, checkerboard titration identified the optimal antigen concentration as 70 µg/mL and the optimal serum dilution as 1:100,000. A cut-off value was established for the assay, and testing 300 duck serum samples using the SP-based ELISA identified 41 positive samples (14%) and 259 negative samples (86%). The SP-based ELISA exhibited 100% sensitivity and specificity, achieving a perfect agreement score of 1.0 in comparison with the serum neutralization test. Additionally, specificity testing using antibodies specific to Japanese Encephalitis virus (JEV) revealed no cross-reactivity in the ELISA test. Therefore, the developed SP-based ELISA is highly effective for screening and monitoring DTMUV outbreaks in duck farms, significantly reducing the risk of viral spread and enabling the timely implementation of disease control measures.

鸭坦布苏病毒（Duck Tembusu virus, DTMUV）属于黄病毒科（Flaviviridae）黄病毒属（Orthoflavivirus），可引发鸭只发病，累及神经系统并显著降低产蛋量。2013年泰国首次报道DTMUV暴发，疫情在多个地区广泛扩散。然而，由于DTMUV抗体与其他黄病毒存在交叉反应，其血清学诊断颇具挑战。为解决这一问题，本研究开发了基于亚病毒颗粒（subviral particles, SP）的酶联免疫吸附试验（ELISA）。研究人员将DTMUV KPS54A61株的膜前体基因与包膜基因克隆至带有OSF标签的pCAGGS载体中，转染HEK-293T细胞以制备亚病毒颗粒。通过抗DTMUV E蛋白抗体与抗Strep-tag抗体进行免疫印迹检测转染细胞上清中的亚病毒颗粒，成功观测到约59 kDa的蛋白条带。电镜观察确认存在直径约35 nm的颗粒。为优化SP-ELISA，通过棋盘格滴定确定最佳抗原浓度为70 μg/mL，最佳血清稀释度为1:100000，并确定了该检测方法的临界值（cut-off值）。使用该SP-ELISA检测300份鸭血清样本，共检出41份阳性样本（占比14%），259份阴性样本（占比86%）。该ELISA的敏感性与特异性均达100%，与血清中和试验相比一致率为1.0。此外，使用日本脑炎病毒（Japanese Encephalitis virus, JEV）特异性抗体开展特异性检测，未发现该ELISA存在交叉反应。综上，本研究开发的SP-ELISA可高效用于鸭场DTMUV暴发的筛查与监测，能够显著降低病毒传播风险，助力及时落实疫病防控措施。