Yang Qiqi, Wei Jing, Ye Chuantao, Pei Jiawei, Pei Xuemin, Wang Yuan, Dong Yangchao, Zhang Hui, Jiang Dongshen, Yang Xiaojing, Ma Hongwei, Cheng Linfeng, Liu He, Zhang Liang, Lei Yingfeng, Xu Zhikai, Yu Pengbo, Zhang Fanglin, Ye Wei
Department of Microbiology, School of Basic Medicine, Air Force Medical University: Fourth Military Medical University, Xi' An, Shaanxi, China.
Shaanxi Provincial Center for Disease Control and Prevention, Xi'an, Shaanxi, China.
Virology. 2025 Jul;608:110542. doi: 10.1016/j.virol.2025.110542. Epub 2025 Apr 17.
Rodent-borne orthohantavirus causes severe hemorrhagic fever worldwide, with hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus cardiopulmonary syndrome (HCPS) in the Amrerica. In East Asia, Hantaan orthohantavirus (HTNV) is the main pathogen responsible for severe HFRS, with a case fatality rate up to 10 % with no specific treatment available. The antisera or neutralizing antibody (NAb) is able to block virus infection, however, the traditional NAb titer measuring based on focus reduction neutralization test (FRNT) is quite labour-extensive and takes 7-10 days. This study aims to shorten the measuring time of NAb neutralization efficiency by 1-2 days based on quantitative RT-PCR. For this purpose, we developed an in vitro transcripted viral RNA standard and generated a viral RNA copy number standard curve. Using this standard curve, we compared the HTNV propagation kinetics between viral RNA copy numbers and secreted infectious virion. The detection limit and suitable timeframe and condition for qRT-PCR based viral RNA copy numbers measuring was also determined. In addition, when applying this method to measuring the NAb neutralization efficiency of HFRS convalescent serum samples, we could obtain the NAb neutralization efficiency within 1 or 2 days. Furthermore, this method was also nicely correlated with the FRNT - based NAb measurement. To conclude, we established a rapid and convenient viral RNA transcripts copy reduction neutralization test (VcRNT) to measure NAb neutralization efficiency that could finish within 1 or 2 days, and provided a reliable and efficient alternative for FRNT.
啮齿动物传播的正汉坦病毒在全球范围内引发严重出血热，在欧亚大陆导致肾综合征出血热（HFRS），在美洲导致汉坦病毒心肺综合征（HCPS）。在东亚，汉滩正汉坦病毒（HTNV）是导致严重HFRS的主要病原体，在没有特效治疗方法的情况下，病死率高达10%。抗血清或中和抗体（NAb）能够阻断病毒感染，然而，基于蚀斑减少中和试验（FRNT）的传统NAb滴度测定相当耗费人力，需要7至10天。本研究旨在基于定量逆转录聚合酶链反应（RT-PCR）将NAb中和效率的测定时间缩短1至2天。为此，我们开发了一种体外转录的病毒RNA标准品，并生成了病毒RNA拷贝数标准曲线。利用该标准曲线，我们比较了病毒RNA拷贝数与分泌的感染性病毒粒子之间的HTNV增殖动力学。还确定了基于qRT-PCR的病毒RNA拷贝数测定的检测限、合适的时间范围和条件。此外，当将该方法应用于测量HFRS康复期血清样本的NAb中和效率时，我们能够在1或2天内获得NAb中和效率。此外，该方法与基于FRNT的NAb测量也具有良好的相关性。总之，我们建立了一种快速便捷的病毒RNA转录本拷贝减少中和试验（VcRNT）来测量NAb中和效率，该试验可在1或2天内完成，并为FRNT提供了一种可靠且高效的替代方法。
