正交病毒(汉塔病毒科,OrderBunyavirales)可在人类中引起两种严重的综合征:肾综合征出血热(HFRS),与旧世界正病毒有关,汉坦病毒心肺综合征(HCPS),与美洲的正交病毒有关。在欧洲,四种不同的正切病毒(DOBV,PUUV,SEOV,和TULV)与人类疾病相关。由于正畸病毒物种之间的疾病严重程度和人畜共患来源不同,通过RT-PCR或比较病毒中和试验(VNT)确定感染物种是很重要的。目前,焦点减少中和试验(FRNT)被认为是正感染病毒VNT的“黄金标准”,然而,这个测试是费力和耗时的。因此,需要更多的高通量替代品。在这项研究中,我们开发了一种比较性正畸病毒微中性化试验(MNT),包括在欧洲传播的所有4种人类致病性正畸病毒。使用RT-PCR确认的啮齿动物(n=17)和人血清(n=17)验证该测定。怀疑有DOBV的人血清(n=3)和正感冒病毒阴性啮齿动物(n=3)和人血清(n=85)的队列。16/17RT-PCR确认的啮齿动物血清和18/20RT-PCR确认和DOBV疑似人血清成功分型,而对于其余的啮齿动物(n=1)和人血清(n=2),无法检测到中和滴度。所有阴性对照血清在MNT中测试为阴性。随后使用50名正坦病毒患者的临床队列评估该测定。在所有50名患者中都证实了正坦病毒感染,和47/50(94%)血清分型成功,确认PUUV是荷兰正坦病毒感染的主要原因。值得注意的是,使用MNT诊断了2013年之前未识别的两例SEOV病例,在诊断设置中强调MNT的附加值。总之,我们证明了成功开发和临床实施了比较欧洲正坦病毒MNT,以确定欧洲HFRS患者中的感染病毒种类。需要确定致病物种才能做出适当的公共卫生反应,并可以支持个别患者的护理。对于许多实验室来说,实施正坦病毒中和试验并不是一个简单的过程.这个问题将通过向多个欧洲实验室推出比较MNT来解决,以支持患者诊断。监测和公共卫生对策。
Orthohantaviruses (family Hantaviridae, order Bunyavirales) can cause two serious syndromes in humans: hemorrhagic fever with renal syndrome (HFRS), associated with the Old World orthohantaviruses, and hantavirus cardiopulmonary syndrome (HCPS), associated with orthohantaviruses in the Americas. In Europe, four different orthohantaviruses (DOBV, PUUV, SEOV, and TULV) are associated with human disease. As disease severity and zoonotic source differ between orthohantavirus species, conclusive determination of the infecting species by either RT-PCR or comparative virus neutralization test (VNT) is of importance. Currently, the focus reduction neutralization test (FRNT) is considered the \'Gold Standard\' for orthohantavirus VNTs, however this test is laborious and time-consuming. Consequently, more high-throughput alternatives are needed. In this study, we developed a comparative orthohantavirus microneutralization test (MNT) including all four human pathogenic orthohantavirus species circulating in Europe. The assay was validated using RT-PCR-confirmed rodent (n=17) and human sera (n=17), DOBV-suspected human sera (n=3) and cohorts of orthohantavirus-negative rodent (n=3) and human sera (n=85). 16/17 RT-PCR-confirmed rodent sera and 18/20 of the RT-PCR-confirmed and DOBV-suspected human sera were serotyped successfully, while for the remaining rodent (n=1) and human sera (n=2) no neutralizing titers could be detected. All negative control sera tested negative in the MNT. The assay was subsequently evaluated using a clinical cohort of 50 orthohantavirus patients. Orthohantavirus infection was confirmed in all 50 patients, and 47/50 (94%) sera were serotyped successfully, confirming PUUV as the major cause of orthohantavirus infections in Netherlands. Notably, two previously unrecognized SEOV cases from 2013 were diagnosed using the MNT, underlining the added value of the MNT in a diagnostic setting. In conclusion, we demonstrate the successful development and clinical implementation of a comparative European orthohantavirus MNT to determine the infecting virus species in European HFRS patients. Identification of the causative species is needed for an adequate Public Health response and can support individual patient care. For many labs, the implementation of orthohantavirus neutralization tests has not been a straightforward procedure. This issue will be addressed by the rollout of the comparative MNT to multiple European laboratories to support patient diagnostics, surveillance and Public Health responses.