Hart Lucas M, MacKenzie Ashley, Purcell Maureen K, Powers Rachel L, Hershberger Paul K
a U.S. Geological Survey , Western Fisheries Research Center, Marrowstone Marine Field Station , 616 Marrowstone Point Road, Nordland , Washington 98358 , USA.
b U.S. Geological Survey , Western Fisheries Research Center , 6505 Northeast 65th Street, Seattle , Washington 98358 , USA.
J Aquat Anim Health. 2017 Jun;29(2):74-82. doi: 10.1080/08997659.2017.1285369.
Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations. Received November 7, 2016; accepted January 14, 2017.
针对太平洋鲱（Clupea pallasii）血浆中病毒性出血性败血症病毒（VHSV）中和活性的检测与定量，对蚀斑中和试验（PNT）方法进行了优化。该PNT试验依赖补体，因为中和活性会因热灭活而减弱；此外，通过添加来自无特定病原体的太平洋鲱的外源性补体，中和活性大多得以恢复。优化方法包括将VHSV等分试样与含有内源性补体的全试验血浆进行系列稀释（从1:16开始）并孵育过夜。然后使用96孔微孔板中的病毒蚀斑试验对所得病毒滴度进行计数。血清中和活性具有病毒特异性，因为病毒性出血性败血症（VHS）幸存者的血浆对传染性造血器官坏死病毒（一种密切相关的弹状病毒）仅表现出可忽略不计的反应性。在暴露于VHSV后存活的太平洋鲱中，早在暴露后37天就在血浆中检测到中和活性，并在暴露后约64天达到峰值。相对于在较高温度（9.9°C和13.1°C）下饲养的鱼，在7.4°C下饲养的鱼的中和活性开始时间略有延迟；然而，在所有温度处理中，中和活性在暴露后至少持续345天。预计这种评估太平洋鲱中VHSV中和活性的新能力将能够对先前的VHS感染与同龄鱼补充失败之间进行回顾性比较。此外，优化后的PNT可作为一种预测工具，用于识别野生太平洋鲱种群未来发生VHS流行病的可能性。2016年11月7日收到；2017年1月14日接受。
