Wildlife Epidemiology Laboratory, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
Wildlife Epidemiology Laboratory, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
J Virol Methods. 2018 Apr;254:40-45. doi: 10.1016/j.jviromet.2018.01.006. Epub 2018 Jan 31.
Blanding's turtles (Emydoidea blandingii), an endangered species in Illinois, have experienced range-wide declines because of habitat degradation and fragmentation, predation, and road mortality. While ongoing studies are crucial to a thorough understanding of the natural history and demographics in these disjointed Illinois populations, infectious disease threats have been largely unevaluated. Herpesvirus outbreaks have been associated with high morbidity and mortality in populations of captive tortoises and turtles worldwide, including the family Emydidae (pond and box turtles). However, novel herpesviruses including Terrapene herpesvirus 1, Emydid herpesvirus 1 and 2, and Glyptemys herpesvirus 1 and 2, have recently been identified in apparently healthy free-ranging freshwater turtles. In 2015, 20 free-ranging Blanding's turtles in DuPage County, Illinois were screened for a herpesvirus using consensus PCR. A novel herpesvirus species (Emydoidea herpesvirus 1; EBHV1) was identified in two animals and shared a high degree of sequence homology to other freshwater turtle herpesviruses. Two quantitative real-time PCR assays, using EBHV1 primer-1 and primer-2, were developed to target an EBHV1-specific segment of the DNA-dependent DNA polymerase gene and validated. Both assays performed with high efficiency (slope = -3.2; R = 1), low intra-assay variability, and low inter-assay variability (coefficient of variation <2% at all dilutions). However, EBHV1 primer-2 displayed less variation and was selected to test clinical samples and five closely related herpesvirus control samples. Results indicate that this assay is specific for EBHV1, has a linear range of detection from 10 to 10 viral copies per reaction, and can categorically detect as few as 1 viral copy per reaction. This qPCR assay provides a valuable diagnostic tool for future characterization of EBHV1 epidemiology.
Blanding's 龟（Emydoidea blandingii）是伊利诺伊州的濒危物种，由于栖息地退化和破碎化、捕食和道路死亡率，其分布范围已经全面减少。虽然正在进行的研究对于彻底了解这些分散的伊利诺伊州种群的自然历史和人口统计学至关重要，但传染病威胁在很大程度上尚未得到评估。疱疹病毒爆发与世界各地圈养龟鳖类动物种群的高发病率和死亡率有关，包括 Emydidae 科（池塘和箱龟）。然而，最近在显然健康的自由放养淡水龟鳖中发现了包括 Terrapene herpesvirus 1、Emydid herpesvirus 1 和 2 以及 Glyptemys herpesvirus 1 和 2 在内的新型疱疹病毒。2015 年，伊利诺伊州杜佩奇县的 20 只自由放养的 Blanding's 龟被筛选用于疱疹病毒使用共识 PCR。在两种动物中发现了一种新型疱疹病毒物种（Emydoidea herpesvirus 1；EBHV1），与其他淡水龟疱疹病毒具有高度序列同源性。开发了两种使用 EBHV1 引物-1 和引物-2 的定量实时 PCR 检测方法，以针对 DNA 依赖性 DNA 聚合酶基因的 EBHV1 特异性片段进行靶向，并进行了验证。两种检测方法均具有高效率（斜率=-3.2；R=1）、低内检测变异性和低间检测变异性（在所有稀释度下变异系数均<2%）。然而，EBHV1 引物-2 显示出更小的变异性，并被选中用于测试临床样本和五个密切相关的疱疹病毒对照样本。结果表明，该检测方法对 EBHV1 具有特异性，检测范围从每个反应 10 到 10 个病毒拷贝具有线性，并且可以明确检测到每个反应低至 1 个病毒拷贝。该 qPCR 检测方法为未来 EBHV1 流行病学的特征提供了有价值的诊断工具。
