Hick Paul, Tweedie Alison, Whittington Richard J
Faculty of Veterinary Science, University of Sydney, 425 Werrombi Road, Camden NSW 2570 Australia.
J Vet Diagn Invest. 2011 May;23(3):465-75. doi: 10.1177/1040638711404147.
An optimized culture method for detection of infection of fish with the Red spotted grouper nervous necrosis virus (RGNNV) genotype of betanodavirus in striped snakehead (SSN-1, Channa striatus) cells is described. Inoculation of fish tissue homogenates at the same time or within 4 hr of seeding the SSN-1 cells was as sensitive as the method recommended by the World Organization for Animal Health, where homogenates were adsorbed onto an established cell monolayer. Such modification halved the time required and the costs of consumables, and reduced the potential for error when processing large numbers of samples. Positive culture results were obtained from 88.3% of 392 fish tissue homogenates in which RGNNV was detected using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay; 99.7% of 943 tissue homogenates, which were qRT-PCR negative, were cell culture negative. Cytopathic effect (CPE) was characterized by large intracytoplasmic vacuoles in 0.1-60% of cells. Detachment of affected cells from the culture surface resulting in progressive disruption of the monolayer occurred in 46.4% of primary cultures and 96.0% of subcultures of positive samples. Identification of CPE that did not disrupt the cell monolayer increased estimates of the 50% tissue culture infective dose (TCID(50)) by 1.07-2.79 logs (95% confidence interval). The predicted mean TCID(50)/ml was 3.3 logs higher when cells were inoculated less than 36 hr after subculture at less than 80% confluence compared to cells inoculated at greater than 80% confluence and more than 36 hr after subculture (P < 0.05).
本文描述了一种优化的培养方法，用于检测条纹鳢（SSN-1，Channa striatus）细胞中感染了β-诺达病毒红斑石斑鱼神经坏死病毒（RGNNV）基因型的鱼类。在接种SSN-1细胞的同时或接种后4小时内接种鱼组织匀浆，其敏感性与世界动物卫生组织推荐的方法相同，即匀浆吸附到已建立的细胞单层上。这种改进将所需时间和消耗品成本减半，并减少了处理大量样品时的误差可能性。在392份使用定量逆转录聚合酶链反应（qRT-PCR）检测出RGNNV的鱼组织匀浆中，88.3%获得了阳性培养结果；在943份qRT-PCR阴性的组织匀浆中，99.7%细胞培养呈阴性。细胞病变效应（CPE）的特征是0.1%-60%的细胞内出现大的胞质空泡。46.4%的原代培养阳性样品和96.0%的传代培养阳性样品中，受影响的细胞从培养表面脱落，导致单层逐渐破坏。识别未破坏细胞单层的CPE可使50%组织培养感染剂量（TCID(50)）的估计值提高1.07-2.79对数（95%置信区间）。与在汇合度大于80%且传代后超过36小时接种的细胞相比，在汇合度小于80%且传代后小于36小时接种的细胞，预测的平均TCID(50)/ml高3.3对数（P < 0.05）。
