Li Caiwen, Shields Jeffrey D
Virginia Institute of Marine Science, The College of William and Mary, P.O. BOX 1346, SR 1208, Gloucester Point, VA 23062, USA.
J Invertebr Pathol. 2007 Jan;94(1):48-55. doi: 10.1016/j.jip.2006.08.011. Epub 2006 Oct 10.
Primary cultures of hemocytes from the Caribbean spiny lobster Panulirus argus were developed for studies on the in vitro propagation of Panulirus argus Virus 1 (PaV1). A modified Leibovitz L-15 medium supported the best survival of hemocytes in in vitro primary cultures. However, degradation of the cultures occurred rapidly in the presence of granulocytes. A Percoll step gradient was used to separate hemocytes into three subpopulations enriched in hyalinocytes, semigranulocytes, and granulocytes, respectively. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability ( approximately 80%) after 18 days incubation compared with granulocytes, which degraded over 2-3 days. Susceptibility of the cell types was investigated in challenge studies with PaV1. Hyalinocytes and semigranulocytes were susceptible to PaV1. Cytopathic effects (CPE) were observed as early as 12h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 24h of infection. The presence of virus within cells was further confirmed by in situ hybridization using a specific DNA probe. The probe gave a unique staining pattern to cells infected with PaV1 24-h post-inoculation. Cells in the control treatment were intact and negative to hybridization. This assay was further applied to the quantification of infectious virus in hemolymph using a 50% tissue culture infectious dose assay (TCID(50)) based on CPE. These tools will now allow the quantification of PaV1 using established culture-based methods.
为了研究黄斑龙虾病毒1（PaV1）的体外增殖，开发了来自加勒比刺龙虾（Panulirus argus）血细胞的原代培养物。改良的Leibovitz L - 15培养基在体外原代培养中支持血细胞的最佳存活。然而，在粒细胞存在的情况下，培养物迅速降解。使用Percoll梯度分离法将血细胞分为三个亚群，分别富含透明细胞、半颗粒细胞和颗粒细胞。单独培养时，与在2 - 3天内降解的颗粒细胞相比，透明细胞和半颗粒细胞在培养18天后保持较高的活力（约80%）。在用PaV1进行的攻毒研究中，研究了这些细胞类型的易感性。透明细胞和半颗粒细胞对PaV1敏感。接种后12小时就观察到细胞病变效应（CPE），随着感染的进展，CPE变得更加明显，并在接种培养物中出现细胞碎片和细胞渗出物。感染后24小时内细胞裂解明显。使用特异性DNA探针通过原位杂交进一步证实细胞内病毒的存在。接种后24小时，该探针在感染PaV1的细胞上呈现独特的染色模式。对照处理中的细胞完整，杂交呈阴性。该测定法进一步应用于使用基于CPE的50%组织培养感染剂量测定法（TCID(50)）对血淋巴中感染性病毒进行定量。这些工具现在将允许使用既定的基于培养的方法对PaV1进行定量。
