DETECTION AND CHARACTERIZATION OF HCV RNA SEQUENCES IN IMMUNE GLOBULINS

因为担心美国许可的免疫球蛋白的安全性 (IG)，我们继续监测FDA发布的批次丙型肝炎病毒RNA水平 聚合酶链式反应方法(见上一年度报告)。在25批肌注免疫球蛋白中 其中包括一些特定的IG，23批中的14批由源等离子体制备 (SP)阳性，回收血浆(RP)2批阴性。 丙型肝炎病毒核糖核酸的范围为30~500PCRU/g IgG。11例患者中还检测到了丙型肝炎病毒RNA 从6个厂家生产的32批SP静脉注射免疫球蛋白中筛选出一批。全 阳性批次来自同一家制造商；14批中的11批 在2~250个PCRU/g抗体水平范围内呈阳性。然而，6 由相同工艺和相同制造商由RP制备的批次 不是。另外6批由另外两家制造商用RP制成 也是阴性的。这可能归因于抗-丙型肝炎病毒反应性较低。 因此，RP的病毒载量较低。等离子体的作用 丙种球蛋白中丙型肝炎病毒RNA水平的抗-丙型肝炎病毒筛查仍不清楚 目前还没有完全由筛选出的血浆制成的产品。 在免疫球蛋白中检测到的丙型肝炎病毒RNA似乎不是以游离态(未包裹)的形式存在 单链RNA，因为它不能被核糖核酸酶A(40)消化 单位/毫升，37℃作用3小时)。此外，它不是作为一种 从下游序列开始的片段RNA，即推定的NS3和NS5 也检测到了丙型肝炎病毒基因组的编码区 这两个区域除了来自5‘非编码区的区域外，还有 用于聚合酶链式反应。在PCR阳性免疫球蛋白中，丙型肝炎病毒的浮力密度(BD)为 经蔗糖梯度超速离心法测定为1.16g/cm3， 与在单一供体血浆样本中测定的相似，即1.15 G/cm3。当血浆样品用25%乙醇处理(在4度下处理22h C)，丙型肝炎病毒的BD为1.20，而0.1%NP40处理后， BD为1.28。因此，乙醇可能会在血浆中改变病毒的包膜。 分馏，但不像NP40那么大，这可能会暴露 丙型肝炎病毒的核心。丙型肝炎病毒核糖核酸检测在免疫中的性质及意义 球蛋白需要进一步阐明。

Because of concern about the safety of U. S. licensed immune globulins (IG), we continued to monitor HCV RNA levels in FDA released lots by our PCR method (see previous annual report). Among 25 lots of intramuscular IG which include some specific IG, 14 of 23 lots prepared from Source Plasma (SP) were positive while 2 lots from recovered plasma (RP) were negative. HCV RNA ranged from 30 to 500 PCR U/g IgG. HCV RNA was also detected in 11 of 32 lots of intravenous IG prepared from SP by 6 manufacturers. All positive lots were derived from the same manufacturer; 11 of the 14 lots were positive with levels ranging from 2 to 250 PCR U/g IgG. However, 6 lots prepared from RP by the same process and by the same manufacturer were negative. An additional 6 lots prepared from RP by 2 other manufacturers were also negative. This may be attributed to the lower anti-HCV reactive rate, and hence, the lower viral load in RP. The effect of plasma screening by anti-HCV on the level of HCV RNA in IG remains unknown since products manufactured solely from screened plasma are not yet available. HCV RNA detected in IG appeared not to exist as a free (not encapsulated) single-stranded RNA since it was not digestible by ribonuclease A (40 units/ml at 37 degrees C for 3 h). In addition, it was not present as a fragmented RNA since downstream sequences, i.e., the putative NS3 and NS5 coding regions of the HCV genome, were also detected when primers from those two regions in addition to those from the 5' non-coding region were used for PCR. The buoyant density (bd) of HCV in a PCR positive IG was determined by sucrose gradient ultracentrifugation to be 1.16 g/cm3, similar to that determined in a single donor plasma sample, i.e., 1.15 g/cm3. When a plasma sample was treated with 25% ETOH (22 h at 4 degrees C), the bd of HCV was 1.20, whereas treatment with 0.1% NP40 resulted in a bd of 1.28. Hence, ethanol may alter the viral envelope during plasma fractionation, but not to as great an extent as does NP40, which may expose the core of HCV. The nature and significance of HCV RNA detected in immune globulins requires further elucidation.