DETECTION AND CHARACTERIZATION OF HCV RNA SEQUENCES IN IMMUNE GLOBULINS

由于担心美国的安全。S.许可免疫球蛋白 (IG)，我们继续监测FDA发布的批次中的HCV RNA水平， PCR法（见上一份年度报告）。 在25批肌内注射用IG中 其中包括一些特定的IG，23个批次中有14个批次由源血浆制备 (SP)回收血浆（RP）的2个批次为阳性，而回收血浆（RP）的2个批次为阴性。 HCV RNA范围为30至500 PCR U/g IgG。 11例患者同时检出HCVRNA 对6个厂家32批SP制备的静脉注射用IG进行了分析。 所有 阳性批次来自同一生产商; 14个批次中的11个 结果表明，在20~250 PCR U/g IgG范围内，均为阳性。 然而，6 通过相同工艺和相同生产商由RP制备的批次 阴性 另外6个批次由其他2个生产商从RP制备 也是消极的。 这可能是由于较低的抗HCV反应性， 率，因此，RP中的病毒载量较低。 等离子体的影响 通过抗HCV对IG中HCV RNA水平的筛查仍然未知， 仅由筛选过的血浆制造的产品还不能获得。 在IG中检测到的HCV RNA似乎不以游离形式存在（未包封） 单链RNA，因为它不能被核糖核酸酶A消化（40 单位/ml在37 ° C下3小时）。 此外，它没有作为一个 片段化的RNA，因为下游序列，即，假定的NS3和NS5 HCV基因组的编码区，也被检测到时，引物从 除了来自5 '非编码区的那些之外，这两个区域是 用于PCR。 PCR阳性IG中HCV的浮力密度（bd）为 通过蔗糖梯度超离心测定为1.16 g/cm 3， 类似于在单个供体血浆样品中测定的，即，1.15 g/cm 3。 当血浆样品用25%ETOH处理（4 ℃下22 h C），HCV的bd为1.20，而用0.1%NP 40处理导致HCV的bd为1.20。 BD为1.28。 因此，乙醇可能会改变血浆中的病毒包膜， 分馏，但不像NP 40那样大，这可能会暴露 HCV的核心 免疫组化检测HCV RNA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.