Rawal B D, Vyas G N
Department of Laboratory Medicine, University of California, San Francisco 94143-0134, USA.
Biologicals. 1996 Jun;24(2):113-6. doi: 10.1006/biol.1996.0014.
The mechanism of in vitro inactivation of cell-free human immunodeficiency virus (CFHIV) with ascorbic acid (M) or Congo red (CR) was investigated with specific regard to the impact of an excess of magnesium ions on the viral inactivation. Quadruplicate reaction mixtures containing CFHIV were mixed with a virus-inactivating dose of 500 micrograms/ml ascorbic acid in RPMI medium devoid of fetal bovine serum and incubated for 3 h at 4 degrees C in two parallel sets of experiments. AA-free CFHIV and virion-free AA were included in each experiment as the positive and negative controls, respectively. After adding 10(6) MT2 cells to capture the surviving virons, the mixtures were incubated for 1 h at 37 degrees C. The cells from the first set were washed three times with Hanks balanced salt solution (HBSS) only, and those from the second set were washed with HBSS fortified with MgCl2 (1.0 mg/ml). Similarly, inactivation of CFHIV by increasing amounts of CR ranging between 12.5-100 micrograms/ml was also tested for the effect of MgCl2, except that (i) the assay was performed in subdued light, (ii) CFHIV-CR mixtures were incubated at 37 degrees C for 1 h in the dark and (iii) H9 cells were used instead of the MT-2 cells to capture the surviving virions in the test mixtures. The cells were cultured in RPMI with 20% FBS for 5 days at 37 degrees C. The absence of p24 antigen in the culture supernatant of MT2 or H9 cells indicated HIV inactivation by AA or CR, respectively. Remarkably, the cultured cells that were washed with HBSS + MgCl2 consistently expressed p24 antigen at levels comparable with those from the untreated virus control. Therefore, the apparent in vitro inactivation of CFHIV by either AA or CR was reversible as validated by washing of the cells with HBSS + MgCl2 following capture of the virions from CFHIV-AA or CFHIV-CR inactivation mixtures. These observations underscore the need for including extra magnesium ions as a control in validating various protocols used for assessing the in vitro virucidal activity of reverse transcriptase inhibitors, membrane binding dyes, or other candidate chemical agents.
研究了抗坏血酸（M）或刚果红（CR）对无细胞人类免疫缺陷病毒（CFHIV）的体外灭活机制，特别关注过量镁离子对病毒灭活的影响。在不含胎牛血清的RPMI培养基中，将含有CFHIV的一式四份反应混合物与500微克/毫升抗坏血酸的病毒灭活剂量混合，并在4℃下孵育3小时，进行两组平行实验。在每个实验中，分别将不含抗坏血酸的CFHIV和不含病毒体的抗坏血酸作为阳性和阴性对照。加入10⁶个MT2细胞以捕获存活的病毒体后，混合物在37℃下孵育1小时。第一组的细胞仅用Hanks平衡盐溶液（HBSS）洗涤三次，第二组的细胞用添加了MgCl₂（1.0毫克/毫升）的HBSS洗涤。同样，还测试了12.5 - 100微克/毫升范围内不同浓度CR对CFHIV的灭活作用以及镁离子的影响，不同之处在于：（i）测定在弱光下进行；（ii）CFHIV - CR混合物在黑暗中于37℃孵育1小时；（iii）使用H9细胞代替MT - 2细胞来捕获测试混合物中存活的病毒体。细胞在含有20%胎牛血清的RPMI培养基中于37℃培养5天。MT2或H9细胞培养上清液中不存在p24抗原分别表明抗坏血酸或刚果红使HIV失活。值得注意的是，用HBSS + MgCl₂洗涤的培养细胞始终表达与未处理病毒对照相当水平的p24抗原。因此，在从CFHIV - 抗坏血酸或CFHIV - 刚果红灭活混合物中捕获病毒体后，用HBSS + MgCl₂洗涤细胞验证了抗坏血酸或刚果红对CFHIV的体外明显灭活是可逆的。这些观察结果强调了在验证用于评估逆转录酶抑制剂、膜结合染料或其他候选化学试剂的体外杀病毒活性的各种方案时，需要加入额外的镁离子作为对照。
