Jahrling P B, Beall J L
J Clin Microbiol. 1977 Sep;6(3):238-43. doi: 10.1128/jcm.6.3.238-243.1977.
Hydroxylapatite column chromatography methods were developed to characterize selected alphavirus populations. Different conditions of pH and phosphate molarity were required to obtain satisfactory elution profiles and separations for Western equine encephalomyelitis virus strains, compared with Eastern equine encephalomyelitis virus and Semliki Forest virus strains. Raising the pH of the buffers effected earlier elutions of all viruses. Selection of phosphate gradients with more gentle slopes and adjustment to the proper pH effected better separations of virus subpopulations. Elution profiles were not affected by 0.85% NaCl, 10% fetal calf serum, or 1% bovine serum albumin, which are common constituents of virus-stabilizing diluents. Passage of Western equine encephalomyelitis and Semliki Forest viruses in BHK-21, Vero, or duck embryo cell cultures or in suckling mouse brains did not usually affect elution profiles, unless passage also resulted in a shift in the plaque size marker. Essentially all infectious virus applied to the column was recoverable in appropriate fractions. This permitted accurate determinations of heterogeneity within alphavirus populations. For Western equine encephalomyelitis large-plaque (LP) and small-plaque (SP) virus populations, it was possible to detect ratios of 1 LP in a population of 10(6) SP, and 1 SP in 10(3) LP by using linear phosphate gradients. When stepwise elution procedures were used, it was possible to detect ratios of 1 SP in a population of 10(5) LP. Hydroxylapatite column chromatography therefore appears to be a useful tool for characterizing alphaviruses and for isolating minority subpopulations of viruses of biological or epidemiological importance from apparently homogeneous virus stocks.
开发了羟基磷灰石柱色谱法来表征选定的甲病毒群体。与东部马脑脊髓炎病毒和塞姆利基森林病毒株相比，需要不同的pH值和磷酸盐摩尔浓度条件，才能获得满意的洗脱图谱，并对西部马脑脊髓炎病毒株进行分离。提高缓冲液的pH值会使所有病毒更早洗脱。选择斜率更平缓的磷酸盐梯度并将pH值调整到合适的值，能更好地分离病毒亚群。0.85% NaCl、10%胎牛血清或1%牛血清白蛋白是病毒稳定稀释剂的常见成分，它们不会影响洗脱图谱。西部马脑脊髓炎病毒和塞姆利基森林病毒在BHK-21、Vero或鸭胚细胞培养物中或在乳鼠脑中传代，通常不会影响洗脱图谱，除非传代还导致蚀斑大小标记发生变化。基本上所有应用于柱的感染性病毒都能在适当的馏分中回收。这使得能够准确测定甲病毒群体内的异质性。对于西部马脑脊髓炎大蚀斑（LP）和小蚀斑（SP）病毒群体，通过使用线性磷酸盐梯度，可以检测到10⁶个SP群体中有1个LP，以及10³个LP中有1个SP的比例。当使用分步洗脱程序时，可以检测到10⁵个LP群体中有1个SP的比例。因此，羟基磷灰石柱色谱法似乎是一种有用的工具，可用于表征甲病毒，并从看似均匀的病毒储备中分离出具有生物学或流行病学重要性的病毒少数亚群。
