Jackwood D J, Hanes G, Miller S H
Department of Veterinary Preventive Medicine, Ohio State University, Wooster 44691, USA.
Avian Dis. 1996 Apr-Jun;40(2):457-60.
The preservation of viable infectious agents for future studies could create complicated logistic problems, and at times it is not feasible. Methods for preserving the genetic integrity of inactivated agents would not only facilitate these studies but would also make it possible to transport inactivated preparations around the world. In this report, the effect of inactivation on the genetic material of infectious bursal disease virus (IBDV) was studied. Tissues from the bursa of Fabricius of birds experimentally infected 3 days earlier with the classic STC strain of IBDV were collected and immediately placed in a solution of phenol:chloroform:isoamyl alcohol (25:24:1) for 24, 48, 72, or 96 hr. Infected bursal tissue not treated with the phenol:chloroform solution and uninfected phenol: chloroform bursal tissue were used as controls. In a separate experiment, bursal tissues collected 5 days following infection of specific-pathogen-free birds with the classic STC or variant 1084-E strain were placed in the phenol:chloroform solution for 2 wk. All bursal samples were tested for viable IBDV following phenol:chloroform treatment. The tissues were washed in phosphate-buffered saline to remove phenol and then homogenized. Viability of the viruses in homogenized bursal tissue was examined by inoculation of embryonated chicken eggs. Viable IBDV was not observed in any phenol:chloroform-treated bursal tissue but was observed in the infected but non-phenol:chloroform-treated control bursa. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to test the integrity of the viral RNA. Viral RNA from the nontreated control and all infected bursal samples treated with phenol:chloroform solution at all the time points examined were transcribed into DNA, and a 394-bp fragment of the VP2 gene was amplified using specific primers in the PCR. The RT/PCR assay was negative using the phenol:chloroform-treated uninfected bursal tissue. This study clearly demonstrated that phenol:chloroform treatment of infected bursal tissue inactivated the classic and variant IBDV strains tested and preserved the viral RNA for use in the RT/PCR assay.
保存有活力的感染因子以供未来研究可能会产生复杂的后勤问题，而且有时是不可行的。保存灭活因子遗传完整性的方法不仅会促进这些研究，还将使在全球范围内运输灭活制剂成为可能。在本报告中，研究了灭活对传染性法氏囊病病毒（IBDV）遗传物质的影响。收集3天前用IBDV经典STC株实验感染的鸟类法氏囊组织，并立即置于苯酚：氯仿：异戊醇（25:24:1）溶液中24、48、72或96小时。未用苯酚：氯仿溶液处理的感染法氏囊组织和未感染的苯酚：氯仿法氏囊组织用作对照。在另一个实验中，将无特定病原体鸟类用经典STC或变异1084 - E株感染5天后收集的法氏囊组织置于苯酚：氯仿溶液中2周。所有法氏囊样品在苯酚：氯仿处理后检测是否存在有活力的IBDV。将组织用磷酸盐缓冲盐水洗涤以去除苯酚，然后匀浆。通过接种鸡胚检查匀浆法氏囊组织中病毒的活力。在任何经苯酚：氯仿处理的法氏囊组织中均未观察到有活力的IBDV，但在感染但未用苯酚：氯仿处理的对照法氏囊中观察到了。逆转录酶/聚合酶链反应（RT/PCR）用于检测病毒RNA的完整性。在所有检测时间点，来自未处理对照以及所有经苯酚：氯仿溶液处理的感染法氏囊样品的病毒RNA都被转录成DNA，并在PCR中使用特异性引物扩增VP2基因的394 bp片段。用苯酚：氯仿处理的未感染法氏囊组织进行RT/PCR检测为阴性。这项研究清楚地表明，用苯酚：氯仿处理感染的法氏囊组织可使所测试的经典和变异IBDV株失活，并保存病毒RNA用于RT/PCR检测。
