反复冻融次数和不同保存温度对伪狂犬病毒的神经环路标记效果的影响

伪狂犬病毒(pseudorabies virus， PRV)是常用于神经环路示踪研究的嗜神经病毒工具之一，然而目前缺乏 PRV的保存条件对其感染滴度以及在体神经环路示踪效果影响的研究.首先，将2 h内在－80℃及室温下同一批次的PRV分别进行1～4次冻融，利用噬斑法在BHK-21细胞上检测其滴度，研究结果显示1～3次冻融对PRV的滴度改变无显著影响，而第4次冻融使PRV病毒的滴度显著下降.其次，从－80℃冰箱中取出同一批次的两份PRV，将其冻融1次，分别在－80℃和－20℃保存两周后，利用噬斑法在BHK-21细胞上检测其滴度；同时，将两份PRV利用在体立体定位分别注射于小鼠齿状回(dentate gyrus， DG)区，取相同的感染时间点，对DG输入环路标记效果进行分析，研究得出不同温度下长期保存对PRV的滴度及标记效果有较大影响.该研究结果对于PRV病毒的保存及其在神经环路标记中的应用具有一定的参考意义.

Effects of repeated freezing and thawing times and different storage temperatures of pseudorabies virus on mapping neural circuits

Pseudorabies virus (PRV) is one of the commonly used neurotropical viruses for neural circuits tracing studies. However， the affects of preservation conditions of PRV on its infection titer and in vivo infection efficiency remains unclear. In this study， 2 μL aliquots of PRV with 1-4 times of freeze-thaw cycles at －80℃ or room temperature within 2 h were respectively tittered on BHK21 cells using plaque methods. The results showed that 1-3 times of freeze-thaw cycles had no significant effect on the PRV titers， while the fourth freeze-thaw cycles significantly reduced the PRV titers. Then， 2 μL PRV aliquots were subjected to one freezing-thawing cycle and stored at －80℃ and －20℃， respectively. Two weeks later， they were separately tittered on BHK21 cells and then stereotacticly injected into the dentate gyrus (DG) area of mice for in vivo neural circuit tracing analyzation. We found that the viral titer and in vivo tracing efficiency of PRV were significantly affected by the long-time storage temperatures. Our results may provide some helpful instructions for the preservation of PRV virus and its applications in neural circuit mapping.