不同毒力水貂阿留申病毒在猫肾细胞中的增殖特性及凋亡特性的比较研究

试验旨在对不同毒力水貂阿留申病毒（Aleutian mink disease virus，AMDV）在猫肾细胞（feline kidney cell，CRFK）中的增殖规律及其诱导细胞凋亡情况进行比较研究。将标准毒株AMDV-G及分离到的野毒株AMDV-DL124、AMDV-DL125、AMDV-QD2、AMDV-QD3、AMDV-ZJ3接种CRFK细胞，应用间接免疫荧光、实时荧光定量PCR、TCID₅₀测定技术研究病毒在细胞中的复制及表达情况，同时检测病毒诱导的细胞凋亡情况。间接免疫荧光结果显示，5株野毒株荧光着色趋势差异不大，均在感染后12 h出现荧光，随感染时间延长荧光增多，AMDV-G荧光出现时间比野毒株晚，但病毒感染后72 h几乎所有细胞均出现荧光；实时荧光定量PCR结果显示，基因组复制趋势大致相同，AMDV-DL125感染后3 h复制开始，AMDV-G感染后24 h复制才开始并呈快速增长趋势，但感染后72 h均达到峰值。TCID₅₀检测结果表明，0~12 h为病毒感染潜伏期，AMDV-G感染后60 h达到峰值，野毒株均在感染后72 h达到峰值，但是6株病毒均能在感染后48~72 h维持较高的感染滴度，其后随细胞崩解而降低。SPSS 23.0统计软件分析凋亡检测结果显示，与对照组相比，野毒株感染细胞后2~12 h诱导细胞凋亡差异显著（P<0.05），AMDV-G诱导细胞凋亡差异明显低于野毒株，但是诱导细胞凋亡时间较野毒株长，在感染后24 h仍对细胞凋亡有较明显的诱导作用，但是各病毒诱导的细胞凋亡主要集中在2~12 h。该结果为AMDV的培养、鉴定及致病机理研究提供一定参考。

Comparative Study of Proliferation and Apoptosis Characteristics of Different Virulence of Aleutian Mink Disease Virus in CRFK Cells

This study was aimed to compare the proliferation and apoptosis characteristics of different virulence of Aleutian mink disease virus(AMDV) in feline kidney cell (CPFK). CRFK cells were infected separately with the standard strain of AMDV-G and the wild strains of AMDV-DL124, AMDV-DL125, AMDV-QD2, AMDV-QD3 and AMDV-ZJ3. Indirect immunofluorescence,Real-time quantitative PCR and TCID₅₀ were used to detect the replication and expression of the viruses in the cells, at the same time the apoptosis induced by the viruses was detected. The results of IFA showed that CRFK cells which infected separately by wild strains appeared the fluorescence after 12 h of infection. With the extension of the infection time, the fluorescence of cells increased, but the fluorescence of AMDV-G was later than the wild strains, almost all of the cells were appeared the fluorescence after 72 h of infection. The Real-time quantitative PCR results showed that the tendency of genome replication of different virus were approximately the same. The genome replication began at 3 h after the AMDV-DL125 infection,the rapid increase of genome replication of AMDV-G occurred at 24 h after the infection, the genome replication of whole viruses reached a peak after 72 h of infection. The results of TCID₅₀ showed that the latent period of viruses infection were 0 to 12 h after infection, AMDV-G reached the peak after 60 h of infection. However, the wild strains at 48 to 72 h could maintain a higher infection titer and reached peak at 72 h after infection, then decreased with the cell disintegration. Apoptosis detection results which analysis by SPSS 23.0 statistical analysis software showed that, compared with control group,cell apoptosis was significantly higher after 2 to 12 h of cells infected by wild strains induced (P<0.05), cell apoptosis of AMDV-G was significantly lower than wild strains, however, the time of cell apoptosis induced by AMDV-G was longer,the apoptosis was still significant after 24 h of infection. But the apoptosis caused by virus infection was mostly concentrated in 2 to 12 h after infection. The results would provide some references for the study on the culture and identification and pathogenic mechanism of AMDV.