BVDV与IBRV感染MDBK细胞的病毒复制与细胞凋亡研究

发布时间：2018-01-06 05:32

本文关键词：BVDV与IBRV感染MDBK细胞的病毒复制与细胞凋亡研究　出处：《黑龙江八一农垦大学》2017年硕士论文　论文类型：学位论文

【摘要】：牛传染性鼻气管炎病毒(IBRV)和牛病毒性腹泻病毒(BVDV)是两种重要的牛病毒性传染病毒。两种病毒均可造成机体的免疫抑制。临床中存在BVDV与IBRV共同感染、BVDV与其它呼吸道病原混合感染或继发感染的情况。本研究在体外进行了两种病毒感染MDBK细胞后的病毒复制和凋亡研究,为研究病毒感染的分子机制和开发疫苗奠定了基础。本研究首先进行了CP型的BVDV与IBRV的增殖,然后选择0.2 MOI、1.0MOI、2.0 MOI的BVDV感染MDBK细胞,50%细胞病变时再分别感染IBRV(0.1 MOI),同时设单独感染BVDV、IBRV的对照组和空白细胞对照。共感染24h后收集细胞提取病毒核酸,采用实时荧光定量PCR和间接免疫荧光等方法进行了病毒感染、载量和细胞活性等检测,确定了MDBK细胞感染两种病毒后的病毒复制和细胞活性。应用CCK-8细胞增殖试验、TUNEL检测法、流式细胞仪等进行了病毒定位与凋亡等检测,确定病毒共感染对细胞凋亡的影响。结果表明,当BVDV 0.2MOI、1.0MOI时,IBRV的病毒拷贝数与IBRV单独感染组差异不显著。当BVDV 2.0 MOI时,IBRV病毒拷贝数明显低于IBRV单独感染组。间接免疫荧光检测显示BVDV与IBRV可以共同感染MDBK细胞并造成细胞凋亡,且随细胞感染BVDV病毒量的增加,细胞病变逐渐加剧。细胞活性检测结果显示,两个病毒共同感染后对细胞造成了较大影响,随着病毒量的增多,细胞活性降低,且共同感染组细胞活性低于单独BVDV感染组细胞活性。流式细胞仪检测感染率与凋亡率结果均表明,BVDV与IBRV共感染细胞的感染率均高于BVDV单独感染的细胞,且此试验中的BVDV对IBRV的复制有显著影响。综上所述,研究表明感染BVDV的MDBK细胞再次感染IBRV时,两个病毒均可在细胞中进行复制,但高感染复数的BVDV会加剧MDBK细胞的病变,进而影响IBRV的复制。BVDV与IBRV共同感染引起细胞活性明显降低,对细胞造成的损伤大于病毒单独感染,并造成更严重的细胞凋亡。
[Abstract]:Bovine infectious rhinotracheitis virus (IBRV) and bovine viral diarrhea virus (BVV). It is an important bovine viral infection virus. Both viruses can cause immunosuppression. There are co-infection of BVDV and IBRV in clinic. In this study, the replication and apoptosis of two viruses infected with MDBK cells were studied in vitro. In order to study the molecular mechanism of virus infection and develop vaccine, we first carried out the proliferation of CP type BVDV and IBRV, and then selected 0.2 MOI 1.0 moi. 2. 0 MOI BVDV infected 50% of MDBK cells with cytopathic changes, and IBRV(0.1 moi was infected separately, and only BVDV was infected. IBRV control group and blank cell control group. After 24 hours of co-infection, the virus nucleic acid was extracted from the cells, and the virus infection was carried out by real-time fluorescence quantitative PCR and indirect immunofluorescence. The viral replication and cell activity of MDBK cells infected with two viruses were determined by the assay of load and cell activity. CCK-8 cell proliferation test and Tunel assay were used to detect the viral replication and cell activity. The virus localization and apoptosis were detected by flow cytometry to determine the effect of virus co-infection on cell apoptosis. The results showed that when BVDV 0.2 MOI was 1.0 MOI. The number of viral copies of IBRV was not significantly different from that of IBRV alone, when BVDV 2.0 MOI. The copy number of IBRV virus was significantly lower than that of IBRV alone. Indirect immunofluorescence assay showed that BVDV and IBRV could co-infect MDBK cells and induce apoptosis. With the increase of BVDV virus infection, the cytopathic changes were gradually aggravated. The results of cell activity test showed that the co-infection of the two viruses had a great impact on the cells, with the increase of the amount of virus. The cell activity of the co-infected group was lower than that of the group of BVDV infection alone. The results of flow cytometry showed that the infection rate and apoptosis rate were higher than those in the co-infection group. The infection rate of BVDV and IBRV co-infected cells was higher than that of BVDV alone, and the BVDV in this test had a significant effect on the replication of IBRV. Studies have shown that BVDV infected MDBK cells re-infected with IBRV, the two viruses can be replicated in the cells, but highly infected plural BVDV will aggravate the pathological changes of MDBK cells. Furthermore, the co-infection of IBRV, BVDV and IBRV resulted in a significant decrease in cell activity, which caused more damage to cells than virus infection alone, and resulted in more severe cell apoptosis.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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