鹿流行性出血热病毒的分离与鉴定

发布时间：2019-05-25 03:02

【摘要】：[目的]鹿流行性出血热是由库蠓等媒介昆虫吸血叮咬传播鹿流行性出血热病毒引起的一种非接触性病毒性传染病,该病毒属于环状病毒属,呼肠孤病毒科,主要感染野生和家养动物。本研究课题主要是为了了解设立在山西省阳城县蓝舌病监控牛场和羊场的监控动物虫媒病毒感染情况,因此,于2013年5月到2014年6月期间,在山西省阳城县采集监控动物牛羊的血清、肝素抗凝血对虫媒病毒进行分离和鉴定。[方法]采用竞争酶联免疫吸附(C-ELISA)试验分别检测牛羊血清中的BTV和EHDV抗体,采用微量中和试验检测牛羊血清中病毒抗体进行病毒型特异性确定,采用细胞培养、RT-PCR和测序分析,对病毒进行分离和鉴定。[结果]在无临床症状的监控牛血液中分离到1株阳性分离物,株阳性分离物只在BHK-21细胞上产生规律性细胞病变,在C6/36细胞上不会出现CPE;对这株阳性分离物细胞液过滤后测其TCID50,滴度分别为104.5TCID50/100μL,采用环状病毒属特异性引物RT-PCR扩增检测,与阳性对照扩增片段相似；采用随机RT-PCR进行扩增测序,将这株病毒的VP2和VP7序列在NBCI上进行BLAST比对,同源性达到99.0%；微量中和试验结果和分子生物学试验确定该毒株的血清学为EHDV-1。[结论]本次研究首次从山西省的黄牛血液中分离到鹿流行性出血热病毒,为今后山西省虫媒病毒的分离与鉴定提供一个重要的参考,为虫媒病毒的防治奠定了理论基础。
[Abstract]:[objective] Deer epidemic hemorrhagic fever (EHF) is a non-contact viral infectious disease caused by blood sucking and bites by vector insects such as Kucoides. The virus belongs to the genus annular virus and the family Enterovirus. Mainly infected with wild and domestic animals. The main purpose of this study is to understand the monitoring of animal arbovirus infection in cattle and sheep farms with blue tongue disease in Yangcheng County, Shanxi Province. therefore, from May 2013 to June 2014, The serum of monitored animal cattle and sheep was collected in Yangcheng County, Shanxi Province, and heparin anticoagulant was used to isolate and identify the arbovirus. [methods] BTV and EHDV antibodies in bovine sheep serum were detected by competitive enzyme-linked immunosorbent assay (C-ELISA), viral antibodies in bovine sheep serum were determined by microneutralization test, and cell culture was used. RT-PCR and sequencing analysis were used to isolate and identify the virus. [results] A positive isolate was isolated from bovine blood without clinical symptoms. The positive isolate only produced regular cytopathic effect on BHK-21 cells, but no CPE; appeared on C636 cells. The TCID50, titers of the positive isolate were 100 渭 L 104.5TCID50/ after filtration, and the amplified fragments were similar to those of the positive control by RT-PCR amplification with annular virus specific primers. Random RT-PCR was used to amplify and sequence the VP2 and VP7 sequences of the virus on NBCI, and the homology was 99.0%. The serological results of microneutralization test and molecular biology test confirmed that the serology of the strain was EHDV-1.. [conclusion] Deer epidemic hemorrhagic fever virus was isolated from the blood of yellow cattle in Shanxi Province for the first time, which provides an important reference for the isolation and identification of arbovirus in Shanxi Province in the future, and lays a theoretical foundation for the prevention and control of arbovirus.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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