水貂源犬瘟热病毒的分离及鉴定
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摘要
为了研究当前犬瘟热的流行毒株,试验选取来自辽宁省大连市疑似犬瘟热发病死亡水貂的肺脏、脾脏、肾脏等组织,将研磨匀浆后的上清液接种于稳定表达犬瘟热SLAM受体的非洲猕猴肾细胞系-Vero/DogSLAM(VDS),通过病料的处理、VDS细胞的复苏与培养、病毒的分离与培养、病毒TCID_(50)的测定、反转录-聚合酶链式反应(RT-PCR)的方法检测感染细胞中的病毒核酸,用序列比对的方法与已发表的犬瘟热病毒H蛋白(CDV-H)和SLAM受体结合区(RBS)序列进行比对分析,用CDV-H蛋白间接免疫荧光(IFA)的方法检测荧光蛋白表达。结果表明:在Vero/DogSLAM(VDS)细胞上接种病毒培养24 h后,80%～90%的VDS细胞出现合胞体病变(CPE),分离到的病毒TCID_(50)达到1×10~(5. 23)/(100μL),同时检测的CDV-H和SLAM受体结合区(RBS)序列比对分析一致,同源性为98%以上,在细胞质中可观察到抗CDV-H蛋白的特异性绿色荧光,RT-PCR成功扩增出H基因部分片段。说明本试验成功分离并鉴定出1株犬瘟热毒株,将其命名为LNDL(17) M4株。
In order to investigate the current CD epidemic strains,epidemic samples of lung,spleen,kidney and other tissues were collected from the susceptible diseased minks in Dalian and Liaoning Province. The supernatant of the grinding homogenate was inoculated into the African macaque kidney cell line in which the SLAM receptor-Vero/Dog SLAM( VDS) of canine distemper can be stably expressed. Then the viral nucleic acid of infected cells was detected by the pathological material treatment and the methods of resuscitation and culture of VDS cells,virus isolation and culture,determination of virus TCID_(50),reverse transcription-polymerase chain reaction( RT-PCR). Also,sequence alignment method was used to compare the identified sequences with the published H protein( CDV-H) and SLAM receptor binding domain( RBS) sequences of canine distemper virus. In addition,fluorescent protein expression was detected by indirect immunofluorescence( IFA). The results showed that after 24 hours' culture of transfected Vero/Dog SLAM( VDS) cells,syncytial lesions( CPE) was observed in 80% to 90% VDS cells. TCID_(50) of isolated virus reached to 1 × 10~(5. 23)/( 100 μL). Meanwhile,the sequence alignment analysis of CDV-H and SLAM receptor binding domain( RBS) was consistent,with a homology of more than 98%. Furthermore,specific green fluorescence of CDV-H protein was observed in cytoplasm,and partial fragments of H gene were successfully amplified by RT-PCR. The results indicated that one strains of canine distemper virus was successfully isolated and identified,and named as LNDL( 17) M4-VDS strain.
In order to investigate the current CD epidemic strains,epidemic samples of lung,spleen,kidney and other tissues were collected from the susceptible diseased minks in Dalian and Liaoning Province. The supernatant of the grinding homogenate was inoculated into the African macaque kidney cell line in which the SLAM receptor-Vero/Dog SLAM( VDS) of canine distemper can be stably expressed. Then the viral nucleic acid of infected cells was detected by the pathological material treatment and the methods of resuscitation and culture of VDS cells,virus isolation and culture,determination of virus TCID_(50),reverse transcription-polymerase chain reaction( RT-PCR). Also,sequence alignment method was used to compare the identified sequences with the published H protein( CDV-H) and SLAM receptor binding domain( RBS) sequences of canine distemper virus. In addition,fluorescent protein expression was detected by indirect immunofluorescence( IFA). The results showed that after 24 hours' culture of transfected Vero/Dog SLAM( VDS) cells,syncytial lesions( CPE) was observed in 80% to 90% VDS cells. TCID_(50) of isolated virus reached to 1 × 10~(5. 23)/( 100 μL). Meanwhile,the sequence alignment analysis of CDV-H and SLAM receptor binding domain( RBS) was consistent,with a homology of more than 98%. Furthermore,specific green fluorescence of CDV-H protein was observed in cytoplasm,and partial fragments of H gene were successfully amplified by RT-PCR. The results indicated that one strains of canine distemper virus was successfully isolated and identified,and named as LNDL( 17) M4-VDS strain.
引文
[1]刘玉秀.犬瘟热病毒敏感细胞系和感染性克隆的构建与初步应用研究[D].长春:吉林大学,2014.
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[9] TAN B, WEN Y J,WANG F X, et al. Pathogenesis and phylogenetic analyses of canine distemper virus stain ZJ7 isolate from domestic dogs in China[J]. Virol J,2011,8:520. doi:10. 1186/1743-422X-8-520.
[10] WOMA T Y,VAN VUUREN M. Isolation of canine distemper viruses from domestic dogs in South Africa using Vero/Dog SLAM cells and its application to diagnosis[J]. African J Microbiol Res,2009,3(3):111-118.
[11]赵建军,张海玲,闫喜军,等.稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立与应用[J].微生物学报,2012,52(12):1515-1523.
[12]魏凤,管宇,王金良,等.犬瘟热病毒在不同组织细胞上增殖特性的研究[J].动物医学进展,2010,31(2):83-86.
[2] CARPENTER M A,APPEL M J,ROELKE-PARKER M E,et al.Genetic characterization of canine distemper virus in Serengeti carnivores[J]. Vet Immunol Immunopathol, 1998, 65(2/3/4):259-266.
[3] APPEL M J,YATES R A,FOLEY G L,et al. Canine distemper epizootic in lions,tigers,and leopards in North America[J]. Vet Diagn Invest,1994,6(3):277-288.
[4] YOSHIKAWA Y,OCHIKUB F,MATSUBARA Y,et al. Natural infection with canine distemper virus in a Japanese monkey Macaca fuscat[J]. Vet Microbiol,1989,20(3):193-205.
[5] WILSON S C,EYBATOV T M,AMANO M,et al. The role of canine distemper virus and persistent organic pollutants in mortality patterns of Caspian seals(Pusa caspica)[J]. PLoS One,2014,9(7):e99265.
[6]曾智勇,周莉,汤德元,等.犬瘟热病毒贵州株CDV-GZ1的分离与鉴定[J].贵州农业科学,2012,40(2):109-111.
[7] WHETSTONE C A,BUNN T O,GOURLAY J A. Canine distemper virus titration in ferret peritoneal macrophages[J]. Cornell Veterinarian,1981,71(2):144-148.
[8]田克恭,遇秀玲.犬瘟热病毒强毒株的分离与鉴定[J].畜牧兽医学报,1998,29(2):151-154.
[9] TAN B, WEN Y J,WANG F X, et al. Pathogenesis and phylogenetic analyses of canine distemper virus stain ZJ7 isolate from domestic dogs in China[J]. Virol J,2011,8:520. doi:10. 1186/1743-422X-8-520.
[10] WOMA T Y,VAN VUUREN M. Isolation of canine distemper viruses from domestic dogs in South Africa using Vero/Dog SLAM cells and its application to diagnosis[J]. African J Microbiol Res,2009,3(3):111-118.
[11]赵建军,张海玲,闫喜军,等.稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立与应用[J].微生物学报,2012,52(12):1515-1523.
[12]魏凤,管宇,王金良,等.犬瘟热病毒在不同组织细胞上增殖特性的研究[J].动物医学进展,2010,31(2):83-86.