Construction of a Chimeric Infectious Clone of Chinese Equine Infectious Anemia Virus by Partial LTR Substitution

WEI Li-li; WANG Xiao-jun; WANG Ying; LIANG Hua; SHEN Tao; LI Jing-peng; ZHANG Xiao-yan; XIANG Wen-hua; SHAO Yi-ming; SHEN Rong-xian

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April 2005

WEI Li-li, WANG Xiao-jun, WANG Ying, LIANG Hua, SHEN Tao, LI Jing-peng, ZHANG Xiao-yan, XIANG Wen-hua, SHAO Yi-ming and SHEN Rong-xian. Construction of a Chimeric Infectious Clone of Chinese Equine Infectious Anemia Virus by Partial LTR Substitution[J]. Virologica Sinica, 2005, 20(2): 149-154.

Citation: WEI Li-li, WANG Xiao-jun, WANG Ying, LIANG Hua, SHEN Tao, LI Jing-peng, ZHANG Xiao-yan, XIANG Wen-hua, SHAO Yi-ming, SHEN Rong-xian. Construction of a Chimeric Infectious Clone of Chinese Equine Infectious Anemia Virus by Partial LTR Substitution .VIROLOGICA SINICA, 2005, 20(2) : 149-154.

马传染性贫血病毒基因非编码区LTR嵌合克隆的构建

魏丽丽 ^1,2 ,
王晓钧 ¹ ,
王盈 ² ,
粱华 ³ ,
沈韬 ³ ,
李景鹏 ² ,
张晓燕 ³ ,
相文华 ^1* ,
邵一鸣 ¹ ,
沈荣显 ¹

1.
1.黑龙江哈尔滨中国农业科学院哈尔滨兽医研究所 2. 东北农业大学生命科学院 3.黑龙江哈尔滨中国疾病预防控制中心性病艾滋病预防控制中心北京

摘要

在已有全长感染性克隆pLGFD3 8 和pD70344 的基础上,根据马传贫弱毒疫苗致弱过程中不同代次毒株LTR序列的分析,在LTR U3区选取特定的酶切位点对EIAV非编码区LTR基因进行了部分替换。将替换的全基因克隆转染驴胎皮肤细胞(FDD)并以驴白细胞(DL)传代,用逆转录酶活性检测、RT PCR方法及Real time RT PCR验证其感染性。结果发现,其衍生病毒感染上述两种细胞均出现明显的细胞病变;细胞培养上清可检测到RT酶活性和RT PCR阳性。电镜下可见大量典型的EIAV颗粒。pLGFD9 12 嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3 8具有相似的复制水平,pLGFD9 12嵌合克隆衍生病毒在DL细胞上复制水平略高于FDD细胞。此结果为进一步深入研究LTR对马传染性贫血病毒复制水平和毒力的影响奠定了基础。
- 马传染性贫血病毒
- , 基因替换
- , 嵌合病毒
- , LTR

Construction of a Chimeric Infectious Clone of Chinese Equine Infectious Anemia Virus by Partial LTR Substitution

Abstract

According to mutations occurred in the passages during the Equine infectious anemia virus (EIAV) vaccine attenuation, a full length chimeric gene clone, pLGFD9-12, was constructed successfully at a backbone of clone pLGFD3-8 by substitution with LTR U3 region of a virulent EIAV strain. The pLGFD9-12 was used to transfect fetal donkey dermal (FDD) cells and passaged in donkey leukocyte (DL) culture. The cell cultures were monitored by RT-PCR, reverse transcriptase activity assay and real-time RT-PCR. The results of RT activity and RT-PCR were positive in the supernatant of cell cultures and viruses particles were also clearly observed under electron microscope. The replication of pLGFD9-12 chimeric viruses and its parental virus pLGFD3-8 has no obvious differences. The level of replication of the pLGFD9-12 chimeric clone cultured in DL cells was higher than that in FDD cells. The characteristics on virulence and replication ability of pLGFD9-12 will be evaluated in vivo.
- Equine infectious anemia virus
- , Gene substitution
- , Chimeric virus
- , LTR

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Construction of a Chimeric Infectious Clone of Chinese Equine Infectious Anemia Virus by Partial LTR Substitution

Abstract: According to mutations occurred in the passages during the Equine infectious anemia virus (EIAV) vaccine attenuation, a full length chimeric gene clone, pLGFD9-12, was constructed successfully at a backbone of clone pLGFD3-8 by substitution with LTR U3 region of a virulent EIAV strain. The pLGFD9-12 was used to transfect fetal donkey dermal (FDD) cells and passaged in donkey leukocyte (DL) culture. The cell cultures were monitored by RT-PCR, reverse transcriptase activity assay and real-time RT-PCR. The results of RT activity and RT-PCR were positive in the supernatant of cell cultures and viruses particles were also clearly observed under electron microscope. The replication of pLGFD9-12 chimeric viruses and its parental virus pLGFD3-8 has no obvious differences. The level of replication of the pLGFD9-12 chimeric clone cultured in DL cells was higher than that in FDD cells. The characteristics on virulence and replication ability of pLGFD9-12 will be evaluated in vivo.