整合REV LTR序列的MDV弱毒株的分离鉴定与生物学特性

发布时间：2018-02-21 22:24

本文关键词： 马立克氏病病毒 GD株 LTR序列　出处：《微生物学报》2014年06期 　论文类型：期刊论文

【摘要】：【目的】从非免疫健康三黄鸡中分离到一株马立克氏病病毒(MDV),命名为MDV GD06株,本工作系统研究了其生物学特性。【方法】PCR扩增GD06株Meq基因及短末端重复区(RS)序列,进行序列分析,并用间接免疫荧光试验进行血清型特异性鉴定;通过接种SPF鸡和鸡胚成纤维细胞(CEF)来判定GD06株体内外的增殖能力;为确定GD06株的致病性,用1日龄SPF鸡进行攻毒试验。【结果】GD06株为MDV血清I型病毒,其基因组中自然整合禽网状内皮增殖症病毒(REV)的长末端重复序列(LTR),Meq基因富含脯氨酸的结构域比参考强毒株Md5多59个氨基酸,与MD商品化疫苗CVI988/Rispens和814株相符,具有弱毒株的特征。在接种CEF细胞96、120、144、168、192 h,GD06株病毒滴度分别为1.9×105、3.9×105、6.1×105、6.5×105、5.8×105PFU,明显比CVI988/Rispens(病毒滴度分别为1.3×105、3.5×105、5.0×105、5.7×105、4.7×105PFU)高(P0.05);接种SPF鸡21、28天,GD06株在鸡体内的病毒滴度分别为740和350 PFU,明显比CVI988/Rispens株(病毒滴度分别为460、216 PFU)高(P0.05),结果表明,GD06株在CEF细胞和鸡体内具有比CVI988/Rispens更快的增殖能力。人工接种攻毒实验表明,GD06株对SPF鸡没有致病性,不引免疫抑制。【结论】研究结果表明,MDV GD06株为国内首次分离的自然整合有REV LTR序列的重组MDV弱毒株。
[Abstract]:[objective] to isolate a Marek's disease virus (MDV) strain named MDV GD06 strain from non-immune healthy Sanhuang chicken, and to study its biological characteristics systematically. [methods] the Meq gene and short terminal repeats of GD06 strain were amplified by PCR and sequenced. In order to determine the pathogenicity of GD06 strain, the specific serotype was identified by indirect immunofluorescence assay, and the proliferation ability of GD06 strain was determined by inoculation of GD06 chicken and chicken embryo fibroblasts in vitro and in vivo. One day old SPF chicken was used to attack the virus. [results] the GD06 strain was MDV serotype I virus. The long terminal repeats of the naturally integrated avian reticuloendotheliosis virus (Rev) gene contained 59 amino acids more than that of the reference virulent strain Md5, which was consistent with the MD commercial vaccine CVI988/Rispens and 814 strains. The titer of GD06 strain was 1.9 脳 10 53.9 脳 10 55.10 5 脳 10 55.8 脳 10 5 PFU at 96,120,144,168,192 h after inoculation with CEF cells, which was significantly higher than that of CVI 988% Rispensus (virus titer was 1.3 脳 10 555 5 脳 10 55.0 脳 10 55.0 脳 10 55.0 脳 10 55.70 脳 10 55.47 脳 10 5 5 SPF), and the titer of GD06 strain inoculated with SPF chicken was 740 U and 350 U, respectively, compared with that of CVI988/Rispens strain (1. 3 脳 10 55.50 脳 10 55.0 脳 10 55.57 脳 10 55.70 脳 10 557 脳 10 557). The titer of GD06 strain inoculated with SPF chicken was 740 and 350U, respectively (P < 0.05), which was higher than that of CVI988/Rispens strain (P < 0.05). The titer of the virus was 460,216 PFU, respectively. The results showed that GD06 strain could proliferate faster than CVI988/Rispens in CEF cells and chickens, and the inoculation test showed that GD06 strain had no pathogenicity to SPF chickens. [conclusion] the results showed that GD06 strain was the first isolated recombinant MDV attenuated strain with REV LTR sequence.
【作者单位】：华南农业大学兽医学院;农业部动物疫病防控生物技术与制品创制重点实验室;广东省兽用生物制品生物技术研究与应用企业重点实验;
【分类号】：R373

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