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【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(P<0.05);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(P<0.05),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。
【OBJECTIVE】 A MDV strain named MDV GD06 was isolated from three non-immune healthy three yellow chickens. The biological characteristics of this MDV strain were studied systematically in this work. 【Methods】 Meq gene and short terminal repeat (RS) sequences of GD06 strain were amplified by PCR. Sequence analysis was performed and the serotypes were identified by indirect immunofluorescence assay. SPF chickens and chicken embryo fibroblasts (CEF) To determine the ability of GD06 to proliferate both in vitro and in vivo. To determine the pathogenicity of GD06 strain, one-day-old SPF chickens were used for challenge test. 【Results】 The GD06 strain was MDV serotype I virus. The GD06 strain naturally integrated the long terminal repeat (LTR) of the reticulovirus reticulovirus (REV) in the genome. The proline rich domain of the Meq gene was more virulent than the reference virulent strain The strain Md5 was 59 amino acids in length, consistent with the MD vaccine CVI988 / Rispens and 814, with the characteristics of a virulent strain. The virus titer of GD06 strain was 1.9 × 105,3.9 × 105,6.1 × 105,6.5 × 105,5.8 × 105PFU at 96, 120, 144, 168, 192 h after inoculation of CEF cells, which was significantly lower than that of CVI988 / Rispens (P <0.05). The virus titer of GD06 strain in chickens was 21 and 28 days after inoculation of SPF chicken was 740 And 350 PFU significantly higher than that of CVI988 / Rispens strains (460 and 216 PFU, respectively) (P <0.05). The results showed that GD06 strain had faster proliferation ability than CVI988 / Rispens in CEF cells and chickens. Artificial inoculation challenge experiments show that, GD06 strains of SPF chickens is not pathogenic, does not lead to immunosuppression. 【Conclusion】 The results showed that MDV GD06 strain was the first attenuated recombinant MDV strain in China that was naturally integrated with REV LTR sequence.