整合REV LTR序列的MDV弱毒株的分离鉴定与生物学特性

Isolation and characterization of a non-pathogenic Marek’s disease virus with a naturally inserted REV LTR sequence

摘要:

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、5.8×105 PFU，明显比CVI988/Rispens(病毒滴度分别为1.3×105、3.5×105、5.0×105、5.7×105、4.7×105 PFU)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。

Abstract:

Abstract:［Objective］Marek’s disease virus (MDV)，GD06 strain，was isolated from healthy Three-Yellow chickens without MD vaccine．In this study，we characterized GD06 strain/［Methods］Meq gene and repeat short region of GD06 strain were amplified and analyzed，and the serotype was determined by indirect immunofluorescence assay．The growth characteristics of GD06 stain were evaluated in Specific Pathogen Free (SPF) chickens and chicken embryo fibroblasts．The pathogenicity of GD06 strain was assessed by challenge test using SPF chickens.［Results］GD06 strain was MDV-1 serotype with a naturally inserted reticuloendotheliosis virus long terminal repeat sequence．Similar to the vaccinated strains CVI988/Rispens and 814，the Meq gene of strain GD06 contained a 59-aa insertion in the proline-rich domain compared with very virulent strain Md5．On 96，120，144，168 and 192 hours post infection，the virus titers of GD06 were higher than that of CVI988/Rispens in vitro (P＜0.05)，and the viremia levels in GD06-infected group were significantly higher than that of CVI988/Rispens group on 21 and 42 days post inoculation (P＜0.05)．The result of challenge test demonstrated that GD06 remained nonpathogenic for chickens and induced little immunosuppressive effects.［Conclusion］GD06 is an MDV-1 non-pathogenic strain naturally integrated with reticuloendotheliosis virus and long terminal repeat sequence．