Development of new vaccine strategies against Marek's disease virus with increased virulence

本研究的目的是对近年来在野外引起疫苗破损的马立克氏病病毒分离株进行分子特征分析，为研制新型超强毒疫苗提供新的疫苗策略。从白额鹅等野鸟和疫苗破损的家鸡中分离出不同毒力的马立克氏病病毒，并与已知的疫苗破损株进行核苷酸序列比较。日本新近分离的MDV毒株的MEQ基因(MDV的候选癌基因)的核苷酸序列与已知的vvMDV有很高的同源性，但也发现了一些以前未发现的突变。此外，在北海道分离株的糖蛋白B基因中发现了一个额外的EcoRI位点，而在几个野鸟和鸡分离株中发现了vvMDV中常见的糖蛋白L基因的一个小的缺失。在…中在接下来的实验中，我们分析了这些突变对MEQ基因产物MEQ转录激活的影响。毒力增强的MDV毒株表现出更高的MEQ转录激活活性，MEQ转录激活结构域的突变可能是MDV毒株毒力的决定因素。然而，具有新发现的突变的北海道株的MEQ并没有表现出更高的转录激活活性。L-meq基因产物(L-meq)存在于非致病性MDV疫苗株中，它既能抑制meq的转录激活，又能抑制vvMDV的体外复制，提示L-meq在维持潜伏期中起重要作用。我们还发现了一种新的meq基因转录变异体，称为Δmeq，它在马立克氏病肿瘤细胞中的表达在诱导细胞凋亡时上调。Δ-MEQ对MEQ和L-MEQ的转录激活均有抑制作用，提示Δ-MEQ可作为MEQ的负性调节因子，对潜伏期的细胞凋亡或再激活起到抑制作用。因此，ΔMEQ可用于开发一种新的疫苗，该疫苗只在vvMDV转化的细胞中诱导细胞程序性死亡。较少

The aim of this research is molecular characterization of recent isolates of Marek's disease virus (MDV) which can cause vaccine breaks in the field, in order to develop new vaccine strategies against theses very virulent MDV (vvMDV) by using a new MDV vaccine with the modified L-meq gene.Strains of vvMDV were isolated from wild birds such as white-fronted geese or from domestic chickens with vaccine breaks, and their nucleotide sequences were compared with those of known vvMDVs. The nucleotide sequences of the meq gene (a candidate oncogene of MDV) of recent isolates of MDVs in Japan showed high homologies with those of known vvMDVs, but some mutations previously unidentified were also found in these isolates. In addition, an additional EcoRI site was identified in the glycoprotein B gene of an isolate, strain Hokkaido, while a small deletion in the glycoprotein L gene, which had been frequently observed in vvMDV, was found in several isolates from wild birds and from chickens. In the … More next experiment, we analyzed the effects of these mutations on the transcriptional activation by the meq gene product, MEQ. MDV strains with increased virulence showed higher transcriptional activation by their MEQ, and mutations in the transcriptional activation domain of MEQ could be a determinant for the virulence of MDV strains. However, MEQ of strain Hokkaido, with newly identified mutations, did not display higher transcriptional activation by their MEQ. The L-meq gene product (L-MEQ), present in vaccine strains of apathogenic MDV, was shown to suppress both transcriptional activation by MEQ and replication in vitro of vvMDV, suggesting important roles of L-MEQ on the maintenance of latent phase. We also identified a new transcription variant of the meq gene, termed Δmeq, whose expression is up-regulated in Marek's disease tumor cells when apoptosis is induced. Δmeq inhibited the transcriptional activation by MEQ and L-MEQ, showing that Δmeq could play roles as a negative regulator of MEQ on apoptosis or reactivation of MDV from latent phase. Thus, Δmeq can be used to develop a new vaccine which induces programmed cell death only in transformed cells by vvMDV. Less