禽白血病病毒准种多样性及其致病性在我国鸡群中的演变

In the early period of more than 10 years since subgroup J Avian leukosis virus (ALV-J) emerged in 1988, it spread only in white meat-type chickens and caused high mortality with tumors. In twenty years after ALV-J was transferred in China, however, it gradually adapted to infect different types of chickens with quite different genetic background and also caused high mortality with tumors. To understand if such epidemic phenomenon is related to some specific mutations in ALV-J genomes and to predict its further evolution and spread trends, this application will inoculate chickens of different types with 3 representative ALV-J strains isolated from white meat-type chickens, layers and Chinese indigenous breeds. Infectivity and pathogenicity of different strains to different types of chickens will be compared. Natural vertical and horizontal infections will be imitated as continuous passages conducted in chickens generation by generation. In the process, a plenty of serum samples were collected in different periods of infection passages for deep sequencing to follow and explore multiplicity of quasspecies and evolution trend of the dominant quasispecies in gp85 gene and 3'-UTR of ALV-J in sera of chickens at different infection passages. Infectious clones of ALV-J will be constructed based on the sequences of gp85 and 3'-UTR of new dominant quasispecies, then used challenge chickens of different types to explore or confirm the relationship between the mutations at specific sites on ALV-J and its pathogenicity to chickens with different genetic characteristic. It could be used to predict evolution trend of ALV-J in chicken population in China and provide epidemic information for improvement of its prevention and control measures.

ALV-J在全球流行最初十多年,仅对白羽肉鸡高度易感。但在传进我国后，ALV-J逐渐适应遗传背景差异很大的鸡群，在不同类型鸡群中诱发很高的肿瘤死亡率。为阐明这一现象是否与ALV-J的特定变异相关，并预测该病毒进一步演变和传播趋势，本申请项目将从遗传背景差异很大的白羽肉鸡、蛋鸡和中国地方品种鸡分离到的3个ALV-J代表株接种不同类型鸡，连传8代，比较不同毒株致病性与鸡群的遗传性间相互关系。在模拟自然垂直和横向感染过程的不同阶段采集大量样品，用现代高通量测序技术，跟踪和探索不同感染代次鸡血清中ALV-J的gp85基因和3'-UTR的准种多样性及其优势准种的演变趋势。将演变出的代表性优势准种，构建不同感染性克隆，再对不同类型鸡做感染试验，以探索ALV-J基因组上特定位点变异与其对不同遗传特性鸡致病性间相互关系。为预测ALV-J在我国鸡群中的演变趋势，改进对ALV的防控措施提供分子流行病学依据。

本项目证明了，高通量测序比常规测序能更准确地揭示不同ALV-J感染鸡群体、个体、组织器官、独立样品间甚至同一样品内病毒gp85高变区和LTR调控区的基因序列演变的高度多样性及其演变规律。研究发现，不仅ALV-J感染的不同鸡群和个体间可能存在着基因序列的差异，而且同一个体同一脏器样品中也同样存在着突变株，其同源性可低至94%左右。研究还发现了，ALV-J感染的不同生态系统如鸡群的遗传背景、细胞培养还是鸡体内、有无免疫抗体及不同脏器的组织亲嗜性等对病毒基因组随机突变的选择作用，证明了这一选择作用会促进病毒向某一个方向变异，表现为某一位点发生氨基酸稳定突变的准种的优势程度显著升高，如垂直传播的病毒主要来自卵泡中的优势准种。已发表SCI论文5篇，培养博士研究生三名。