Sequencing of hypermutated Marek's Disease Virus Y547S genomes isolated from different passages, replicates in CEC or T7 chicken intestinal epithelial cells

Mareks disease virus (MDV) is a ubiquitous and highly oncogenic alphaherpesvirus of chickens that causes significant economic losses in the poultry industry. In contrast to most RNA viruses, large DNA viruses feature high-fidelity genome replication, mainly achieved through a proofreading function of the viral DNA polymerase. We have generated one mutant Y547S within the conserved exonuclease domain (Exo) III, by replacing a conserved amino acid tyrosine with serine. The Y547S mutant proved to be a slow hypermutator, exhibiting a mutation frequency about three times higher than wild type virus (WT) in chicken embryo cell (CEC) culture, which makes this particular mutant an interesting model to study the correlation between mutation frequency and adaptation. To address this question, we adapted both WT and Y547S not only to long-term propagation in permissive CEC culture, but also to growth in a naturally non-permissive cell line. We then determined the phenotype of adapted viruses both in vitro and in vivo using chickens, the natural host of MDV.

马立克氏病病毒（Marek's disease virus, MDV）是一种广泛分布且具有强致癌性的鸡源α疱疹病毒（alphaherpesvirus），给家禽养殖业造成了显著的经济损失。与多数RNA病毒不同，大型DNA病毒（large DNA viruses）具备高保真基因组复制能力，该特性主要通过病毒DNA聚合酶的校对功能实现。本研究构建了保守核酸外切酶结构域（exonuclease domain, Exo）III内的Y547S突变体，即将其中的保守氨基酸酪氨酸替换为丝氨酸。实验证实，该Y547S突变体属于慢发超突变体，在鸡胚细胞（chicken embryo cell, CEC）培养体系中，其突变频率约为野生型病毒（wild type virus, WT）的3倍，因此该突变体可作为研究突变频率与适应性进化相关性的理想模型。为探究该科学问题，本研究对野生型病毒与Y547S突变体分别开展了两类适应性驯化：一是在允许性鸡胚细胞培养体系中进行长期增殖传代，二是在天然非允许性细胞系中实现适应性生长。随后通过体外实验以及以MDV天然宿主鸡为对象的体内实验，对适应性进化后病毒的表型进行了测定与分析。