Pandey Utsav, Bell Andrew S, Renner Daniel W, Kennedy David A, Shreve Jacob T, Cairns Chris L, Jones Matthew J, Dunn Patricia A, Read Andrew F, Szpara Moriah L
Department of Biochemistry and Molecular Biology, Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, USA.
Center for Infectious Disease Dynamics, Departments of Biology and Entomology, Pennsylvania State University, University Park, Pennsylvania, USA.
mSphere. 2016 Oct 5;1(5). doi: 10.1128/mSphere.00132-16. eCollection 2016 Sep-Oct.
The intensification of the poultry industry over the last 60 years facilitated the evolution of increased virulence and vaccine breaks in Marek's disease virus (MDV-1). Full-genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high-quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown. Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last half century, we do not yet understand the genetic basis of its pathogenicity. Our knowledge of genome-wide variation between strains of this virus comes exclusively from isolates that have been cultured in the laboratory. MDV-1 isolates tend to lose virulence during repeated cycles of replication in the laboratory, raising concerns about the ability of cultured isolates to accurately reflect virus in the field. The ability to directly sequence and compare field isolates of this virus is critical to understanding the genetic basis of rising virulence in the wild. Our approaches remove the prior requirement for cell culture and allow direct measurement of viral genomic variation within and between hosts, over time, and during adaptation to changing conditions.
在过去60年里，家禽业的集约化发展促进了马立克氏病病毒1型（MDV-1）毒力增强和疫苗失效情况的演变。全基因组序列对于理解这种演变为何以及如何发生至关重要，但目前所知的MDV全基因组变异情况均来自实验室培养。为纠正这一状况，我们开发了直接从野外样本中获取高质量基因组序列的方法，无需在测序前采用基于序列的富集策略。我们将此方法应用于对野外MDV-1基因组的首次特征分析，且未事先进行培养。这些病毒是从接种过疫苗的宿主中收集的，这些宿主感染了自然传播的野外MDV-1毒株，且未出现疾病爆发。这反映了当前困扰家禽业的问题，即尽管接种了疫苗，毒力强的野外毒株仍在传播，且由于缺乏明显的疾病症状而可能未被发现。我们发现，来自相邻野外地点的病毒基因组具有高度的总体DNA一致性，并且尽管有强烈的纯化选择证据，但在与毒力和宿主免疫操纵相关的蛋白质中存在编码变异。我们的方法增强了生态野外监测能力，能够确定病毒毒力和疫苗失效的基础，并且可用于从其他已知和未知的大型DNA病毒的临床样本中获取全基因组。尽管临床和实验室数据均显示，在过去半个世纪里MDV-1野外分离株的毒力有所增强，但我们尚未了解其致病性的遗传基础。我们对该病毒不同毒株间全基因组变异的了解完全来自在实验室中培养的分离株。MDV-1分离株在实验室中反复传代复制过程中往往会丧失毒力，这引发了人们对培养分离株能否准确反映野外病毒情况的担忧。直接对该病毒的野外分离株进行测序和比较的能力对于理解野外毒力上升的遗传基础至关重要。我们的方法消除了先前对细胞培养的需求，能够直接测量病毒在宿主内和宿主间、随时间推移以及在适应不断变化的条件过程中的基因组变异。
