Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.IMPORTANCE EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.

中文翻译：

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宿主内选择压力在种马生殖道中持续感染期间驱动马动脉炎病毒进化。

马动脉炎病毒（EAV）是马病毒性动脉炎（EVA）的病原体，EVA是马的生殖和呼吸系统疾病。自然感染后，被感染的种马中有10％至70％可以成为EAV的携带者，并继续在精液中散发病毒。在这项研究中，从鼻分泌物，血沉棕黄层细胞和精液中分离出的连续病毒，对七个被实验感染和两个自然感染的EAV携带者种马进行了深度测序，以阐明单个传播事件后宿主内部的微进化过程。鼻分泌物和血沉棕黄层细胞变异的分析缺乏广泛的阳性选择。但是，这两种样品类型的突变谱特征不同。相比之下，急性感染过程中最初的精液病毒种群经历了选择性瓶颈，如种群数量减少和病毒基因组多个位点的多样化选择所反映的。此外，在持续感染期间，广泛的全基因组纯化选择塑造了种马生殖道中的变异体多样性。总体而言，持续感染过程中EAV进化的非随机性质是由宿主内部主动选择压力驱动的。在病毒基因组内的开放阅读框中，ORF3，ORF5和ORF1a的nsp2编码区在持久性过程中积累了大多数核苷酸取代，其中ORF3和ORF5具有最高的宿主内进化速率。此处提出的发现提供了对EAV进化机制的新颖见解，并确定了可能与种马生殖道持续感染的建立和维持有关的病毒基因组关键区域。重要意义EAV可以在被感染种马的生殖道中持续存在，因此，长期的携带者种马构成其唯一的天然资源库。先前的研究表明，输精管的壶腹是种马生殖道中病毒持续存在的主要部位，而持续存在与无法清除感染的明显炎症反应有关。这是第一项使用下一代测序和当代序列分析技术描述种马生殖道急性和长期持续感染过程中EAV全长基因组进化的研究。该数据提供了对急性和持续感染期间EAV宿主内部进化的新颖见解，并证明了持续感染的特征在于广泛的全基因组纯化选择和宿主内部选择性压力介导的非随机进化模式，其中重要的核苷酸取代发生在ORF1a（区域编码nsp2），ORF3和ORF5。