Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A.
Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A.
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DOI：
10.1128/mbio.01451-15
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发表时间：
2016-01-05
期刊：
影响因子：
6.4
通讯作者：
Baric R
中科院分区：
文献类型：
--
作者：
Beall A;Yount B;Lin CM;Hou Y;Wang Q;Saif L;Baric R
猪流行性腹泻病毒（PEDV）是一种高致病性的冠状病毒。在美国，高毒力PEDV毒株在哺乳仔猪中造成80%至100%的死亡率，并在动物和农场之间迅速传播。为了研究控制PEDV发病和传播的遗传因素，我们建立了PEDV PC22A的分子克隆。传染性克隆衍生的PEDV （icPEDV）在细胞培养和猪体内的复制效率与亲本病毒一样高，导致体内致命疾病。重要的是，重组PEDV通过间接接触迅速传播给未接种的猪，在复制野生型病毒表型的同时，显示出毒性和有效传播。利用反向遗传学，我们去除开放阅读框3 (ORF3)，并用红色荧光蛋白（RFP）基因替换该区域，生成icPEDV-ΔORF3-RFP。icPEDV-ΔORF3-RFP在体外和体内都能有效复制，在猪之间有效传播，并产生致命的疾病结果。然而，icPEDV-ΔORF3-RFP-infected猪的腹泻评分低于野生型病毒或icPEDV感染猪，并且该病毒形成的斑块比PC22A小。总之，这些数据描述了一个强大的反向遗传学平台的发展，用于识别调节致病结果和体内传播效率的遗传因素，为在临床环境中开发和评估减毒活疫苗和治疗方法的功效提供了关键的基础设施发展。猪流行性腹泻病毒（PEDV）于2013年在美国出现，此后导致美国10%的农场猪死亡。尽管该疾病已经在国际上流行了几十年，但缺乏快速的反向遗传学平台来操纵PEDV并识别影响传播和毒力的遗传因素，阻碍了对这一重要农业疾病的研究。在这里，我们提出了一个基于dna的感染克隆系统，该系统在体外和仔猪中复制了循环美国菌株PC22A的发病机制。这种传染性克隆既可用于研究PEDV冠状病毒的遗传、毒力和传播，也可用于研制PEDV减毒活疫苗。
Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting. Porcine epidemic diarrhea virus (PEDV) emerged in the United States in 2013 and has since killed 10% of U.S. farm pigs. Though the disease has been circulating internationally for decades, the lack of a rapid reverse-genetics platform for manipulating PEDV and identifying genetic factors that impact transmission and virulence has hindered the study of this important agricultural disease. Here, we present a DNA-based infectious-clone system that replicates the pathogenesis of circulating U.S. strain PC22A both in vitro and in piglets. This infectious clone can be used both to study the genetics, virulence, and transmission of PEDV coronavirus and to inform the creation of a live attenuated PEDV vaccine.