Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

ABSTRACT 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 pathog...

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Autores principales:	Anne Beall, Boyd Yount, Chun-Ming Lin, Yixuan Hou, Qiuhong Wang, Linda Saif, Ralph Baric
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1c00b780fa4a4817a98226c0103b8b96
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spelling	oai:doaj.org-article:1c00b780fa4a4817a98226c0103b8b962021-11-15T15:49:39ZCharacterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A10.1128/mBio.01451-152150-7511https://doaj.org/article/1c00b780fa4a4817a98226c0103b8b962016-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01451-15https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE 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.Anne BeallBoyd YountChun-Ming LinYixuan HouQiuhong WangLinda SaifRalph BaricAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 1 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Anne Beall Boyd Yount Chun-Ming Lin Yixuan Hou Qiuhong Wang Linda Saif Ralph Baric Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A
description	ABSTRACT 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. IMPORTANCE 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.
format	article
author	Anne Beall Boyd Yount Chun-Ming Lin Yixuan Hou Qiuhong Wang Linda Saif Ralph Baric
author_facet	Anne Beall Boyd Yount Chun-Ming Lin Yixuan Hou Qiuhong Wang Linda Saif Ralph Baric
author_sort	Anne Beall
title	Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A
title_short	Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A
title_full	Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A
title_fullStr	Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A
title_full_unstemmed	Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A
title_sort	characterization of a pathogenic full-length cdna clone and transmission model for porcine epidemic diarrhea virus strain pc22a
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/1c00b780fa4a4817a98226c0103b8b96
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Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

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