Dataset Information

Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence.

ABSTRACT: The recently emerged highly virulent variants of porcine epidemic and diarrhea virus (PEDV) remain a huge threat to the worldwide swine industry. Here, we describe the development of a bacterial artificial chromosome (BAC) reverse genetics system for PEDV based on two recent Chinese field isolates, namely CHM2013 and BJ2011C. Phylogenetically, CHM2013 is closely related to the vaccine strain SM98 whereas the isolate BJ2011C belongs to the GIIb group, a cluster that contains many recent pandemic strains. The full-length cDNA clones of the two isolates were constructed into BAC under the control of CMV promoter. The rescued viruses rBJ2011C and rCHM2013 were found to replicate at the kinetics similar to their respective parental viruses in cell culture. When tested in the 2-day-old pig model, rBJ2011C caused severe diarrhea of piglets with extensive damages to the intestinal epithelium, leading to 100% fatality within 48 hours. In contrast, the rCHM2013-inoculated piglets all survived with only very minor tissue damage observed. Thus, we have successfully established a convenient platform for PEDV genome manipulation. This study also represents the first description of a DNA-launched reverse genetics system for the highly virulent PEDV.

SUBMITTER: Li J

PROVIDER: S-EPMC5354467 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence.

Li Jie J Jin Zhonghui Z Gao Yueyi Y Zhou Lei L Ge Xinna X Guo Xin X Han Jun J Yang Hanchun H

PloS one 20170316 3

The recently emerged highly virulent variants of porcine epidemic and diarrhea virus (PEDV) remain a huge threat to the worldwide swine industry. Here, we describe the development of a bacterial artificial chromosome (BAC) reverse genetics system for PEDV based on two recent Chinese field isolates, namely CHM2013 and BJ2011C. Phylogenetically, CHM2013 is closely related to the vaccine strain SM98 whereas the isolate BJ2011C belongs to the GIIb group, a cluster that contains many recent pandemic ...[more]

PMID: 28301551

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Project description:UNLABELLED: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-?ORF3-RFP. icPEDV-?ORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-?ORF3-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.

Project description:The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Dataset Information

Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence.

Publications

Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence.

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