Dataset Information

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

ABSTRACT: Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-?ORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-?ORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.

SUBMITTER: Peng Q

PROVIDER: S-EPMC7113866 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

Peng Qi Q Fang Liurong L Ding Zhen Z Wang Dang D Peng Guiqing G Xiao Shaobo S

Journal of virological methods 20191108

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ11 ...[more]

PMID: 31712093

Similar Datasets

Project description:Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.

Dataset Information

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

Publications

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets