Comparative Characterization and Pathogenicity of a Novel Porcine Epidemic Diarrhea Virus (PEDV) with a Naturally Occurring Truncated ORF3 Gene Coinfected with PEDVs Possessing an Intact ORF3 Gene in Piglets

Abstract

Coinfection caused by various genotypes of porcine epidemic diarrhea virus (PEDV) is a new disease situation. We previously reported the coexistence of PEDV strains containing different ORF3 genotypes in China. In this study, the PEDV strains 17GXCZ-1ORF3d and 17GXCZ-1ORF3c were isolated and plaque-purified from the same piglet, which had a natural large deletion at the 172–554 bp position of the ORF3 gene or possessed a complete ORF3 gene, respectively. Meanwhile, 17GXCZ-1ORF3d had >99% nt identity with 17GXCZ-1ORF3c in the 5′UTR, ORF1a/1b, S, E, M, N and 3′UTR regions but only demonstrated low nucleotide identities (80.5%) in the ORF3 gene. To elucidate the pathogenicity, 7-day-old piglets were infected. Piglets infected with these two PEDV strains exhibited severe clinical signs and shed the virus at the highest level within 96 hpi. Compared with the piglets inoculated with the 17GXCZ-1ORF3c strain, the piglets inoculated with the 17GXCZ-1ORF3d strain had higher mortality rates (75% vs. 50%), an earlier onset of clinical signs with a significantly higher diarrhea score, lower VH:CD ratios and a higher percentage of PEDV-positive enterocytes. This study is the first to report PEDV coinfections with different ORF3 genotypes, and a PEDV strain with a large deletion in the ORF3 gene might have the advantage of a potential genetic marker, which would be useful during vaccine development.

Genetic Characteristics and Pathogenicity of a Novel Porcine Epidemic Diarrhea Virus with a Naturally Occurring Truncated ORF3 Gene

“…Nevertheless, the truncated ORF3 HN2021 strain belonging to G2 genotype appeared to be highly virulent to suckling piglets. Similar results emerged in the 17GXCZ-1ORF3d strain belonging to G2 genotype, which has a naturally truncated ORF3 gene containing a continuous 382 nucleotide deletions from 172–554 nt and has been verified as a virulent strain causing severe diarrhea and high mortality in suckling piglets [ 18 ]. The genotype of PEDV is closely related to pathogenicity and immune protection [ 38 ].…”

“…The cDNA was screened by RT-qPCR (quantitative real-time RT-PCR), the primers targeting the PEDV M gene were designed ( Table S1 ), and RT-qPCR was conducted by using the Premix Ex Taq™ kit (TaKaRa) on a real-time thermocycler (CFX96™ Optics Module; BIO-RAD; California, CA, USA). For ORF3 amplification, the primers against ORF3 gene ( Table S1 ) were used [ 18 ]. Then, a total of 20 pairs of primers were designed to amplify the complete genome of the PEDV strain by conventional RT-PCR ( Table S1 ), and the positive cDNA was selected and amplified for complete genome.…”

One-Step Assembly of a Porcine Epidemic Diarrhea Virus Infectious cDNA Clone by Homologous Recombination in Yeast: Rapid Manipulation of Viral Genome With CRISPR/Cas9 Gene-Editing Technology

“…The first reverse genetics system of SARS-CoV-2 assembled in yeast using a YAC-based vector (pVC604) remained stable in yeast; however, their stability in E. coli was not evaluated (Thi Nhu Thao et al, 2020) In a previous study, CRISPR/Cas9 technology was applied to edit the PEDV genome with a BAC-based infectious cDNA clone (Peng et al, 2020), which was demonstrated to be a simple and rapid method. Deletions within ORF3 were detected in vaccine strains and many field PEDV strains (Cui et al, 2020;Lu et al, 2020bLu et al, , 2021, suggesting that ORF3 may be non-essential for viral replication. Using reverse genetics systems, ORF3 was demonstrated to be not critical for PEDV replication and can be successfully replaced with reporter genes (Beall et al, 2016;Kaewborisuth et al, 2018;Peng et al, 2020).…”

Phylogenetic and Spatiotemporal Analyses of Porcine Epidemic Diarrhea Virus in Guangxi, China during 2017–2022

“…PEDV has an enveloped single-stranded RNA genome that is approximately 28 kilobases (kb) long with a 5′ untranslated UTR region as well as a 3′ UTR [ 8 ]. The PEDV genome encodes two replicase polyproteins (ORFs 1a and 1b), four structural proteins (spike S, envelope E, membrane M and nucleocapsid N) and one accessory protein encoded by ORF3 [ 13 , 14 ]. According to the difference of the S gene, PEDV can be divided into two types: G1 and G2 [ 15 ].…”