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.

Highlights

  • Porcine epidemic diarrhea (PED), characterized by severe acute watery diarrhea, vomiting, dehydration and growth retardation in pigs of all ages, especially newborn piglets, causes tremendous economic losses to the swine industry worldwide [1,2,3]

  • We found that two ORF3 genotypes were present in the same piglet, one of which had a naturally truncated ORF3 gene that contained a continuous 382 nucleotides deletion from

  • To further characterize the porcine epidemic diarrhea virus (PEDV) detected by the ORF3 sequencing, both strains of the plaque-purified viruses were subjected to next-generation sequencing

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Summary

Introduction

Porcine epidemic diarrhea (PED), characterized by severe acute watery diarrhea, vomiting, dehydration and growth retardation in pigs of all ages, especially newborn piglets, causes tremendous economic losses to the swine industry worldwide [1,2,3]. The PEDV ORF3 is a single accessory protein gene that is genus-specific for coronaviruses [15]. The coinfections caused by the two S gene genotypes of PEDV were first reported in Japan in 2017 with a frequency rate of 32.7% [20]. Viral replication is normally enhanced during the coinfection of the two S genotypes of PEDV in piglets, and the clinical symptoms caused by the co-infection were as severe as those of the highly virulent PEDV alone [22,23]. Two purified PEDV strains with different ORF3 genotypes were obtained from the same piglet, and the biological and genetic characteristics as well as the pathogenicity in 7-day-old piglets were investigated

Sample Collection
PEDV Diagnosis and ORF3 Amplification
PEDV Isolation and Purification
Indirect Immunofluorescence Assay
Multi-Step Growth Curves of Viruses
Whole Genome Sequencing of PEDV
Multiple Alignments and Phylogenetic Analyses
Experimental Design of Infection
Results
Pathogenicity of 17GXCZ-1ORF3d and 17GXCZ-1ORF3c Strains in Piglets
Pathogenicity analysis of of the the PEDV
Conclusions
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