Attenuation of a Virulent Porcine Deltacoronavirus Strain DHeB1 via Serial Passage in LLC-PK1 Cells.

Porcine deltacoronavirus (PDCoV) is a newly discovered enteropathogenic coronavirus primarily responsible for diarrhea and mortality in piglets, with the potential to infect humans, thereby posing a significant threat to both human health and the global pig industry. Currently, there is no commercially available live-attenuated vaccine for PDCoV. In this study, an isolated virulent PDCoV strain, DHeB1, was continuously passaged in LLC-PK1 cells for up to 110 passages. The virus growth kinetics in cell culture and complete genome sequences of various passages (F11, F40, F70, F90, and F110) were determined. The results indicated significant increases in virus titers at passages F40 and F90. Sequence analysis revealed that only a few single-nucleotide mutations (some of which resulted in amino acid changes) and one nucleotide insertion were observed throughout successive passages. Notably, the eight and seven amino acid mutations that emerged in F40 and F70, respectively, remained stable in subsequent passages and were predominantly located in the S glycoprotein. The pathogenicity of F11, F40, F70, and F90 was assessed in 5-day-old piglets, revealing markedly reduced clinical symptoms, histopathological lesions, and intestinal PDCoV antigen distributions in piglets inoculated with F70 or F90. Importantly, F90 exhibited little to no virulence in piglets. The immunogenicity of F70, F90, and F110 was further evaluated in weaned piglets, with results indicating that the neutralizing antibody titers induced by F70 and F90 were comparable and significantly higher than those induced by F110. Collectively, these findings suggest that the PDCoV strain DHeB1 has been attenuated and can be used to develop a live-attenuated vaccine against PDCoV.