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Open AccessArticle

Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus

1
Department of Biochemistry and Molecular Biology, School of Medicine, Zhejiang University, Hangzhou 310058, China
2
Ruminant Diseases Research Center, Key Laboratory of Animal Resistant Biology of Shandong, College of Life Sciences, Shandong Normal University, Jinan 250014, China
3
Shandong Provincial Key Laboratory of Animal Diseases Control and Breeding, Shandong Academy of Agricultural Sciences, Jinan 250100, China
4
Shandong Key Laboratory of Poultry Diseases Diagnosis and Immunology, Shandong Academy of Agricultural Sciences, Jinan 250023, China
5
China Institute of Veterinary Drug Control, Beijing 100081, China
6
National Agricultural Science and Technology Center, Chengdu 610000, China
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Viruses 2020, 12(4), 391; https://doi.org/10.3390/v12040391
Received: 21 February 2020 / Revised: 26 March 2020 / Accepted: 27 March 2020 / Published: 1 April 2020
(This article belongs to the Section Animal Viruses)
A highly virulent porcine epidemic diarrhea virus (PEDV) appeared in China and spread rapidly to neighbor countries, which have led to great economic losses to the pig industry. In the present study, we isolated a PEDV using Vero cells and serially propagated 100 passages. PEDV SDSX16 was characterized in vitro and in vivo. The viral titers increased to 107.6 TCID50/mL (100th) by serial passages. The spike (S) gene and the whole gene of the SDSX16 virus was fully sequenced to assess the genetic stability and relatedness to previously identified PEDV. Along with successive passage in vitro, there were 18 nucleotides (nt) deletion occurred in the spike (S) gene resulting in a deletion of six amino acids when the SDSX16 strain was passaged to the 64th generation, and this deletion was stable until the P100. However, the ORF1a/b, M, N, E, and ORF3 genes had only a few point mutations in amino acids and no deletions. According to growth kinetics experiments, the SDSX16 deletion strain significantly enhanced its replication in Vero cells since it was passaged to the 64th generation. The animal studies showed that PEDV SDSX16-P10 caused more severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis than SDSX16-P75, indicating that SDSX16-P10 is enteropathogenic in the natural host, and the pathogenicity of SDSX16 decreased with successive passage in vitro. However, SDSX16-P10 was found to cause lower levels of cytokine expression than SDSX16-P75 using real-time PCR and flow cytometry, such as IL1β, IL6, IFN-β, TNF-α, indicating that SDSX16-P10 might inhibit the expression of cytokines. Our data indicated that successive passage in vitro resulted in virulent attenuation in vivo of the PEDV variant strain SDSX16. View Full-Text
Keywords: porcine epidemic diarrhea virus; virus isolate; variant; pathogenicity; cytokines porcine epidemic diarrhea virus; virus isolate; variant; pathogenicity; cytokines
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Zhao, P.; Wang, S.; Chen, Z.; Yu, J.; Tang, R.; Qiu, W.; Zhao, L.; Liu, Y.; Guo, X.; He, H.; Xu, G.; Li, J.; Wu, J. Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus. Viruses 2020, 12, 391.

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