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.

中文翻译：

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猪流行性腹泻病毒变异体在体外的连续传代导致毒力降低和滴度升高。


我国出现高毒力猪流行性腹泻病毒（PEDV）并迅速向周边国家蔓延，给养猪业造成巨大经济损失。在本研究中，我们使用 Vero 细胞分离出 PEDV，并连续繁殖 100 代。 PEDV SDSX16 在体外和体内进行了表征。通过连续传代，病毒滴度增加至 107.6 TCID50/mL（第 100 次）。对 SDSX16 病毒的刺突 (S) 基因和整个基因进行了完整测序，以评估其遗传稳定性以及与先前鉴定的 PEDV 的相关性。随着体外连续传代，SDSX16菌株传至第64代时，spike（S）基因发生18个核苷酸（nt）缺失，导致6个氨基酸缺失，并且这种缺失一直稳定到P100 。然而，ORF1a/b、M、N、E和ORF3基因只有少数氨基酸点突变，没有缺失。根据生长动力学实验，SDSX16缺失菌株自传代至第64代以来，在Vero细胞中的复制显着增强。动物研究表明，PEDV SDSX16-P10比SDSX16-P75引起更严重的腹泻、呕吐、排粪和急性萎缩性肠炎，表明SDSX16-P10在自然宿主中具有肠道致病性，并且SDSX16的致病性随着在体内的连续传代而降低。体外。然而，使用实时PCR和流式细胞术发现SDSX16-P10导致细胞因子表达水平低于SDSX16-P75，例如IL1β、IL6、IFN-β、TNF-α，表明SDSX16-P10可能抑制表达细胞因子。我们的数据表明，PEDV 变种株 SDSX16 的体外连续传代导致体内毒力减弱。