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Abstract：
Porcine deltacoronavirus (PDCoV) is an important enteric coronavirus that has caused enormous economic losses in the pig industry worldwide. However, no commercial vaccine is currently available. Therefore, developing a safe and efficacious live-attenuated vaccine candidate is urgently needed. In this study, the PDCoV strain CH/XJYN/2016 was continuously passaged in LLC-PK cells until passage 240, and the virus growth kinetics in cell culture, pathogenicity in neonatal piglets, transcriptome differences after LLC-PK infection, changes in the functional characteristics of the spike (S) protein in the high- and low-passage strains, genetic variation of the virus genome, resistance to pepsin and acid, and protective effects of this strain when used as a live-attenuated vaccine were examined. The results of animal experiments demonstrated that the virulent PDCoV strain CH/XJYN/2016 was completely attenuated and not pathogenic in piglets following serial cell passage. Genome sequence analysis showed that amino acid mutations in nonstructural proteins were mainly concentrated in Nsp3, structural protein mutations were mainly concentrated in the S protein, and the N, M, and E genes were conserved. Transcriptome comparison revealed that compared with negative control cells, P10-infected LLC-PK cells had the most differentially expressed genes (DEGs), while P0 and P240 had the least number of DEGs. Analysis of trypsin dependence and related structural differences revealed that the P10 S protein interacted more strongly with trypsin and that the P120 S protein interacted more strongly with the APN receptor. Moreover, the infectivity of P240 was not affected by pepsin but was significantly decreased after exposure to low pH. Furthermore, the P240-based live-attenuated vaccine provided complete protection to piglets against the challenge of virulent PDCoV. In conclusion, we showed that a PDCoV strain was completely attenuated through serial passaging in vitro. These results provide insights into the potential molecular mechanisms of PDCoV attenuation and the development of a promising live-attenuated PDCoV vaccine.IMPORTANCEPorcine deltacoronavirus (PDCoV) is one of the most important enteropathogenic pathogens that cause diarrhea in pigs of various ages, especially in suckling piglets, and causes enormous economic losses in the global commercial pork industry. There are currently no effective measures to prevent and control PDCoV. As reported in previous porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus studies, inactivated vaccines usually elicit less robust protective immune responses than live-attenuated vaccines in native sows. Therefore, identifying potential attenuation mechanisms, gene evolution, pathogenicity differences during PDCoV passaging, and immunogenicity as live-attenuated vaccines is important for elucidating the mechanism of attenuation and developing safe and effective vaccines for virulent PDCoV strains. In this study, we demonstrated that the virulence of the PDCoV strain CH/XJYN/2016 was completely attenuated following serial cell passaging in vitro, and changes in the biological characteristics and protection efficacy of the strain were evaluated. Our results help elucidate the mechanism of PDCoV attenuation and support the development of appropriate designs for the study of live PDCoV vaccines.
摘要：
猪deltacoronavirus(PDCoV)是一种重要的肠道冠状病毒,在世界范围内给养猪业造成了巨大的经济损失。然而,目前没有商业疫苗。因此,迫切需要开发一种安全有效的减毒活疫苗.在这项研究中,PDCoV株CH/XJYN/2016在LLC-PK细胞中连续传代至240代，细胞培养中的病毒生长动力学，对新生仔猪的致病性，LLC-PK感染后的转录组差异，高传代菌株和低传代菌株中穗（S）蛋白功能特性的变化，病毒基因组的遗传变异，对胃蛋白酶和酸的抗性，并检查了该菌株用作减毒活疫苗时的保护作用。动物实验结果表明，连续细胞传代后，PDCoV毒株CH/XJYN/2016完全减毒，在仔猪中无致病性。基因组序列分析表明,非结构蛋白的氨基酸突变主要集中在Nsp3,结构蛋白的突变主要集中在S蛋白,N,M,E基因是保守的。转录组比较显示，与阴性对照细胞相比，P10感染的LLC-PK细胞具有最多的差异表达基因（DEGs），而P0和P240的DEG数量最少。对胰蛋白酶依赖性和相关结构差异的分析表明，P10S蛋白与胰蛋白酶的相互作用更强，而P120S蛋白与APN受体的相互作用更强。此外，P240的感染性不受胃蛋白酶的影响，但在暴露于低pH值后明显降低。此外，基于P240的减毒活疫苗为仔猪提供了完全保护,使其免受强毒PDCoV的攻击.总之,我们表明，通过体外连续传代，PDCoV菌株完全减毒。这些结果为PDCoV减毒的潜在分子机制和有希望的减毒活活PDCoV疫苗的开发提供了见解。重要的猪deltacronavirus（PDCoV）是引起不同年龄猪腹泻的最重要的肠病原之一，尤其是乳猪,并在全球商业猪肉行业造成巨大的经济损失。目前尚无有效的预防和控制PDCoV的措施。如先前的猪流行性腹泻病毒(PEDV)和传染性胃肠炎病毒研究报告,在本地母猪中，灭活疫苗通常引起的保护性免疫反应不如减毒活疫苗。因此,识别潜在的衰减机制，基因进化,PDCoV传代过程中的致病性差异，和免疫原性作为减毒活疫苗对于阐明减毒机制和开发安全有效的PDCoV毒株疫苗具有重要意义。在这项研究中,我们证明了PDCoV菌株CH/XJYN/2016的毒力在体外连续细胞传代后完全减弱，并对菌株的生物学特性和保护效力的变化进行了评价。我们的结果有助于阐明PDCoV减弱的机制，并支持开发用于研究活PDCoV疫苗的适当设计。
