猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）是属于冠状病毒科α-冠状病毒属的一种单股正链RNA病毒。PEDV感染引起的猪流行性腹泻（porcine epidemic diarrhea，PED）是一种以急性腹泻、呕吐、脱水为主要特征的肠道传染病，引起7日龄以下仔猪的高死亡率，给全球养猪业造成了巨大的经济损失。由于PEDV基因组的高变异特性，现有商品化疫苗难以提供持续有效的免疫保护，因此深入研究PEDV的致病机制和防控策略具有重要意义。冠状病毒S蛋白表面的N-聚糖在维持蛋白质折叠与稳定性、调节病毒的细胞嗜性等方面具有重要作用。PEDV S蛋白是病毒进入宿主细胞的关键介质，其表面也存在N-糖基化修饰，目前尚不清楚该修饰对PEDV复制和致病性的影响。本研究聚焦于PEDV S蛋白的N-糖基化修饰，旨在探究其对病毒感染和致病性的影响，为开发新型防控策略提供依据。具体内容如下：

PEDV S蛋白N-糖基化修饰对病毒复制及致病性的影响

为了探究PEDV S蛋白N-糖基化在病毒感染中的作用，本研究首先通过使用四种内质网中的N-糖基化途径抑制剂（tunicamycin、NGI-1、miglustat和celgosivir）处理非洲绿猴肾细胞（Vero）和猪肾上皮细胞（LLC-PK1），通过RT-qPCR、Western blot和TCID₅₀测定PEDV感染后抑制剂处理组PEDV基因转录、蛋白表达和病毒滴度，分析糖基化对病毒复制的影响。结果显示N-糖基化途径被抑制后能够显著降低病毒在两种细胞中的复制水平，且对细胞活力无显著影响。通过糖苷酶进一步验证了PEDV S蛋白糖基化现象的发生。

为了深入解析PEDV S蛋白N-糖基化修饰在病毒复制及致病性中的作用，本研究利用细菌人工染色体（bacterial artificial chromosome, BAC）为载体构建了PEDV反向遗传平台，并利用CRISPR/Cas9技术对潜在的糖基化位点进行突变，构建多个单糖基化位点突变的重组病毒以研究突变位点对病毒复制和致病性的影响。结果显示，成功构建出感染性克隆质粒pBAC-PEDV，转染Vero细胞后拯救出重组病毒，经IFA、Western blot、蚀斑试验和滴度试验鉴定，发现该拯救病毒的生长特性与亲本病毒相比没有明显差异。19株N-糖基化位点缺失突变PEDV毒株得到成功拯救，并具有较好的传代稳定性。将19株N-糖基化缺失突变病毒及野生病毒以相同剂量感染Vero细胞，通过PFU和蚀斑试验测定糖基化位点对病毒在细胞中感染和复制的影响，结果显示，N118、N216、N726、N1232和N1249位点的N-糖基化缺失显著降低了病毒在细胞中的复制水平，并减小了病毒形成的蚀斑。随后通过qPCR方法检测N-糖基化位点突变对病毒的吸附、入胞及复制阶段的影响，结果发现N118、N216和N726位点突变影响病毒吸附；N1232和N1249位点突变影响了病毒的入侵阶段。为了进一步分析N-糖基化对病毒致病性的影响，选择在细胞中复制能力发生变化的突变毒N118Q、N216Q、N726Q、N1232Q、N1249Q口服仔猪，进行了仔猪攻毒实验，同时设定野生型（WT）病毒对照组，通过临床症状观察、病理变化、病毒载量等指标评估了不同病毒的致病性。试验结果显示，感染WT病毒的仔猪出现腹泻、食欲不振症状，剖检可见肠壁变薄且肠道有黏液状消化物；N118Q、N216Q和N726Q突变病毒组仔猪出现轻度腹泻，部分肠段肠壁变薄；而N1232Q和N1249Q突变病毒组仔猪粪便正常，肠道没有明显变化。病毒载量检测结果发现，N1232Q和N1249Q组粪便及肠道组织中的病毒RNA含量显著降低。肠道组织病理学与免疫荧光检测显示，WT感染组小肠绒毛发生萎缩融合，N118Q、N216Q和N726Q组也有一定损伤，而N1232Q和N1249Q组则无明显变化且无抗原阳性信号。以上结果说明N1232和N1249两个糖基化位点突变导致病毒致病性显著降低，说明了S蛋白糖基化影响PEDV复制和致病性，且部分糖基化位点尤为重要，该研究为新型疫苗设计提供了分子基础。

STT3B通过调控PEDV S蛋白N-糖基化修饰促进病毒复制

有研究报道，寡糖基转移酶（oligosaccharyltransferase，OST）复合物能够催化预先组装好的寡糖链转移到新生多肽的天冬酰胺残基上。为了阐明PEDV S蛋白N-糖基化形成机制，本研究采用shRNA技术敲低Vero细胞中OST关键催化亚基STT3A和STT3B的表达，感染病毒后分析两个亚基对病毒复制和S蛋白迁移率的影响，结果发现仅STT3B敲低可显著抑制PEDV N蛋白的表达及病毒滴度，并加快了S蛋白的迁移率，显著降低了S蛋白的糖基化水平。进一步在LLC-PK1细胞中验证发现，STT3B敲低使病毒增殖效率明显下降。通过免疫共沉淀（Co-IP）和激光共聚焦试验发现STT3B蛋白与S蛋白具有相互作用并共同定位在内质网中，且敲低STT3B后，PEDV S蛋白在细胞中的定位明显改变。为探究STT3B的酶活性在PEDV复制中的作用，构建了STT3B酶活性失活体（WWDYG序列突变为WAAYG），通过在STT3B缺失细胞中转染野生型（WT）和催化失活型STT3B质粒，发现STT3B的酶活性缺失会降低PEDV的复制水平及S蛋白的N-糖基化形成。通过N-糖基化缺陷病毒进行进一步验证，发现STT3B缺失对N-糖基化缺陷型病毒复制的影响较小。以上结果说明，PEDV S蛋白在内质网中的STT3B的催化作用下进行N-糖基化修饰。

综上所述，本研究利用BAC系统构建了PEDV反向遗传系统，在此基础上，构建多个S蛋白糖基化位点突变病毒，通过细胞感染和仔猪感染实验，证明了N-糖基化修饰在病毒复制中的关键作用，并进一步阐明了STT3B通过调控PEDV S蛋白糖基化修饰促进病毒复制，丰富了PEDV的致病机制的研究，并为PEDV的防控提供了潜在的靶点。

Porcine Epidemic Diarrhea Virus (PEDV) is a single-stranded, positive-sense RNA virus belonging to the genus Alphacoronavirus within the family Coronaviridae. PEDV causes Porcine Epidemic Diarrhea (PED), an acute enteric infectious disease characterized by severe diarrhea, vomiting, and dehydration, leading to high mortality rates in piglets under 7 days of age and causing significant economic losses to the global swine industry. The PEDV genome is highly variable, and currently available commercial vaccines are not fully effective in controlling PEDV infections. Therefore, in-depth research on the pathogenic mechanisms and control strategies of PEDV is of great significance. N-glycans on the surface of coronaviral spike (S) proteins play crucial roles in maintaining protein folding and stability, as well as regulating viral tropism. The PEDV S protein serves as a critical mediator for viral entry into host cells. This study focuses on the N-glycosylation modifications of the PEDV S protein, aiming to investigate their impacts on viral replication and pathogenicity, thereby providing a basis for developing novel prevention and treatment strategies. The main contents are listed as follows:

Effects of N-glycosylation modification of PEDV S protein on virus replication and pathogenicity

To explore the role of N-glycosylation in PEDV infection, Vero and LLC-PK1 cells were treated with four endoplasmic reticulum N-glycosylation inhibitors (tunicamycin, NGI-1, miglustat, and celgosivir). RT-qPCR, Western blot, and TCID₅₀ assays demonstrated that inhibition of N-glycosylation significantly reduced viral gene transcription, protein expression, and viral titers in both cell lines without affecting cell viability. Glycosidase treatment further confirmed the occurrence of S protein glycosylation.

A bacterial artificial chromosome (BAC) based reverse genetics system was established to generate recombinant PEDV mutants with single N-glycosylation site deletions via CRISPR/Cas9. Nineteen glycosylation-deficient mutants were rescued, exhibiting stable propagation. Infection assays revealed that mutations at N118, N216, N726, N1232, and N1249 significantly impaired viral replication and reduced plaque sizes. qPCR analysis indicated that N118Q, N216Q, and N726Q mutations disrupted viral attachment, while N1232Q and N1249Q mutations hindered viral entry. Pathogenicity evaluation in piglets demonstrated that wild-type (WT) PEDV caused severe diarrhea, intestinal villus atrophy, and high viral loads. In contrast, N1232Q and N1249Q mutants induced no clinical symptoms, exhibited normal intestinal histology, and showed significantly reduced viral RNA levels. N118Q, N216Q, and N726Q mutants elicited mild diarrhea and moderate intestinal damage. These findings highlight the critical roles of N1232 and N1249 glycosylation sites in PEDV pathogenicity.

STT3B promotes PEDV replication by regulating the N-Glycosylation of the S protein

Mechanistic studies revealed that knockdown of the oligosaccharyltransferase (OST) catalytic subunit STT3B, but not STT3A, in Vero and LLC-PK1 cells significantly suppressed PEDV replication, reduced S protein glycosylation, and altered its electrophoretic mobility. Co-immunoprecipitation and confocal microscopy confirmed the interaction and co-localization of STT3B with S protein in the endoplasmic reticulum. Reconstitution of STT3B-deficient cells with a catalytically inactive STT3B mutant (WAAYG) failed to restore viral replication or S protein glycosylation. Furthermore, STT3B depletion minimally affected replication of N-glycosylation-deficient mutants, confirming its enzymatic role in modifying S protein.

In conclusion, this study established a BAC-based reverse genetics platform for PEDV and identified critical N-glycosylation sites governing viral replication and pathogenicity. The discovery of STT3B-mediated N-glycosylation of S protein advances our understanding of PEDV pathogenesis and provides potential targets for vaccine design and antiviral strategies.