猪流行性腹泻病毒（Porcine epidemic diarrhea virus, PEDV）是引起哺乳仔猪腹泻的主要病原，因其高致病性和高致死率，已在全球范围内给养猪业造成了严重的经济损失。该病毒主要侵染仔猪小肠黏膜，引起急性水样腹泻和脱水性死亡。PEDV在肠道内的感染过程受到局部免疫应答的严密调控，其中，肠道黏膜天然免疫在病毒感染初期发挥关键的抑制作用。因此，系统阐明PEDV与宿主天然免疫系统之间的相互作用，对于深入解析病毒致病机制并开发新型抗病毒策略具有重要意义。在天然免疫应答的过程中，模式识别受体识别病原体相关分子模式（Pathogen-associated molecular patterns，PAMPs）激活信号通路，其分子特征直接影响免疫信号通路的激活效率和应答特异性。然而，目前PEDV的关键PAMP仍不明确，其激活模式识别受体识别功能引起宿主天然免疫应答发生的分子机制尚未阐明。因此，本研究首先基于单细胞测序技术，并结合体内外模型，鉴定模式识别受体视黄酸诱导基因I（Retinoic acid-inducible gene I, RIG-I）在识别PEDV感染过程中发挥关键作用，并探明其在哺乳仔猪肠道的组织分布特点。随后，利用RNA结合蛋白免疫沉淀（RNA-Binding Protein Immunoprecipitation, RIP）试验，揭示RIG-I识别的PEDV关键PAMP，并阐明其分子识别机制。最后，在体内外验证PEDV PAMP激活天然免疫能力的基础上，进一步系统评价其不仅能够抵御病毒感染，还能够配合灭活流感病毒使用增强机体特异性保护力。本研究主要分为以下三个部分：

1、PEDV主要模式识别受体的鉴定

本研究首先基于单细胞测序结果，结合体内外试验，发现肠上皮细胞中模式识别受体RIG-I的转录水平与PEDV感染具有较高的相关性。进一步利用荧光原位杂交技术，同样揭示了RIG-I虽然在生理状态下主要分布于仔猪肠道固有层抗原呈递细胞中，但在PEDV口服感染后肠道上皮层的表达也显著上调。在此基础上，通过过表达与干扰RIG-I试验，发现RIG-I在识别PEDV激活干扰素信号通道诱导天然免疫的过程中发挥关键作用。此外，本研究还发现RIG-I还参与识别另一种猪肠道冠状病毒——传染性胃肠炎病毒（Transmissible gastroenteritis virus, TGEV）触发免疫应答的过程。

2、RIG-I识别PEDV PAMP的机制探究

在鉴定RIG-I有效识别PEDV激活天然免疫应答的基础上，本研究使用RIP-seq，发现RIG-I主要识别PEDV基因组正链375-1900 bp区域，位于5′端ORF1a基因起始段，其识别具有A/C及C/G碱基序列偏爱性。通过对该区域的截短探究其与免疫识别之间的关系，进一步发现了PEDV基因组375-760bp激活干扰素应答最为显著，在激活天然免疫反应中具有关键作用，可认为是PEDV关键PAMP。基于对该片段的二级结构分析发现主要含有三个茎环，其免疫激活功能依赖于茎环结构之间的相互作用。RIG-I根据功能特征分为N端CARD信号域、中央ATP酶解旋酶域和C端结构域，本研究分别构建了RIG-I的不同功能域缺失体，利用RNA下拉（pull-down）试验证实了RIG-I的C端结构域结合PEDV关键PAMP。

3、PEDV关键PAMP增强机体抗病毒功能的初探

PAMP能够被模式识别受体识别以诱导下游干扰素信号通路活化，激活天然免疫反应。因此，本研究通过体内外模型，证实PEDV关键PAMP能够有效激活天然免疫反应的基础上，进一步发现其不仅能够有效抑制PEDV的复制，其抑制效率高达80%，还能够发挥广谱抗病毒活性（对TGEV、PDCoV、H1N1抑制率高达60%）。体内试验也进一步证实，PEDV PAMP能够显著激活仔猪肠道天然免疫反应，降低了仔猪肠道病毒载量，增强了仔猪对PEDV的抵抗能力。同时，本研究还发现PEDV PAMP有效增强了灭活流感病毒诱导的小鼠体内免疫保护效果，并降低了流感病毒的感染率，其保护效率高达80%。

综上所述，本研究阐明了RIG-I识别PEDV的关键作用及其分子机制，并验证了PEDV关键PAMP的天然免疫激活效果，发现其不仅能够发挥抗病毒活性，还能够增强灭活病毒免疫保护力，具有较好的免疫增强剂潜力，为PEDV等RNA病毒防控提供了有效手段。
 

Porcine epidemic diarrhea virus (PEDV) is the primary pathogen causing diarrhea in suckling piglets. Due to its high pathogenicity and mortality, it has caused severe economic losses to the global pig farming industry. The virus mainly infects the mucosa of the small intestine in piglets, causing acute watery diarrhea and dehydration-related death. The infection process of PEDV in the gut is strictly regulated by local immune responses, among which the intestinal mucosal innate immune system plays a key inhibitory role in the early stage of viral infection. Therefore, systematically elucidating the interaction between PEDV and the host innate immune system is of great significance for in-depth understanding of the viral pathogenesis and developing new antiviral strategies. During the process of innate immune response, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) to activate signaling pathways, and the molecular characteristics of PAMPs directly affect the activation efficiency and response specificity of immune signaling pathways. However, the key PAMPs of PEDV remain unclear, and the molecular mechanisms of PAMPs activating PRRs to trigger host innate immune responses are still not well understood. Therefore, this study first identified that the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) plays a key role in recognizing PEDV infection based on single-cell sequencing technology and combined with in vitro and in vivo models, and explored its tissue distribution characteristics in the intestines of suckling piglets. Subsequently, using the RNA-binding protein immunoprecipitation (RIP) assay, the key PAMP of PEDV recognized by RIG-I was revealed, and its molecular recognition mechanism was clarified. Finally, based on verifying the ability of PEDV PAMP to activate innate immunity in vitro and in vivo, its ability to not only resist viral infection but also enhance specific protection in combination with inactivated influenza virus was further evaluated. This study is mainly divided into the following three parts:

1. Identification of the major pattern recognition receptor for PEDV

This study first identified a high correlation between the transcription level of the pattern recognition receptor RIG-I and PEDV infection based on single-cell sequencing results and combined in vitro and in vivo experiments. Further investigation using fluorescence in situ hybridization revealed that although RIG-I is primarily located in the antigen-presenting cells of the intestinal lamina propria of piglets under physiological conditions, its expression in the intestinal epithelial layer is significantly upregulated following oral PEDV infection. Subsequently, overexpression and knockdown experiments of RIG-I demonstrated that RIG-I plays a key role in recognizing PEDV to activate the interferon signaling pathway and induce innate immunity. In addition, this study also found that RIG-I is involved in the process of recognizing another porcine intestinal coronavirus—Transmissible gastroenteritis virus (TGEV)—and triggering immune responses.

2. Mechanism of RIG-I Recognition of PEDV PAMP

Based on the identification that RIG-I effectively recognizes PEDV to activate innate immune responses, this study used RIP-seq and found that RIG-I mainly recognizes the 375-1900 bp region of the PEDV genomic positive strand, located at the beginning of the 5′ end ORF1a gene, with a preference for A/C and C/G base sequences. Further investigation of the truncated regions revealed that the 375-760 bp region of the PEDV genome most significantly activates the interferon response and plays a key role in innate immune activation, which can be considered a critical PAMP of PEDV. Secondary structure analysis of this segment showed that it mainly contains three stem-loop structures, and its immune-activating function depends on the interaction between these stem-loop structures. RIG-I is divided into three functional domains based on its characteristics: the N-terminal CARD signaling domain, the central ATPase helicase domain, and the C-terminal domain. This study constructed different RIG-I domain deletion mutants and used RNA pull-down assays to confirm that the C-terminal domain of RIG-I binds to the critical PEDV PAMP

3. Preliminary Exploration of the Antiviral Function of PEDV PAMP

PAMPs can be recognized by pattern recognition receptors to induce the activation of downstream interferon signaling pathways and activate innate immune responses. Therefore, based on the confirmation that the critical PEDV PAMPs effectively activate innate immune responses, this study further discovered that they not only significantly inhibit PEDV replication (with an inhibition efficiency of up to 80%) but also exhibit broad-spectrum antiviral activity (with inhibition rates of up to 60% against TGEV, PDCoV, and H1N1). In vivo experiments further confirmed that PEDV PAMPs significantly activate innate immune responses in the intestines of piglets, reduce viral loads in the intestines, and enhance piglets' resistance to PEDV. Additionally, this study found that PEDV PAMPs effectively enhance the immune protection induced by inactivated viruses in mice and reduce influenza virus infection, with a protection efficiency of up to 80%.

In summary, this study elucidated the key role of RIG-I in recognizing PEDV and its molecular mechanism and verified the innate immune activation effect of PEDV key PAMPs. It was found that PEDV PAMPs not only exhibit antiviral activity but also enhance the immune protection of inactivated viruses, showing potential as vaccine adjuvants and providing effective means for the prevention and control of PEDV and other RNA viruses.