MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
MOE Joint International Research Laboratory of Animal Health and Food Safety , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing , Jiangsu , China , njau.edu.cn
Transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are major enteric coronaviruses responsible for severe diarrhea in neonatal piglets. Retinoic acid-inducible gene I (RIG-I) is a key sensor against RNA viruses, yet its distribution in the porcine intestine and regulatory roles during TGEV and PDCoV infections remain insufficiently understood. In this study, we show that under normal conditions, RIG-I predominantly localizes in lamina propria antigen-presenting cells, with its expression increasing with age. Following viral infection in vivo and in vitro, both TGEV and PDCoV induce RIG-I expression, although TGEV elicits a more robust activation of RIG-I and downstream interferon pathways. Mechanistically, RIG-I overexpression inhibits replication of both viruses, whereas RIG-I knockdown significantly enhances TGEV proliferation only, implying that TGEV primarily depends on RIG-I–mediated immune responses, while PDCoV may rely on other pattern recognition receptors (PRRs). These findings unveil distinct immune regulatory strategies of TGEV and PDCoV and highlight the central role of RIG-I in controlling TGEV infection, offering a theoretical foundation for targeted preventive and therapeutic interventions.
1. Introduction
Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses characterized by high mutation rates and the ability to cross species barriers. Since their discovery in the 1960s, they have caused repeated global outbreaks, posing significant challenges to public health and the livestock industry [1, 2]. While human coronaviruses, such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, have caused recurrent respiratory diseases and continue to threaten public health, porcine enteric coronaviruses similarly cause substantial economic losses in the swine industry. Key pathogens include Porcine Epidemic Diarrhea Virus (PEDV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine acute diarrhea syndrome coronavirus (SADS-CoV) [3]. These viruses spread rapidly via the fecal–oral route, and their high mutation rates and potential for genetic recombination further complicate control measures [4]. TGEV, the first identified porcine enteric coronavirus, belongs to the alpha-coronavirus group and can cause up to 90%–100% mortality in piglets under 2 weeks of age [5]. PDCoV, first reported in Hong Kong in 2012, has demonstrated cross-species transmission potential and can result in 30%–50% mortality [6]. Both viruses primarily target small intestinal epithelial cells, leading to villus atrophy and epithelial cell loss. Mixed infections are common and exacerbate diarrhea, increasing mortality rates. Conventional vaccines are limited in addressing multiple viral strains, and the complex interactions between pathogens and hosts further hinder diagnostic and control efforts. Therefore, a deeper understanding of the infection mechanisms of TGEV and PDCoV, as well as their interactions with host immune responses, is essential for developing effective strategies to control mixed infections and formulate targeted prevention methods.
The host immune system comprises both innate and adaptive immunity, with innate immunity serving as the first line of defense by rapidly recognizing and limiting pathogen spread during the early stages of infection [7]. This response is mediated by a range of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and AIM2-like receptors (ALRs) [8, 9]. Upon recognizing pathogen-associated molecular patterns (PAMPs), these receptors trigger cascade signaling pathways that ultimately lead to the production of interferons (IFNs) and proinflammatory cytokines [10]. RLRs, which include Retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), are responsible for detecting RNA viruses, such as influenza and coronaviruses [11, 12]. During coronavirus infections, RIG-I plays a critical role in the signaling pathway activated by viral dsRNA recognition, initiating a rapid immune response, maintaining immune homeostasis, and facilitating adaptive immune evolution [13]. Upon activation, RIG-I interacts with mitochondrial antiviral signaling protein (MAVS) via its N-terminal CARD domain, recruiting downstream TRAF3/6 and TBK1/IKK complexes, which in turn activate transcription factors, such as NF-κB and IRF3/7, leading to the induction of type I and III IFNs [14]. Although studies have demonstrated that SARS-CoV and SARS-CoV-2 infections can activate the RLR signaling pathway, triggering a large-scale production of interferon-stimulated genes (ISGs) [15, 16], certain structural proteins (e.g., N and M proteins) and nonstructural proteins (e.g., Nsp5 and Nsp7) have been reported to target and inhibit the RLR pathway, blocking signal transduction and suppressing IFN production [17–19]. Thus, RIG-I not only functions as a crucial “sentinel” during coronavirus infections but also serves as a key target for viral immune evasion.
In the study of porcine enteric coronaviruses, TGEV and PDCoV have garnered significant attention due to their ability to cause diarrhea in neonatal piglets, resulting in severe economic losses. Although both viruses belong to the Coronaviridae family, they are classified into different evolutionary branches (TGEV belongs to Alphacoronavirus, while PDCoV belongs to Deltacoronavirus), and they exhibit significant differences in genomic structure, immune activation, and pathological characteristics. By comparing these two viruses within the same research framework, we can better reveal the convergent or divergent mechanisms of different coronaviruses during host adaptation, thereby assessing the pathogenicity of the two viruses and their potential immune evasion strategies. It has been reported that the IFN response following PDCoV infection is significantly lower than that of TGEV [20, 21], suggesting potential differences between the two viruses in host innate immune recognition and interferon signaling pathway regulation. Comparative studies not only deepen our understanding of the immunological characteristics of these two viruses but also provide a scientific basis for the development of broad-spectrum antiviral drugs, cross-protective vaccines, and precise diagnostic technologies. Although the specific key proteins or signaling pathways involved remain unclear. Based on our in vivo and in vitro findings, we observed significant upregulation of RIG-I expression following viral infections. Therefore, this study focuses on the molecular mechanisms by which RIG-I mediates innate immune responses during TGEV and PDCoV infections. Using both intestinal tissue and enterocyte models, we aim to systematically examine the activation of the RIG-I/MAVS/TBK1/IKK signaling axis and its impact on interferon production. Additionally, by employing RIG-I knockdown and overexpression approaches, we will evaluate the regulatory effects of RIG-I on viral replication, cellular damage, and cytokine expression. This study is expected to provide new insights into the pathogenesis of porcine enteric coronaviruses and elucidate the central role of RIG-I in the innate immune regulatory network, laying a theoretical foundation for the development of novel vaccines, antiviral therapies, and targeted strategies to control viral mutations and mixed infections in pigs.
2. Materials and Methods
2.1. Cells and Viruses
Swine testis (ST) cells were cultured in DMEM (Biochannel, China) containing 10% inactivated fetal bovine serum (FBS, Biochannel, China) and 1% penicillin/streptomycin (Biochannel, China) at 37°C with 5% CO2. The TGEV SHXBKP strain (GenBank: KP202848) was provided by our laboratory and propagated in ST cells. The PDCoV CH/JX/JGS/01 strain (GenBank: KY293677) was obtained by Professor Yuxin Tang from Jiangxi Agricultural University and propagated in ST cells.
2.2. Reagents and Antibodies
For in situ hybridization, the RNA FISH kit (Genepharma, China) was utilized. Mouse monoclonal anti-TGEV N antibody was prepared and stored in our laboratory. Mouse monoclonal anti-PDCoV N antibody (1:1000, SD-4-5, Medgene Labs, USA) was used for viral load quantification. To detect RIG-I protein expression, we used RIG-I monoclonal antibody (1:1000, D33H10, Cell Signaling Technology, USA). Additional antibodies included GAPDH monoclonal antibody (1:1000, 30202ES4, Yeasen, China), goat antimouse IgG (1:5000, ab205719, Abcam, USA), goat antirabbit IgG (1:5000, ab150077, Abcam, USA), and AlexaFluor594-conjugated donkey antimouse IgG1 (1:200, 34112ES60, Yeasen, China). Nuclei were stained with DAPI (1:1000, 2313070, Thermo Fisher Scientific, USA) and mounted using antifade reagent (Yeasen, China). For overexpression and knockdown experiments, plasmids were constructed using homologous recombination enzymes (Vazyme, China) and transfected using Lipofectamine 3000 (Invitrogen, USA).
2.3. Animals
All animal procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, China) and followed the National Institutes of Health guidelines. 3-day-old, 7-day-old, 10-day-old, and 21-day-old Duroc/Landrace/Yorkshire piglets were obtained from Jiangsu Nanjing Pig Farm (Nanjing, China), with negative serology for Porcine Reproductive and Respiratory Syndrome Virus, PDCoV, PEDV, and TGEV. All pigs had unrestricted access to water and food.
3-day-old piglets were orally inoculated with 107 plaque-forming units (PFU) of TGEV and PDCoV. Clinical symptoms, including vomiting and watery diarrhea, appeared 24 h postinfection. Pigs were euthanized 48 h postinfection, and samples were collected from the jejunum and ileum. Tissue samples were fixed in 4% paraformaldehyde for 24 h, and dehydrated through a graded ethanol series (75%, 85%, 95%). Subsequently, the samples were dehydrated twice in 100% ethanol for 1 h each. The samples were then immersed in xylene for clearing and embedded in paraffin. For cryopreservation, some tissue samples were frozen for RNA and protein extraction.
2.4. In Situ Hybridization
To investigate the distribution of RIG-I in antigen-presenting cells in the intestine, RIG-I and CD86 probes were synthesized (Supporting Information 1: Table S1). RNA FISH was performed according to the manufacturer’s instructions. Tissue sections were deparaffinized in xylene, rehydrated through a graded ethanol series (100%, 90%, 80%, and 70%, each for 1 min), and digested with proteinase K. After dehydration, the sections were treated with denaturing solution and incubated at 78°C for 8 min. Subsequently, tissue sections were incubated with the 2 μM probe at 37°C for 16 h. Following washing with PBS, sections were stained with DAPI and mounted with antifade reagent.
2.5. Immunofluorescence Staining
Cell samples were fixed with 4% paraformaldehyde, and tissue samples were processed into paraffin sections. Sections were deparaffinized and antigen-retrieved in citrate buffer (pH 6.0) at 90–95°C for 15 min. Both cell samples and tissue sections were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 5% FBS for 2 h at 37°C. TGEV N and PDCoV N antibodies were applied overnight, followed by secondary AlexaFluor594-conjugated donkey antimouse IgG1 antibody. Sections were counterstained with DAPI and mounted with antifade reagent.
2.6. Plasmid Construction and Transfection
To construct the RIG-I overexpression plasmid, total RNA was extracted from ST cells, and RIG-I cDNA was synthesized via reverse transcription and PCR (Supporting Information 1: Table S1). The cDNA was cloned into the PLVX-Flag vector using EcoRI and BamHI restriction sites. The cDNA sequence was confirmed by sequencing. For RIG-I knockdown, siRIG-I was chemically synthesized and used with a control siRNA (Supporting Information 1: Table S1).
Cells were seeded at 2 × 105 cells per well in the complete growth medium. Once cells reached approximately 70% confluence, Lipofectamine 3000 was mixed with siRNA and plasmid DNA, incubated briefly to form complexes, and added to the cells. After 6 h of incubation at 37°C, the medium was replaced, and cells were cultured for 48 h before being harvested for mRNA or protein expression analysis.
2.7. RNA Extraction and RT-qPCR
Total RNA was extracted from cells using TRIzol reagent (Vazyme, China) according to the manufacturer’s protocol. cDNA was synthesized using HiScript IV RT SuperMix for qPCR (+gDNA wiper; Vazyme, China). The reaction consisted of two steps: the first step included 3 μL of 5 × gDNA wiper mix, 1 μg RNA, and RNase-free ddH2O up to 15 μL, followed by incubation at 42°C for 2 min. The second step involved adding 5 μL of 4 × HiScript IV qRT SuperMix to the 15 μL reaction mixture, followed by incubation at 37°C for 15 min and 85°C for 5 s. qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, China) with specific primers (Supporting Information 1: Table S1). Gene expression was analyzed using the 2−ΔΔCT method, with GAPDH as the endogenous control.
2.8. Western Blotting
Proteins were extracted from cells and tissues using RIPA buffer (Biosharp, China) containing PMSF (BioFroxx, China). After adding 5X SDS-PAGE loading buffer and heating to 98°C for 10 min, protein samples were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking with 5% skim milk, membranes were incubated with primary antibodies for RIG-I (overnight at 4°C) and TGEV N or PDCoV N (overnight at 4°C). GAPDH was used as the loading control. Membranes were washed with TBST and incubated with corresponding secondary antibodies for 1 h. Signals were detected using the cECL system (Biosharp, China).
2.9. Virus Infection and TCID50
Cells were grown to 70% confluence in a 12-well plate and washed with PBS. Virus suspension (MOI = 0.1) was added to a serum-free medium and incubated at 37°C for 1 h. After washing with PBS to remove the unbound virus, cells were cultured in a maintenance medium with 2% FBS at 37°C and 5% CO2. Viral titers were measured using the TCID50 method. Virus stocks were serially diluted and added to 96-well plates. After incubation for 4 days at 37°C, TCID50 values were calculated using the Reed-Muench method.
2.10. Statistical Analysis
Data are presented as mean ± standard deviation (SD) and analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Differences between groups were assessed by one-way analysis of variance (ANOVA) or two-sample t-tests. Statistical significance was considered at p < 0.05. Results are based on at least three independent experiments unless stated otherwise.
3. Results
3.1. Expression of RIG-I in the Porcine Intestine at Different Developmental Stages
The immune system of piglets undergoes rapid development during the first few weeks after birth [22]. Given the critical role of RIG-I as an immune sensor, we investigated its distribution in the porcine intestine. Small intestine tissue samples from piglets at various ages (3, 7, 10, and 21 days) were collected for in situ hybridization. As shown in Figure 1A, RIG-I was detected in both the jejunum and ileum, with the majority localized to the lamina propria. The lamina propria, a key component of the intestinal immune system, contains abundant antigen-presenting cells responsible for immune surveillance and regulation [23]. Co-staining with the antigen-presenting cell marker CD86 confirmed RIG-I’s co-localization with these cells within the jejunal and ileal mucosa. At 3 days of age, the immune system is still immature, and RIG-I expression was minimal. Between 7 and 10 days, a critical period for immune development, RIG-I levels increased rapidly, peaking by day 10. At 21 days, around weaning, RIG-I expression stabilized (Figure 1A). Immunofluorescence analysis also showed an age-dependent increase in RIG-I expression in the small intestinal mucosa (Figure 1B).
Spatiotemporal distribution of RIG-I in the intestines of piglets. (A) Distribution of RIG-I in the jejunum and ileum of suckling piglets at 3, 7, 10, and 21 days of age. Nuclei were stained with DAPI (blue). Scale bars: 100 μm (main image) and 20 μm (inset). White arrows indicate RIG-I expression in the lamina propria and antigen-presenting cells. (B) Quantitative analysis of RIG-I and CD86-positive antigen-presenting cells in the jejunum and ileum at different growth stages. The number of positive cells was counted in five randomly selected fields (×10). All data are presented as means ± SD, with comparisons performed using one-way ANOVA.
Spatiotemporal distribution of RIG-I in the intestines of piglets. (A) Distribution of RIG-I in the jejunum and ileum of suckling piglets at 3, 7, 10, and 21 days of age. Nuclei were stained with DAPI (blue). Scale bars: 100 μm (main image) and 20 μm (inset). White arrows indicate RIG-I expression in the lamina propria and antigen-presenting cells. (B) Quantitative analysis of RIG-I and CD86-positive antigen-presenting cells in the jejunum and ileum at different growth stages. The number of positive cells was counted in five randomly selected fields (×10). All data are presented as means ± SD, with comparisons performed using one-way ANOVA.
3.2. Impact of TGEV and PDCoV Infection on RIG-I Expression in Piglet Intestines
To examine how viral infections affect RIG-I distribution and expression, we orally inoculated 3-day-old piglets with TGEV and PDCoV and analyzed jejunal and ileal tissues. Immunofluorescence staining revealed that, compared with the blank control piglets, the infected piglets had abundant viral protein-positive cells in both the jejunum and ileum, predominantly in the cytoplasm of intestinal epithelial cells (Figure 2A,B). RT-qPCR confirmed successful infection of the piglet intestines with both TGEV and PDCoV (Figure 2C,D). In TGEV-infected piglets, RIG-I transcription levels were significantly elevated in both the jejunum and ileum (Figure 2C). In situ hybridization further showed that viral infection increased RIG-I expression in the lamina propria and induced a marked upregulation in the epithelial layer (Figure 2E), suggesting that TGEV infection promotes RIG-I expression in epithelial cells, potentially triggering a stronger innate immune response. In contrast, PDCoV infection resulted in a modest increase in RIG-I expression only in the ileum, with no significant change in the jejunum. Although some RIG-I-positive epithelial cells were observed in the ileum, the overall expression was lower than in the TGEV-infected group (Figure 2E).
Altered expression of RIG-I in piglets infected with TGEV and PDCoV. (A, B) Distribution of viral proteins in intestinal tissues detected by immunofluorescence staining with anti-TGEV (A) and anti-PDCoV (B) monoclonal antibodies (red), with nuclei stained by DAPI (blue). Scale bar = 100 μm. (C, D) Expression of viral RNA and RIG-I in intestinal tissues of TGEV- (C) and PDCoV-infected piglets (D). (E) Distribution of RIG-I in the jejunum and ileum of newborn, TGEV-infected, and PDCoV-infected piglets, stained with a RIG-I probe (green) and DAPI (blue). Scale bars: 100 μm (main image) and 20 μm (inset). White arrows indicate RIG-I in the epithelial layer. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group. ns, no significance.
Altered expression of RIG-I in piglets infected with TGEV and PDCoV. (A, B) Distribution of viral proteins in intestinal tissues detected by immunofluorescence staining with anti-TGEV (A) and anti-PDCoV (B) monoclonal antibodies (red), with nuclei stained by DAPI (blue). Scale bar = 100 μm. (C, D) Expression of viral RNA and RIG-I in intestinal tissues of TGEV- (C) and PDCoV-infected piglets (D). (E) Distribution of RIG-I in the jejunum and ileum of newborn, TGEV-infected, and PDCoV-infected piglets, stained with a RIG-I probe (green) and DAPI (blue). Scale bars: 100 μm (main image) and 20 μm (inset). White arrows indicate RIG-I in the epithelial layer. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group. ns, no significance.
Altered expression of RIG-I in piglets infected with TGEV and PDCoV. (A, B) Distribution of viral proteins in intestinal tissues detected by immunofluorescence staining with anti-TGEV (A) and anti-PDCoV (B) monoclonal antibodies (red), with nuclei stained by DAPI (blue). Scale bar = 100 μm. (C, D) Expression of viral RNA and RIG-I in intestinal tissues of TGEV- (C) and PDCoV-infected piglets (D). (E) Distribution of RIG-I in the jejunum and ileum of newborn, TGEV-infected, and PDCoV-infected piglets, stained with a RIG-I probe (green) and DAPI (blue). Scale bars: 100 μm (main image) and 20 μm (inset). White arrows indicate RIG-I in the epithelial layer. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group. ns, no significance.
Altered expression of RIG-I in piglets infected with TGEV and PDCoV. (A, B) Distribution of viral proteins in intestinal tissues detected by immunofluorescence staining with anti-TGEV (A) and anti-PDCoV (B) monoclonal antibodies (red), with nuclei stained by DAPI (blue). Scale bar = 100 μm. (C, D) Expression of viral RNA and RIG-I in intestinal tissues of TGEV- (C) and PDCoV-infected piglets (D). (E) Distribution of RIG-I in the jejunum and ileum of newborn, TGEV-infected, and PDCoV-infected piglets, stained with a RIG-I probe (green) and DAPI (blue). Scale bars: 100 μm (main image) and 20 μm (inset). White arrows indicate RIG-I in the epithelial layer. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group. ns, no significance.
Altered expression of RIG-I in piglets infected with TGEV and PDCoV. (A, B) Distribution of viral proteins in intestinal tissues detected by immunofluorescence staining with anti-TGEV (A) and anti-PDCoV (B) monoclonal antibodies (red), with nuclei stained by DAPI (blue). Scale bar = 100 μm. (C, D) Expression of viral RNA and RIG-I in intestinal tissues of TGEV- (C) and PDCoV-infected piglets (D). (E) Distribution of RIG-I in the jejunum and ileum of newborn, TGEV-infected, and PDCoV-infected piglets, stained with a RIG-I probe (green) and DAPI (blue). Scale bars: 100 μm (main image) and 20 μm (inset). White arrows indicate RIG-I in the epithelial layer. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group. ns, no significance.
3.3. Impact of TGEV and PDCoV on the Innate Immune Response in ST Cells
To study the impact of TGEV and PDCoV on host cell innate immunity in vitro, we established viral infection models using ST cells. TGEV N protein was detectable at 12 h postinfection, peaked at 24 h, and declined by 36 h, with a similar trend observed in viral RNA levels (Figure 3A,C). PDCoV replication peaked at 24 h (Figure 3B), with a gradual increase in N protein levels over time (Figure 3C), indicating that both viruses can effectively replicate in ST cells.
TGEV and PDCoV trigger RIG-I-mediated innate immune responses in ST Cells. ST cells were infected with TGEV and PDCoV at an MOI of 0.1, and samples were collected at 6, 12, 24, 36, and 48 h. (A, B) qPCR analysis was used to measure mRNA levels of TGEV and PDCoV N proteins. (C) Western blot analysis assessed protein levels of TGEV and PDCoV N proteins. (D–G) qPCR analysis determined mRNA levels of RIG-I, IFN-β, and antiviral molecules (OASL, OAS2, ISG15, ISG56). All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group.
TGEV and PDCoV trigger RIG-I-mediated innate immune responses in ST Cells. ST cells were infected with TGEV and PDCoV at an MOI of 0.1, and samples were collected at 6, 12, 24, 36, and 48 h. (A, B) qPCR analysis was used to measure mRNA levels of TGEV and PDCoV N proteins. (C) Western blot analysis assessed protein levels of TGEV and PDCoV N proteins. (D–G) qPCR analysis determined mRNA levels of RIG-I, IFN-β, and antiviral molecules (OASL, OAS2, ISG15, ISG56). All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group.
TGEV and PDCoV trigger RIG-I-mediated innate immune responses in ST Cells. ST cells were infected with TGEV and PDCoV at an MOI of 0.1, and samples were collected at 6, 12, 24, 36, and 48 h. (A, B) qPCR analysis was used to measure mRNA levels of TGEV and PDCoV N proteins. (C) Western blot analysis assessed protein levels of TGEV and PDCoV N proteins. (D–G) qPCR analysis determined mRNA levels of RIG-I, IFN-β, and antiviral molecules (OASL, OAS2, ISG15, ISG56). All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group.
TGEV and PDCoV trigger RIG-I-mediated innate immune responses in ST Cells. ST cells were infected with TGEV and PDCoV at an MOI of 0.1, and samples were collected at 6, 12, 24, 36, and 48 h. (A, B) qPCR analysis was used to measure mRNA levels of TGEV and PDCoV N proteins. (C) Western blot analysis assessed protein levels of TGEV and PDCoV N proteins. (D–G) qPCR analysis determined mRNA levels of RIG-I, IFN-β, and antiviral molecules (OASL, OAS2, ISG15, ISG56). All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group.
TGEV and PDCoV trigger RIG-I-mediated innate immune responses in ST Cells. ST cells were infected with TGEV and PDCoV at an MOI of 0.1, and samples were collected at 6, 12, 24, 36, and 48 h. (A, B) qPCR analysis was used to measure mRNA levels of TGEV and PDCoV N proteins. (C) Western blot analysis assessed protein levels of TGEV and PDCoV N proteins. (D–G) qPCR analysis determined mRNA levels of RIG-I, IFN-β, and antiviral molecules (OASL, OAS2, ISG15, ISG56). All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group.
TGEV and PDCoV trigger RIG-I-mediated innate immune responses in ST Cells. ST cells were infected with TGEV and PDCoV at an MOI of 0.1, and samples were collected at 6, 12, 24, 36, and 48 h. (A, B) qPCR analysis was used to measure mRNA levels of TGEV and PDCoV N proteins. (C) Western blot analysis assessed protein levels of TGEV and PDCoV N proteins. (D–G) qPCR analysis determined mRNA levels of RIG-I, IFN-β, and antiviral molecules (OASL, OAS2, ISG15, ISG56). All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group.
TGEV and PDCoV trigger RIG-I-mediated innate immune responses in ST Cells. ST cells were infected with TGEV and PDCoV at an MOI of 0.1, and samples were collected at 6, 12, 24, 36, and 48 h. (A, B) qPCR analysis was used to measure mRNA levels of TGEV and PDCoV N proteins. (C) Western blot analysis assessed protein levels of TGEV and PDCoV N proteins. (D–G) qPCR analysis determined mRNA levels of RIG-I, IFN-β, and antiviral molecules (OASL, OAS2, ISG15, ISG56). All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group.
Further analysis of the innate immune response revealed significant activation of antiviral mechanisms, with a marked increase of RIG-I expression (Figure 3D), particularly in TGEV-infected cells. RIG-I activation triggers the interferon signaling pathway, inducing the expression of various antiviral proteins that inhibit viral replication, promote viral particle degradation, and enhance apoptosis, thus constituting a first line of defense against viral infections [24]. RT-qPCR analysis showed that TGEV infection significantly induced IFN-β production (Figure 3E). In contrast, PDCoV also induced an IFN response, but its onset was delayed and the overall response was weaker (Figure 3E). The transcriptional trends of downstream antiviral molecules correlated with IFN expression levels (Figure 3F,G). Despite high levels of type I IFN induction, both TGEV and PDCoV maintained strong replication, suggesting that these viruses may have evolved mechanisms to escape immune surveillance, allowing continued replication in an innate immune environment.
3.4. Role of RIG-I in Recognizing TGEV and Inducing Innate Immunity
To explore the role of RIG-I in TGEV infection, we conducted overexpression and knockdown experiments. We successfully constructed overexpression plasmids and siRNA sequences for RIG-I knockdown, and validated them in ST cells (Supporting Information 2: Figure S1). Overexpression of Flag-RIG-I in ST cells resulted in stable and significant upregulation of RIG-I expression, as confirmed by RT-qPCR and Western blotting (Figure 4A,B). Using this overexpression model, we assessed the regulatory effects of RIG-I on TGEV infection and its influence on the host’s innate immune response. As shown in Figure 4B,C, RIG-I overexpression significantly suppressed TGEV replication in ST cells, as evidenced by an over 80% reduction in viral protein levels and a corresponding decrease in viral titers (Figure 4D). Immunofluorescence staining further confirmed this inhibitory effect, showing a marked reduction in viral fluorescence signals in overexpressing cells (Figure 4E).
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I mediates host immune response to TGEV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F-J) for 24 h, followed by TGEV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and TGEV N protein levels, (C, H) qPCR assessment of TGEV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for TGEV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
To further confirm the essential role of RIG-I in combating TGEV infection, we used specific siRNA to knock down RIG-I expression in ST cells. Compared to control cells, siRNA-mediated knockdown reduced RIG-I mRNA levels by 60% and significantly decreased the transcription of various antiviral genes (Figure 4F). Under RIG-I-deficient conditions, ST cells displayed significantly increased susceptibility to TGEV infection, with higher viral protein levels (Figure 4G,H) and viral titers in the supernatant (Figure 4I). Immunofluorescence analysis also revealed an increase in viral particles (Figure 4J). These results indicate that RIG-I deficiency substantially impairs the host’s ability to resist TGEV infection, confirming RIG-I as a key sensor for TGEV.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
RIG-I does not play a key role in sensing PDCoV infection in ST cells. The blank plasmid and the nontargeting siRNA were transfected into ST cells, respectively, as the negative control (NC) group. The ST cells were transfected with the Flag-RIG-I plasmid (A–E) or RIG-I siRNA (F–J) for 24 h, followed by PDCoV inoculation (MOI 0.1), and samples were collected 36 h postinfection for subsequent analyses, including (A, F) qPCR measurement of RIG-I, IFN-β, OASL, OAS2, ISG15, and ISG56 mRNA levels, (B, G) Western blot detection of RIG-I and PDCoV N protein levels, (C, H) qPCR assessment of PDCoV N protein mRNA, (D, I) TCID50 determination of viral titer in the supernatant, (E, J) and immunofluorescence staining for PDCoV-N protein expression. All data are presented as means ± SD, with comparisons performed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group. ns, no significance.
3.5. Role of RIG-I in Recognizing PDCoV and Inducing Innate Immunity
Next, we investigated the role of RIG-I in recognizing PDCoV, another enteric coronavirus. Compared to TGEV, RIG-I overexpression exerted a more limited inhibitory effect on PDCoV. Although the transcription of ISG15 was significantly increased, other antiviral molecules showed minimal changes (Figure 5A). Despite this, PDCoV replication was still partially suppressed, as indicated by reductions in viral protein levels (Figure 5B,E), viral RNA levels (Figure 5C), and viral titers in the cell supernatant (Figure 5D). These results suggest that RIG-I upregulation can partially limit PDCoV replication, though its inhibitory effect is weaker than that observed with TGEV, indicating that RIG-I plays a less central role in PDCoV infection.
Furthermore, when RIG-I expression was knocked down, no significant increase in PDCoV replication was observed. Antiviral gene transcription remained unchanged (Figure 5F), and viral N protein levels (Figure 5G,H) and viral titers (Figure 5I) were comparable to control cells. Immunofluorescence analysis also confirmed these findings, showing no significant difference in viral particle levels (Figure 5J). These results indicate that while RIG-I exerts some inhibitory effect on PDCoV, it is not a critical factor in PDCoV replication. This suggests that other PRRs or pathways may assume a more dominant role in the host’s immune response to PDCoV.
4. Discussion
RIG-I, a cytoplasmic PRR, recognizes PAMPs such as short dsRNA and 5′-triphosphate RNA, which are produced during RNA virus replication. Upon detection, RIG-I activates signaling pathways, including TBK1/IRF3 and NF-κB, leading to the induction of IFNs and proinflammatory cytokines [25]. Previous studies have underscored RIG-I’s essential role as a “sentinel” in the immune defense against RNA viruses such as influenza, respiratory syncytial virus, and coxsackievirus. When RIG-I is impaired or absent, the host loses the capacity to effectively limit early viral replication and control infection [26–28].
RIG-I expression and function in mucosal tissues are crucial for maintaining immune balance and antiviral responses, making its distribution in immune cells a focal point of investigation. In our study, we observed a gradual increase in RIG-I expression in the porcine intestinal mucosa with age. This finding contrasts with previous observations in the nasal mucosa, where RIG-I expression decreases with age [29]. The upregulation of RIG-I in the lamina propria, rich in antigen-presenting cells, is likely related to the rapid development of the piglet’s intestinal immune system during the first few weeks after birth [22]. Additionally, RIG-I is widely expressed in various immune cells, and its expression is notably increased in endothelial and mucosal epithelial cells upon pathogen exposure, triggering efficient antiviral responses [30]. In our infection models, we confirmed this upregulation: RIG-I activation was evident in both antigen-presenting and intestinal epithelial cells. The functional role of RIG-I in mucosal tissues is vital for maintaining immune homeostasis and defending against viral infections [31]. Interestingly, RIG-I expression increases in human monocytes from neonate to juvenile stages, but declines with age [32]. This suggests that RIG-I expression is regulated by multiple factors, including microbiota composition, mucosal immune maturation, and the host’s response to pathogens. Therefore, studying mucosal antiviral mechanisms requires accounting for dynamic immune and tissue development. This provides new insights into RIG-I’s role in early immune defenses.
Coronaviruses produce dsRNA intermediates with varying lengths and structures during genome replication, which can be recognized by PRRs such as RIG-I and MDA5 [5]. In this study, we compared two porcine enteric coronaviruses, TGEV and PDCoV. Although both viruses generate dsRNA during replication, their effects on RIG-I activation were significantly different. In oral infection experiments with neonatal piglets, TGEV markedly upregulated RIG-I expression in both the jejunum and ileum, whereas PDCoV only induced a moderate increase in RIG-I expression in the ileum. Subsequent in vitro epithelial cell experiments confirmed that TGEV was more efficient in activating RIG-I and its downstream antiviral genes than PDCoV. This difference may reflect distinct viral immune evasion strategies. Previous studies suggest that TGEV, despite upregulating negative regulators like SOCS1/3 to suppress IFN responses, maintains strong IFN induction [33]. In contrast, PDCoV N protein and Nsp5 directly inhibit RIG-I activation, reducing type I IFN production [34, 35]. In RIG-I overexpression and knockdown cell models, we found that TGEV replication is highly dependent on RIG-I. Knockdown of RIG-I significantly enhanced viral replication, while reducing RIG-I levels in PDCoV-infected cells did not boost replication. This suggests that other PRRs, such as MDA5 or TLR3, may play a more prominent role in recognizing PDCoV infection. Other RLR family members have been shown to play significant roles in recognizing coronaviruses like SARS-CoV-2 [36], highlighting the potential involvement of additional PRRs in PDCoV recognition. Moreover, RIG-I preferentially recognizes 5′-triphosphate short dsRNA [11], while PDCoV’s RNA has a more complex secondary structure, including long-range interactions and multiple stem-loop structures, which may affect the stability of its dsRNA intermediates and RIG-I binding efficiency [37]. This could help explain the differences in immune evasion strategies between the two viruses. Although the cell models used in this study are widely employed for research on TGEV and PDCoV replication, they may not fully replicate the complex physiological environment in vivo. For instance, they lack host immune responses and the specific microenvironments of different tissues. This could potentially lead to discrepancies between the experimental results and the actual infection processes in vivo. To address this limitation, we plan to incorporate animal models or organoid co-culture systems for validation in our future work. Nevertheless, the current in vitro studies still provide an essential foundation for exploring mechanisms and offer theoretical support and technical guidance for in vivo research.
5. Conclusion
In conclusion, our study reveals the distribution and developmental regulation of RIG-I in the porcine intestinal mucosa. We also compare the differences between TGEV and PDCoV in terms of RIG-I activation and immune evasion. RIG-I plays a crucial role in recognizing and controlling TGEV infection, while its antiviral effect on PDCoV is relatively weaker. This suggests that PDCoV may rely more on other PRRs to trigger innate immune responses. These findings enhance our understanding of the pathogenicity and immune evasion mechanisms of porcine enteric coronaviruses and provide new insights into the recognition and defense mechanisms of other RNA viruses in mucosal environments. Future research can focus on exploring the activation and function of other PRRs (such as TLR3, TLR7, NLRs, and AIM2) in coronavirus infections. By employing techniques such as RIP and pull-down assays, along with high-resolution structural analysis methods, we aim to uncover the direct binding sites and interaction mechanisms between the virus and these different PRRs. Furthermore, we plan to conduct in vivo validation across different developmental stages and host backgrounds to further elucidate the complexity of immune regulation. Ultimately, these efforts will lay a solid theoretical foundation for the design of novel vaccines, optimization of antiviral strategies, and development of precise diagnostic methods.
Ethics Statement
The animal studies were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health guidelines for the performance of animal experiments.
Conflicts of Interest
The authors declare no conflicts of interest.
Author Contributions
Hui Zeng was responsible for performing the experiments, analyzing the data, and preparing the manuscript. Xuan Wu and Xinyue Zhang participated in the performances of the experiments. Yunlei Cao and Rongfeng Tang were responsible for data verification and manuscript polishing. Yuchen Li and Qian Yang designed the study and revised the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the National Key Research and Development Program of China (Grant No. 2022YFD1801400), the National Natural Science Foundation of China (32473063), the Natural Science Foundation of Jiangsu Province (No. BK20240198), the Fundamental Research Funds for the Central Universities (KJYQ2025004 and KYT2024004), China Postdoctoral Science Foundation funded project (2023M731731), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Supporting Information
Additional supporting information can be found online in the Supporting Information section.
Supporting Information 2 Figure S1. Construction of RIG-I overexpressing and knockdown cells
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