Metformin Hydrochloride Significantly Inhibits Rotavirus Infection in Caco2 Cell Line, Intestinal Organoids, and Mice.

Rotavirus is one of the main pathogens that causes severe diarrhea in children under the age of 5, primarily infecting the enterocytes of the small intestine. Currently, there are no specific drugs available for oral rehydration and antiviral therapy targeting rotavirus. However, metformin hydrochloride, a drug known for its antiviral properties, shows promise as it accumulates in the small intestine and modulates the intestinal microbiota. Therefore, we formulated a hypothesis that metformin hydrochloride could inhibit rotavirus replication in the intestine. To validate the anti-rotavirus effect of metformin hydrochloride, we conducted infection experiments using different models, ranging from in vitro cells and organoids to small intestines in vivo. The findings indicate that a concentration of 0.5 mM metformin hydrochloride significantly inhibits the expression of rotavirus mRNA and protein in Caco-2 cells, small intestinal organoids, and suckling mice models. Rotavirus infections lead to noticeable pathological changes, but treatment with metformin has been observed to mitigate the lesions caused by rotavirus infection in the treated group. Our study establishes that metformin hydrochloride can inhibit rotavirus replication, while also affirming the reliability of organoids as a virus model for in vitro research.

Dietary High Dose of Iron Aggravates the Intestinal Injury but Promotes Intestinal Regeneration by Regulating Intestinal Stem Cells Activity in Adult Mice With Dextran Sodium Sulfate-Induced Colitis.

The effects of excessive dietary iron intake on the body have been an important topic. The purpose of this study was to investigate the effects of high-dose iron on intestinal damage and regeneration in dextran sodium sulfate (DSS)-induced colitis model mice. A total of 72 8-week-old adult C57BL/6 mice were randomly divided into two dietary treatment groups: the basal diet supplemented with 45 (control) and 450 mg/kg iron (high-iron) from ferrous sulfate. The mice were fed different diets for 2 weeks, and then 2.5% DSS was orally administered to all mice for 7 days. Samples of different tissues were collected on days 0, 3, and 7 post administration (DPA). High-iron treatment significantly decreased the relative weight of the large intestine at 7 DPA but not at 0 DPA or 3 DPA. High dietary iron increased the jejunal villus width at 0 DPA, decreased the villus width and the crypt depth of the jejunum at 3 DPA, and decreased the number of colonic crypts at 7 DPA. Meanwhile, high dietary iron decreased the number of goblet cells in the jejunal villi and the Paneth cells in the jejunal crypts at 0 DPA, increased the number of goblet cells per crypt of the colon at 3 DPA, and the number of Paneth cells in the jejunal crypts, the goblet cells in the colon, the Ki67-positive proliferating cells in the colon, and the Sex-determining region Y-box transcription factor 9+ (SOX9) cells in the jejunum crypts and colon at 7 DPA. The organoid formation rate was increased by high-iron treatments at 3 DPA and 7 DPA. High dietary iron treatment decreased the mRNA level of jejunal jagged canonical Notch ligand 2 (Jag-2) at 0 DPA and bone morphogenetic protein 4 (Bmp4) and neural precursor cell-expressed developmentally downregulated 8 (Nedd8) in the jejunum and colon at 7 DPA, whereas it increased the mRNA expression of the serum/glucocorticoid-regulated kinase 1 (Sgk1) in the colon at 3 DPA. The results suggested that a high dose of iron aggravated intestinal injury but promoted intestinal repair by regulating intestinal epithelial cell renewal and intestinal stem cell activity in adult mice with colitis.

Changes in progenitors and differentiated epithelial cells of neonatal piglets.

This study aimed to assess the changes of small intestinal morphology, progenitors, differentiated epithelial cells, and potential mechanisms in neonatal piglets. Hematoxylin and eosin staining of samples from 36 piglets suggested that dramatic changes were observed in the jejunum crypts depth and crypt fission index of neonatal piglets (P < 0.001). The number of intestinal stem cells (ISC) tended to increase (P < 0.10), and a decreased number of enteroendocrine cells appeared in the jejunal crypt on d 7 (P < 0.05). Furthermore, the mRNA expression of jejunal chromogranin A (ChgA) was down-regulated in d 7 piglets (P < 0.05). There was an up-regulation of the adult ISC marker gene of SPARC related modular calcium binding 2 (Smoc2), and Wnt/ß-catenin target genes on d 7 (P < 0.05). These results were further verified in vitro enteroid culture experiments. A mass of hollow spheroids was cultured from the fetal intestine of 0-d-old piglets (P < 0.001), whereas substantial organoids with budding and branching structures were cultured from the intestine of 7-d-old piglets (P < 0.001). The difference was reflected by the organoid budding efficiency, crypt domains per organoid, and the surface area of the organoid. Furthermore, spheroids on d 0 had more Ki67-positive cells and enteroendocrine cells (P < 0.05) and showed a decreasing trend in the ISC and goblet cells (P < 0.10). Moreover, the mRNA expression of spheroids differed markedly from that of organoids, with low expression of intestinal differentiation gene (Lysozyme; P < 0.05), epithelial-specific markers (Villin, E-cadherin; P < 0.05), and adult ISC markers (leucine-rich repeat-containing G protein-coupled receptor 5 [Lgr5], Smoc2; P < 0.001), and up-regulation of fetal marker (connexin 43 [Cnx43]; P < 0.05). The mRNA expression of relevant genes was up-regulated, and involved in Wnt/ß-catenin, epidermal growth factor (EGF), Notch, and bone morphogenetic protein (BMP) signaling on d 7 organoids (P < 0.05). Spheroids displayed low differentiated phenotype and high proliferation, while organoids exhibited strong differentiation potential. These results indicated that the conversion from the fetal progenitors (spheroids) to adult ISC (normal organoids) might largely be responsible for the fast development of intestinal epithelial cells in neonatal piglets.

N-Acetyl-D-glucosamine improves the intestinal development and nutrient absorption of weaned piglets via regulating the activity of intestinal stem cells.

Early weaning in piglets can cause a series of negative effects. This causes serious losses to the livestock industry. N-Acetyl-D-glucosamine (D-GlcNAc) plays an important role in regulating the homeostasis of the intestine. This study aimed to investigate the effects of D-GlcNAc on the growth performance and intestinal function of weaned piglets. Twenty-four weaned piglets ([Yorkshire × Landrace] × Duroc, 6.58 ± 0.15 kg, n = 8) at 21 d old were fed 3 diets supplemented with 0 (control), 1 and 3 g/kg D-GlcNAc. The intestinal organoid model was used to verify the regulatory mechanism of D-GlcNAc on intestinal epithelial cells. On the whole, supplementation of D-GlcNAc in the piglet diet has no significant effect on the growth performance and diarrhoea of weaned piglets (P > 0.05). The apparent digestibility of nutrients and mRNA abundance of nutrient transporters in the 1 g/kg D-GlcNAc group were increased significantly (P < 0.05). D-GlcNAc did not affect villus height (VH) and crypt depth (CD) but resulted in a numerically shorter VH and shallower CD, which lead to an increase in ileal VH:CD ratio (P < 0.05). Cell shedding rates in the ileum villi increased (P < 0.05). The relative length and weight of the small intestine of weaned piglets increased (P < 0.05). In vitro studies found that the budding rates of organoids treated with 0.1 mmol/L D-GlcNAc increased on the d 3 and 5 (P < 0.05). The average budding numbers per budding organoid treated with 0.1 and 10 mmol/L D-GlcNAc increased on d 3 (P < 0.05). D-GlcNAc upregulated leucine rich repeat containing G protein-coupled receptor 5 (Lgr5 + ) and Chromogranin A mRNA abundance in organoids (P < 0.05). Mucin 2 (Muc2) expression increased when treated with 1 and 10 mmol/L D-GlcNAc (P < 0.05). In conclusion, dietary D-GlcNAc cannot improve the growth performance of weaned piglets. However, it can promote the growth and development of the intestinal tract and improve the digestion and absorption capacity of the intestine, which is achieved by affecting the activity of intestinal stem cells.

Dietary Copper Improves Intestinal Morphology via Modulating Intestinal Stem Cell Activity in Pigs.

Copper (Cu) is an essential micronutrient for animals. Many studies have been conducted on the effects of dietary Cu on growth performance, intestinal morphology, and function of piglets. However, the underlying mechanism remains to be explored. Intestinal stem cells (ISC) drive the development and constant renewal of intestinal epithelium. Therefore, we hypothesized that dietary Cu affects piglets' intestinal development via modulating ISC activity. A total of eighty-five 21-day-old piglets were randomly assigned to five groups, where 25, 50, 75, 100, and 125 mg CuSO4/kg on a dry matter basis were supplemented to the basal diet at phase 1 (day 0 to 21). Increasing the dietary Cu concentration decreased (p < 0.05) villus width but increased (p < 0.001) the number of Ki67-positive cells. At phase 2 (day 22 to 163), the other 45 pigs were offered the same diets. Villus height in the 125 mg/kg Cu group was greater (p < 0.001) than in the other groups. Moreover, the effects of Cu on ISC activity in vitro were tested to explore the underlying mechanism. Compared to the control group, 10 µmol/L CuSO4·5H2O increased (p < 0.001) the organoid budding efficiency, crypt depth, and crypts per organoid. Dietary Cu improved the intestinal morphology of finishing pigs via promoting cell proliferation and modulating ISC activity.

Single-Cell Sequencing and Organoids: A Powerful Combination for Modelling Organ Development and Diseases.

The development and function of a particular organ and the pathogenesis of various diseases remain intimately linked to the features of each cell type in the organ. Conventional messenger RNA- or protein-based methodologies often fail to elucidate the contribution of rare cell types, including some subpopulations of stem cells, short-lived progenitors and circulating tumour cells, thus hampering their applications in studies regarding organ development and diseases. The scRNA-seq technique represents a new approach for determining gene expression variability at the single-cell level. Organoids are new preclinical models that recapitulate complete or partial features of their original organ and are thought to be superior to cell models in mimicking the sophisticated spatiotemporal processes of the development and regeneration and diseases. In this review, we highlight recent advances in the field of scRNA-seq, organoids and their current applications and summarize the advantages of using a combination of scRNA-seq and organoid technology to model diseases and organ development.

Rotavirus Infection and Cytopathogenesis in Human Biliary Organoids Potentially Recapitulate Biliary Atresia Development.

Biliary atresia (BA) is a neonatal liver disease characterized by progressive fibroinflammatory obliteration of both intrahepatic and extrahepatic bile ducts. The etiologies of BA remain largely unknown, but rotavirus infection has been implicated at least for a subset of patients, and this causal relation has been well demonstrated in mouse models. In this study, we aim to further consolidate this evidence in human biliary organoids. We obtained seven batches of human biliary organoids cultured from fetal liver, adult liver, and bile duct tissues. We found that these organoids are highly susceptible and support the full life cycle of rotavirus infection in three-dimensional culture. The robust infection triggers active virus-host interactions, including interferon-based host defense mechanisms and injury responses. We have observed direct cytopathogenesis in organoids upon rotavirus infection, which may partially recapitulate the development of BA. Importantly, we have demonstrated the efficacy of mycophenolic acid and interferon alpha but not ribavirin in inhibiting rotavirus in biliary organoids. Furthermore, neutralizing antibody targeting rotavirus VP7 protein effectively inhibits infection in organoids. Thus, we have substantiated the causal evidence of rotavirus inducing BA in humans and provided potential strategies to combat the disease.IMPORTANCE There is substantial evidence indicating the possible involvement of rotavirus in biliary atresia (BA) development, at least in a subset of patients, but concrete proof remains lacking. In a mouse model, it has been well demonstrated that rotavirus can infect the biliary epithelium to cause biliary inflammation and obstruction, representing the pathogenesis of BA in humans. By using recently developed organoids technology, we now have demonstrated that human biliary organoids are susceptible to rotavirus infection, and this provokes active virus-host interactions and causes severe cytopathogenesis. Thus, our model recapitulates some essential aspects of BA development. Furthermore, we have demonstrated that antiviral drugs and neutralizing antibodies are capable of counteracting the infection and BA-like morphological changes, suggesting their potential for mitigating BA in patients.

Drug screening identifies gemcitabine inhibiting rotavirus through alteration of pyrimidine nucleotide synthesis pathway.

Although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, including organ transplantation recipients and cancer patients irrespective of pediatric or adult patients. Since no approved medication against rotavirus infection is available, this study screened a library of safe-in-man broad-spectrum antivirals. We identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of rotavirus infection. We confirmed this effect in 2D cell cultures and 3D cultured human intestinal organoids with both laboratory-adapted rotavirus strains and five clinical isolates. Supplementation of UTP or uridine largely abolished the anti-rotavirus activity of gemcitabine, suggesting its function through inhibition of pyrimidine biosynthesis pathway. Our results support repositioning of gemcitabine for treating rotavirus infection, especially for infected cancer patients.

Dietary vitamin A affects growth performance, intestinal development, and functions in weaned piglets by affecting intestinal stem cells.

Vitamin A (VA) is an important nutrient for weaning piglets. It plays a significant role in the normal formation, development, and maintenance of epithelial cells. Previous studies have shown that VA supplements could improve the host's intestinal barrier function. Therefore, we hypothesized that VA supplements can affect intestinal function in weaned piglets by regulating intestinal stem cells. Thirty-two 21-d-old weaned [(Yorkshire × Landrace) × Duroc] piglets with an average weight of 8.34 ± 0.13 kg were randomly divided into 4 treatment groups, with 1) 2 mg/kg (control), 2) 4 mg/kg, 3) 8 mg/kg, and 4) 16 mg/kg doses of VA, respectively. The experiment lasted for 14 d. Weaned piglets were given ad libitum access to food and water during the test. The ADG (linear, P = 0.020) and G:F (linear, P = 0.005) of the piglets were found to increase significantly from days 8 to 14. The Lgr5+ gene expression (P = 0.012) in the jejunum mucosa of the 16 mg/kg VA group was increased. The jejunum villus height (P = 0.027) and villi surface area (P = 0.035) were significantly increased in the 4 mg/kg VA treatment group. The crypt depth increased significantly in the 4 and 8 mg/kg VA treatment groups (quadratic, P = 0.043), and the ratios of villus height to crypt depth significantly increased in the 16 mg/kg VA group (quadratic, P = 0.015). The maltase (P = 0.032), sucrose (P = 0.041), and alkaline phosphatase activity (linear, P = 0.024) were significantly increased when further supplemented with 4 mg/kg VA. Slc2a2 mRNA abundance was significantly increased in the 2 mg/kg VA group (linear, P = 0.024). Moreover, the budding rates, buddings number per organoid, and Chromogranin A and Muc2 expression of piglet intestinal organoids were significantly reduced (P < 0.05) by VA and its metabolites (retinoic acid). Compared with the control group, the expression of Spp1 and Trop2 increased. These results indicated that VA may increase the stemness of intestinal stem cell in vitro. This study suggested that VA could affect growth performance and intestinal function by regulating intestinal stem cells in the jejunum of weaned piglets.

Mini-gut: a promising model for drug development.

Until recently, major advances in drug development have been hampered by a lack of proper cell and tissue models; but the introduction of organoid technology has revolutionized this field. At the level of the gastrointestinal tract, the so-called mini-gut comprises all major cell types of native intestine and recapitulates the composition and function of native intestinal epithelium. The mini-gut can be classified as an intestinal organoid (IO), derived from pluripotent stem cells, or as an enteroid, consisting only of epithelial cells and generated from adult stem cells. Both classifications have been used as models to develop drugs against cystic fibrosis, cancer and infectious disease, as well as for drug screening, personalized medicine and the development of new medical tools. In this review, we highlight and discuss the importance of mini-guts for drug development and point out their limitations and future prospects.

Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication.

Rotavirus infection remains a great health burden worldwide especially in some developing countries. It causes severe dehydrating diarrhea in infants, young children, as well as immunocompromised and organ transplanted patients. Viral replication heavily relies on the host to supply nucleosides. Thus, host enzymes involved in nucleotide biosynthesis represent potential targets for antiviral development. Dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. In this study, we demonstrated that two specific DHODH enzyme inhibitors, brequinar (BQR) and leflunomide (LFM) robustly inhibited rotavirus replication in conventional human intestinal Caco2 cell line as well as in human primary intestinal organoids. The antiviral effect is conserved in both laboratory strain SA11 and rotavirus strain 2011K isolated from clinical sample. Mechanistic study indicated that BQR and LFM exerted their anti-rotavirus effect through targeting DHODH to deplete pyrimidine nucleotide pool. Therefore, targeting pyrimidine biosynthesis represents a potential approach for developing antiviral strategies against rotavirus.

The Eukaryotic Translation Initiation Factor 4F Complex Restricts Rotavirus Infection via Regulating the Expression of IRF1 and IRF7.

The eIF4F complex is a translation initiation factor that closely regulates translation in response to a multitude of environmental conditions including viral infection. How translation initiation factors regulate rotavirus infection remains poorly understood. In this study, the knockdown of the components of the eIF4F complex using shRNA and CRISPR/Cas9 were performed, respectively. We have demonstrated that loss-of-function of the three components of eIF4F, including eIF4A, eIF4E and eIF4G, remarkably promotes the levels of rotavirus genomic RNA and viral protein VP4. Consistently, knockdown of the negative regulator of eIF4F and programmed cell death protein 4 (PDCD4) inhibits the expression of viral mRNA and the VP4 protein. Mechanically, we confirmed that the silence of the eIF4F complex suppressed the protein level of IRF1 and IRF7 that exert potent antiviral effects against rotavirus infection. Thus, these results demonstrate that the eIF4F complex is an essential host factor restricting rotavirus replication, revealing new targets for the development of new antiviral strategies against rotavirus infection.

Enteroids for Nutritional Studies.

Nutritional studies are greatly hampered by a paucity of proper models. Previous studies on nutrition have employed conventional cell lines and animal models to gain a better understanding of the field. These models lack certain correlations with human physiological responses, which impede their applications in this field. Enteroids are cultured from intestinal stem cells and include enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and stem cells, which mimic hallmarks of in vivo epithelium and support long-term culture without genetic or physiological changes. Enteroids have been used as models to study the effects of diet and nutrients on intestinal growth and development, ion and nutrient transport, secretory and absorption functions, the intestinal barrier, and location-specific functions of the intestine. In this review, the existing models for nutritional studies are discussed and the importance of enteroids as a new model for nutritional studies is highlighted. Taken together, it is suggested that enteroids can serve as a potential model system to be exploited in nutritional studies.

Enteroids: Promising in Vitro Models for Studies of Intestinal Physiology and Nutrition in Farm Animals.

The lack of sophisticated in vitro models limits our current understanding of gastrointestinal functions in farm animals. Conventional 2D cell lines or primary cells fail to recapitulate the physiology of in vivo intestinal epithelium. In contrast stem cell-derived, nontransformed 3D enteroids partially recreate the villus-crypt anatomy of the native intestine and comprise most if not all intestinal cell types including enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and stem cells. This review summarizes the techniques used for generating and culturing enteroids of various farm animal species, focuses on important factors influencing the longevity of enteroids, and provides an overview of their current applications in modeling veterinary pathogens and in developing chemicals and bioactives for treating animal disease and improving production performance. It also mentions current limitations of enteroid models and potential solutions and highlights the opportunities for using these enteroids as a platform in studies regarding veterinary sciences and animal nutrition.

Modeling liver cancer and therapy responsiveness using organoids derived from primary mouse liver tumors.

The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine.

Corrigendum: Organoid and Enteroid Modeling of Salmonella Infection.

[This corrects the article DOI: 10.3389/fcimb.2018.00102.].

Nitazoxanide Inhibits Human Norovirus Replication and Synergizes with Ribavirin by Activation of Cellular Antiviral Response.

Norovirus is the main cause of viral gastroenteritis worldwide. Although norovirus gastroenteritis is self-limiting in immunocompetent individuals, chronic infections with debilitating and life-threatening complications occur in immunocompromised patients. Nitazoxanide (NTZ) has been used empirically in the clinic and has demonstrated effectiveness against norovirus gastroenteritis. In this study, we aimed at uncovering the antiviral potential and mechanisms of action of NTZ and its active metabolite, tizoxanide (TIZ), using a human norovirus (HuNV) replicon. NTZ and TIZ, collectively referred to as thiazolides (TZD), potently inhibited replication of HuNV and a norovirus surrogate, feline calicivirus. Mechanistic studies revealed that TZD activated cellular antiviral response and stimulated the expression of a subset of interferon-stimulated genes (ISGs), particularly interferon regulatory factor 1 (IRF-1), not only in a Huh7 cell-based HuNV replicon, but also in naive Huh7 and Caco-2 cells and novel human intestinal organoids. Overexpression of exogenous IRF-1 inhibited HuNV replication, whereas knockdown of IRF-1 largely attenuated the antiviral activity of TZD, suggesting that IRF-1 mediated TZD inhibition of HuNV. By using a Janus kinase (JAK) inhibitor, CP-690550, and a STAT1 knockout approach, we found that TZD induced antiviral response independently of the classical JAK-signal transducers and activators of transcription (JAK-STAT) pathway. Furthermore, TZD and ribavirin synergized to inhibit HuNV replication and completely depleted the replicons from host cells after long-term treatment. In summary, our results demonstrated that TZD combated HuNV replication through activation of cellular antiviral response, in particular by inducing a prominent antiviral effector, IRF-1. NTZ monotherapy or combination with ribavirin represent promising options for treating norovirus gastroenteritis, especially in immunocompromised patients.

6-Thioguanine inhibits rotavirus replication through suppression of Rac1 GDP/GTP cycling.

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational.

TNF-α exerts potent anti-rotavirus effects via the activation of classical NF-κB pathway.

Active virus-host interactions determine the outcome of pathogen invasions. It has been shown that in isolated dendritic cells (DCs), rotavirus can induce the expression of tumor necrosis factor α (TNF-α), a vital cytokine mediating host immune responses. However, the role of TNF-α in rotavirus infection is unknown. In this study, we demonstrated that TNF-α has potent anti-rotavirus effects, independent of type I interferon production. Blocking of TNF-α by infliximab, a clinically available TNFα antibody, totally abrogated this effect. Mechanistic studies revealed that the anti-rotavirus effect of TNF-α was achieved by NFκB-regulated genes via the activation of classical nuclear factor κB (NF-κB) signaling. Our study reveals the pivotal role and the mechanism-of-actions of TNF-α in the host defense against rotavirus. Thus, this knowledge may contribute to the better understanding of the complexity of rotavirus-host interactions.