Type III and Not Type I Interferons Efficiently Prevent the Spread of Rotavirus in Human Intestinal Epithelial Cells.

Rotavirus infects intestinal epithelial cells and is the leading cause of gastroenteritis in infants worldwide. Upon viral infection, intestinal cells produce type I and type III interferons (IFNs) to alert the tissue and promote an antiviral state. These two types of IFN bind to different receptors but induce similar pathways that stimulate the activation of interferon-stimulated genes (ISGs) to combat viral infection. In this work, we studied the spread of a fluorescent wild-type (WT) SA11 rotavirus in human colorectal cancer cells lacking specific interferon receptors and compared it to that of an NSP1 mutant rotavirus that cannot interfere with the host intrinsic innate immune response. We could show that the WT rotavirus efficiently blocks the production of type I IFNs but that type III IFNs are still produced, whereas the NSP1 mutant rotavirus allows the production of both. Interestingly, while both exogenously added type I and type III IFNs could efficiently protect cells against rotavirus infection, endogenous type III IFNs were found to be key to limit infection of human intestinal cells by rotavirus. By using a fluorescent reporter cell line to highlight the cells mounting an antiviral program, we could show that paracrine signaling driven by type III IFNs efficiently controls the spread of both WT and NSP1 mutant rotavirus. Our results strongly suggest that NSP1 efficiently blocks the type I IFN-mediated antiviral response; however, its restriction of the type III IFN-mediated one is not sufficient to prevent type III IFNs from partially inhibiting viral spread in intestinal epithelial cells. Additionally, our findings further highlight the importance of type III IFNs in controlling rotavirus infection, which could be exploited as antiviral therapeutic measures. IMPORTANCE Rotavirus is one of the most common causes of gastroenteritis worldwide. In developing countries, rotavirus infections lead to more than 200,000 deaths in infants and children. The intestinal epithelial cells lining the gastrointestinal tract combat rotavirus infection by two key antiviral compounds known as type I and III interferons. However, rotavirus has developed countermeasures to block the antiviral actions of the interferons. In this work, we evaluated the arms race between rotavirus and type I and III interferons. We determined that although rotavirus could block the induction of type I interferons, it was unable to block type III interferons. The ability of infected cells to produce and release type III interferons leads to the protection of the noninfected neighboring cells and the clearance of rotavirus infection from the epithelium. This suggests that type III interferons are key antiviral agents and could be used to help control rotavirus infections in children.

Interaction between proteins of the PPARγ and NFκB immune response pathways and rotavirus non-structural proteins.

Cells infected with MA104 rotavirus and/or transfected with plasmids expressing NSP proteins, were analyzed for expression of cellular proteins related to NFκB and PPARγ pathways and evaluated through the ELISA, luminescence, flow cytometry and Western blot techniques. The association between cellular and viral (NSPs) proteins was examined by ELISA, epifluorescence and confocal microscopy techniques. It was observed that NSP1 protein interacts with RXR, NSP1, and NSP3 with PPARγ, NSP2 with p-IKKα/ß and NSP5 with NFκB proteins. We have found that phosphorylated PPARγ is localized in cytoplasm and transcriptional activity of PPRE is diminished. These results lead to the conclusion, that RRV activates the proinflammatory pathway, increasing the expression of NFκB and possibly by PPARγ phosphorylation, its translocation to the nucleus is impeded, thus inactivating the proinflammatory pathway. Keywords: rotavirus; PPARγ; NFκB; NSPs; RRV.

m6A modifications regulate intestinal immunity and rotavirus infection.

N6-methyladenosine (m6A) is an abundant mRNA modification and affects many biological processes. However, how m6A levels are regulated during physiological or pathological processes such as virus infections, and the in vivo function of m6A in the intestinal immune defense against virus infections are largely unknown. Here, we uncover a novel antiviral function of m6A modification during rotavirus (RV) infection in small bowel intestinal epithelial cells (IECs). We found that rotavirus infection induced global m6A modifications on mRNA transcripts by down-regulating the m6a eraser ALKBH5. Mice lacking the m6A writer enzymes METTL3 in IECs (Mettl3ΔIEC) were resistant to RV infection and showed increased expression of interferons (IFNs) and IFN-stimulated genes (ISGs). Using RNA-sequencing and m6A RNA immuno-precipitation (RIP)-sequencing, we identified IRF7, a master regulator of IFN responses, as one of the primary m6A targets during virus infection. In the absence of METTL3, IECs showed increased Irf7 mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in Mettl3ΔIEC mice. Moreover, the global m6A modification on mRNA transcripts declined with age in mice, with a significant drop from 2 weeks to 3 weeks post birth, which likely has broad implications for the development of intestinal immune system against enteric viruses early in life. Collectively, we demonstrated a novel host m6A-IRF7-IFN antiviral signaling cascade that restricts rotavirus infection in vivo.

Enteric virome negatively affects seroconversion following oral rotavirus vaccination in a longitudinally sampled cohort of Ghanaian infants.

Rotavirus vaccines (RVVs) have substantially diminished mortality from severe rotavirus (RV) gastroenteritis but are significantly less effective in low- and middle-income countries (LMICs), limiting their life-saving potential. The etiology of RVV's diminished effectiveness remains incompletely understood, but the enteric microbiota has been implicated in modulating immunity to RVVs. Here, we analyze the enteric microbiota in a longitudinal cohort of 122 Ghanaian infants, evaluated over the course of 3 Rotarix vaccinations between 6 and 15 weeks of age, to assess whether bacterial and viral populations are distinct between non-seroconverted and seroconverted infants. We identify bacterial taxa including Streptococcus and a poorly classified taxon in Enterobacteriaceae as positively correlating with seroconversion. In contrast, both bacteriophage diversity and detection of Enterovirus B and multiple novel cosaviruses are negatively associated with RVV seroconversion. These findings suggest that virome-RVV interference is an underappreciated cause of poor vaccine performance in LMICs.

The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update.

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies.

Single-cell sequencing of rotavirus-infected intestinal epithelium reveals cell-type specific epithelial repair and tuft cell infection.

Intestinal epithelial damage is associated with most digestive diseases and results in detrimental effects on nutrient absorption and production of hormones and antimicrobial defense molecules. Thus, understanding epithelial repair and regeneration following damage is essential in developing therapeutics that assist in rapid healing and restoration of normal intestinal function. Here we used a well-characterized enteric virus (rotavirus) that damages the epithelium at the villus tip but does not directly damage the intestinal stem cell, to explore the regenerative transcriptional response of the intestinal epithelium at the single-cell level. We found that there are specific Lgr5+ cell subsets that exhibit increased cycling frequency associated with significant expansion of the epithelial crypt. This was accompanied by an increase in the number of immature enterocytes. Unexpectedly, we found rotavirus infects tuft cells. Transcriptional profiling indicates tuft cells respond to viral infection through interferon-related pathways. Together these data provide insights as to how the intestinal epithelium responds to insults by providing evidence of stimulation of a repair program driven by stem cells with involvement of tuft cells that results in the production of immature enterocytes that repair the damaged epithelium.

Rotavirus spike protein ΔVP8* as a novel carrier protein for conjugate vaccine platform with demonstrated antigenic potential for use as bivalent vaccine.

Conjugate vaccine platform is a promising strategy to overcome the poor immunogenicity of bacterial polysaccharide antigens in infants and children. A carrier protein in conjugate vaccines works not only as an immune stimulator to polysaccharide, but also as an immunogen; with the latter generally not considered as a measured outcome in real world. Here, we probed the potential of a conjugate vaccine platform to induce enhanced immunogenicity of a truncated rotavirus spike protein ΔVP8*. ΔVP8* was covalently conjugated to Vi capsular polysaccharide (Vi) of Salmonella Typhi to develop a bivalent vaccine, termed Vi-ΔVP8*. Our results demonstrated that the Vi-ΔVP8* vaccine can induce specific immune responses against both antigens in immunized mice. The conjugate vaccine elicits high antibody titers and functional antibodies against S. Typhi and Rotavirus (RV) when compared to immunization with a single antigen. Together, these results indicate that Vi-ΔVP8* is a potent and immunogenic vaccine candidate, thus strengthening the potential of conjugate vaccine platform with enhanced immune responses to carrier protein, including ΔVP8*.

Serological Humoral Immunity Following Natural Infection of Children with High Burden Gastrointestinal Viruses.

Acute gastroenteritis (AGE) is a major cause of morbidity and mortality worldwide, resulting in an estimated 440,571 deaths of children under age 5 annually. Rotavirus, norovirus, and sapovirus are leading causes of childhood AGE. A successful rotavirus vaccine has reduced rotavirus hospitalizations by more than 50%. Using rotavirus as a guide, elucidating the determinants, breath, and duration of serological antibody immunity to AGE viruses, as well as host genetic factors that define susceptibility is essential for informing development of future vaccines and improving current vaccine candidates. Here, we summarize the current knowledge of disease burden and serological antibody immunity following natural infection to inform further vaccine development for these three high-burden viruses.

Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis.

Viral infections lead to expeditious activation of the host's innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin's antiviral action in addition to the intricacy of viperin-virus interaction.

Host miR-4301 promotes rotavirus replication via PPP1R3D in Caco-2 cells.

To investigate the role of miR-4301 in rotavirus (RV)-infected Caco-2 cells. In this experiment, RNAs of RV-infected Caco-2 cells were extracted, and the high-throughput second-generation sequencing was performed to detect the expression profiles of host microRNAs (miRNAs). Synthetic miRNA mimics and inhibitors were examined (quantitative polymerase chain reaction [qPCR], crystalline violet, immunofluorescence and electron microscopy) to evaluate the effect on RV replication. Target genes of miR-4301 were predicted by software analysis. The expression of target genes was evaluated by qPCR and Western blot after transfected with miRNA inhibitor/mimic, and crystalline violet and qPCR were used to detect the downregulation effects of target genes on RV replication. By transfecting miRNA inhibitors/mimics and detecting downstream target genes, the mechanism of miRNA affecting RV replication was analyzed. There were 78 known miRNAs with significant differential expression, including 39 upregulated miRNAs and 39 downregulated miRNAs. The results showed that miR-4301 exerted a key role in enhancing RV replication. PPP1R3D protein which can inhibit RV replication was predicted as the target gene of miR-4301 by software analysis. While upregulating miR-4301 by RV, the expression of PPP1R3D and glycogen synthase (GS) is suppressed. For the first time, the effect of miR-4301 on RV infection, and its influence on GS was investigated. Specifically, RV inhibits host cell glycogen synthesis to utilize the host intracellular glucose for promoting its own replication.

Ligilactobacillus salivarius Strains Isolated From the Porcine Gut Modulate Innate Immune Responses in Epithelial Cells and Improve Protection Against Intestinal Viral-Bacterial Superinfection.

Previously, we constructed a library of Ligilactobacillus salivarius strains from the intestine of wakame-fed pigs and reported a strain-dependent capacity to modulate IFN-ß expression in porcine intestinal epithelial (PIE) cells. In this work, we further characterized the immunomodulatory activities of L. salivarius strains from wakame-fed pigs by evaluating their ability to modulate TLR3- and TLR4-mediated innate immune responses in PIE cells. Two strains with a remarkable immunomodulatory potential were selected: L. salivarius FFIG35 and FFIG58. Both strains improved IFN-ß, IFN-λ and antiviral factors expression in PIE cells after TLR3 activation, which correlated with an enhanced resistance to rotavirus infection. Moreover, a model of enterotoxigenic E. coli (ETEC)/rotavirus superinfection in PIE cells was developed. Cells were more susceptible to rotavirus infection when the challenge occurred in conjunction with ETEC compared to the virus alone. However, L. salivarius FFIG35 and FFIG58 maintained their ability to enhance IFN-ß, IFN-λ and antiviral factors expression in PIE cells, and to reduce rotavirus replication in the context of superinfection. We also demonstrated that FFIG35 and FFIG58 strains regulated the immune response of PIE cells to rotavirus challenge or ETEC/rotavirus superinfection through the modulation of negative regulators of the TLR signaling pathway. In vivo studies performed in mice models confirmed the ability of L. salivarius FFIG58 to beneficially modulate the innate immune response and protect against ETEC infection. The results of this work contribute to the understanding of beneficial lactobacilli interactions with epithelial cells and allow us to hypothesize that the FFIG35 or FFIG58 strains could be used for the development of highly efficient functional feed to improve immune health status and reduce the severity of intestinal infections and superinfections in weaned piglets.

Rotavirus Infection, Illness, and Vaccine Performance in Malnourished Children: A Review of the Literature.

Live, oral rotavirus vaccines are more effective at preventing rotavirus disease in countries with low child mortality compared with high child mortality. Among several hypotheses, poorer protection in malnourished children, who are more prevalent in countries with high child mortality, may partially explain this difference. We conducted a literature search to identify articles with a laboratory-confirmed rotavirus endpoint that evaluated differences by malnutrition status in rotavirus vaccine effectiveness and vaccine efficacy (VE) or the prevalence of rotavirus infection or illness among children <5 years old. We identified 7 analyses from 11 countries published from 2007 to 2019 that stratified rotavirus VE by malnutrition status. Among well-nourished children, VE point estimates ranged from 71% to 84% in observational studies and 26% to 61% in clinical trials. Among malnourished children, they ranged from -28% to 45% in observational studies and -3% to 61% in clinical trials. The relative difference between VE in well-nourished and malnourished children by length-for-age ranged from 37% to 64%, by weight-for-age ranged from 0% to 107%, and by weight-for-height ranged from -65% to 137%. We identified 3 cohort and 6 cross-sectional studies of natural rotavirus infection and illness and none reported that malnourished children were more susceptible to rotavirus infection or illness than well-nourished children. Overall, rotavirus vaccines may offer less protection to children with malnutrition than well-nourished children. As malnourished children often have worse outcomes from diarrhea, high rotavirus vaccine coverage and a better understanding of the performance of oral rotavirus vaccines in this population is important, though our finding that malnourished children may be less susceptible to rotavirus provides important context and information for vaccine evaluation design.

Escherichia coli Nissle 1917 Enhances Innate and Adaptive Immune Responses in a Ciprofloxacin-Treated Defined-Microbiota Piglet Model of Human Rotavirus Infection.

Human rotavirus (HRV) infection is a major cause of gastroenteritis in children worldwide. Broad-spectrum antibiotic-induced intestinal microbial imbalance and the ensuing immune-metabolic dysregulation contribute to the persistence of HRV diarrhea. Escherichia coli Nissle 1917 (EcN), a Gram-negative probiotic, was shown to be a potent immunostimulant and alleviated HRV-induced diarrhea in monocolonized gnotobiotic (Gn) piglets. Our goal was to determine how EcN modulates immune responses in ciprofloxacin (Cipro)-treated Gn piglets colonized with a defined commensal microbiota (DM) and challenged with virulent HRV (VirHRV). Cipro given in therapeutic doses for a short term reduced serum and intestinal total and HRV-specific antibody titers, while EcN treatment alleviated this effect. Similarly, EcN treatment increased the numbers of total immunoglobulin-secreting cells, HRV-specific antibody-secreting cells, activated antibody-forming cells, resting/memory antibody-forming B cells, and naive antibody-forming B cells in systemic and/or intestinal tissues. Decreased levels of proinflammatory but increased levels of immunoregulatory cytokines and increased frequencies of Toll-like receptor-expressing cells were evident in the EcN-treated VirHRV-challenged group. Moreover, EcN treatment increased the frequencies of T helper and T cytotoxic cells in systemic and/or intestinal tissues pre-VirHRV challenge and the frequencies of T helper cells, T cytotoxic cells, effector T cells, and T regulatory cells in systemic and/or intestinal tissues postchallenge. Moreover, EcN treatment increased the frequencies of systemic and mucosal conventional and plasmacytoid dendritic cells, respectively, and the frequencies of systemic natural killer cells. Our findings demonstrated that Cipro use altered immune responses of DM-colonized neonatal Gn pigs, while EcN supplementation rescued these immune parameters partially or completely.IMPORTANCE Rotavirus (RV) is a primary cause of malabsorptive diarrhea in children and is associated with significant morbidity and mortality, especially in developing countries. The use of antibiotics exacerbates intestinal microbial imbalance and results in the persistence of RV-induced diarrhea. Probiotics are now being used to treat enteric infections and ulcerative colitis. We showed previously that probiotics partially protected gnotobiotic (Gn) piglets against human RV (HRV) infection and decreased the severity of diarrhea by modulating immune responses. However, the interactions between antibiotic and probiotic treatments and HRV infection in the context of an established gut microbiota are poorly understood. In this study, we developed a Gn pig model to study antibiotic-probiotic-HRV interactions in the context of a defined commensal microbiota (DM) that mimics aspects of the infant gut microbiota. Our results provide valuable information that will contribute to the treatment of antibiotic- and/or HRV-induced diarrhea and may be applicable to other enteric infections in children.

Rotavirus disease burden pre-vaccine introduction in young children in Rural Southern Mozambique, an area of high HIV prevalence.

BACKGROUND: Rotavirus vaccines have been adopted in African countries since 2009, including Mozambique (2015). Disease burden data are needed to evaluate the impact of rotavirus vaccine. We report the burden of rotavirus-associated diarrhea in Mozambique from the Global Enteric Multicenter Study (GEMS) before vaccine introduction. METHODS: A case-control study (GEMS), was conducted in Manhiça district, recruiting children aged 0-59 months with moderate-to-severe diarrhea (MSD) and less-severe-diarrhea (LSD) between December 2007 and November 2012; including 1-3 matched (age, sex and neighborhood) healthy community controls. Clinical and epidemiological data and stool samples (for laboratory investigation) were collected. Association of rotavirus with MSD or LSD was determined by conditional logistic regression and adjusted attributable fractions (AF) calculated, and risk factors for rotavirus diarrhea assessed. RESULTS: Overall 915 cases and 1,977 controls for MSD, and 431 cases and 430 controls for LSD were enrolled. Rotavirus positivity was 44% (217/495) for cases and 15% (160/1046) of controls, with AF = 34.9% (95% CI: 32.85-37.06) and adjusted Odds Ratio (aOR) of 6.4 p< 0.0001 in infants with MSD compared to 30% (46/155) in cases and 14% (22/154) in controls yielding AF = 18.7%, (95% CI: 12.02-25.39) and aOR = 2.8, p = 0.0011 in infants with LSD. The proportion of children with rotavirus was 32% (21/66) among HIV-positive children and 23% (128/566) among HIV-negative ones for MSD. Presence of animals in the compound (OR = 1.9; p = 0.0151) and giving stored water to the child (OR = 2.0, p = 0.0483) were risk factors for MSD; while animals in the compound (OR = 2.37, p = 0.007); not having routine access to water on a daily basis (OR = 1.53, p = 0.015) and washing hands before cooking (OR = 1.76, p = 0.0197) were risk factors for LSD. CONCLUSION: The implementation of vaccination against rotavirus may likely result in a significant reduction of rotavirus-associated diarrhea, suggesting the need for monitoring of vaccine impact.

A CD22-Shp1 phosphatase axis controls integrin ß7 display and B cell function in mucosal immunity.

The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

Impact of the Rotavirus Vaccination Program in Norway After Four Years With High Coverage.

BACKGROUND: Use of rotavirus vaccines worldwide since 2006 has led to a significant impact on the burden of rotavirus disease. However, only a third of European countries have introduced rotavirus vaccination in their immunization programs. In October 2014, rotavirus vaccination was introduced for Norwegian infants under strict age restrictions. Exclusive use of the monovalent rotavirus vaccine (RV1) and high vaccination coverage from the beginning enabled evaluation of the impact of this vaccine during the first 4 years after introduction. METHODS: Prospective laboratory-based surveillance among children <5 years of age hospitalized for acute gastroenteritis at 5 Norwegian hospitals was used to assess the vaccine effectiveness of 2 vaccine doses against rotavirus hospitalization in a case-control study. We used community controls selected from the national population-based immunization registry, and test-negative controls recruited through hospital surveillance. We also assessed the vaccine impact by using time-series analysis of retrospectively collected registry data on acute gastroenteritis in primary and hospital care during 2009-2018. RESULTS: Vaccine effectiveness against rotavirus-confirmed hospitalization was 76% (95% confidence interval [CI]: 34%-91%) using test-negative controls, and 75% (95% CI: 44%-88%) using community controls. In the postvaccine period, acute gastroenteritis hospitalizations in children <5 years were reduced by 45% compared with the prevaccine years (adjusted incidence rate ratios 0.55; 95% CI: 0.49-0.61). Reduction in hospitalizations was also seen in cohorts not eligible for vaccination. Rates in primary care decreased to a lesser degree. CONCLUSIONS: Four years after introduction of rotavirus vaccination in the national childhood immunization program, we recorded a substantial reduction in the number of children hospitalized for acute gastroenteritis in Norway, attributable to a high vaccine effectiveness.

αvß8 integrin-expression by BATF3-dependent dendritic cells facilitates early IgA responses to Rotavirus.

Secretory intestinal IgA can protect from re-infection with rotavirus (RV), but very little is known about the mechanisms that induce IgA production during intestinal virus infections. Classical dendritic cells (cDCs) in the intestine can facilitate both T cell-dependent and -independent secretory IgA. Here, we show that BATF3-dependent cDC1, but not cDC2, are critical for the optimal induction of RV-specific IgA responses in the mesenteric lymph nodes. This depends on the selective expression of the TGFß-activating integrin αvß8 by cDC1. In contrast, αvß8 on cDC1 is dispensible for steady state immune homeostasis. Given that cDC2 are crucial in driving IgA during steady state but are dispensable for RV-specific IgA responses, we propose that the capacity of DC subsets to induce intestinal IgA responses reflects the context, as opposed to an intrinsic property of individual DC subsets.

Rotavirus Interactions With Host Intestinal Epithelial Cells.

Rotavirus (RV) is the foremost enteric pathogen associated with severe diarrheal illness in young children (<5years) and animals worldwide. RV primarily infects mature enterocytes in the intestinal epithelium causing villus atrophy, enhanced epithelial cell turnover and apoptosis. Intestinal epithelial cells (IECs) being the first physical barrier against RV infection employs a range of innate immune strategies to counteract RVs invasion, including mucus production, toll-like receptor signaling and cytokine/chemokine production. Conversely, RVs have evolved numerous mechanisms to escape/subvert host immunity, seizing translation machinery of the host for effective replication and transmission. RV cell entry process involve penetration through the outer mucus layer, interaction with cell surface molecules and intestinal microbiota before reaching the IECs. For successful cell attachment and entry, RVs use sialic acid, histo-blood group antigens, heat shock cognate protein 70 and cell-surface integrins as attachment factors and/or (co)-receptors. In this review, a comprehensive summary of the existing knowledge of mechanisms underlying RV-IECs interactions, including the role of gut microbiota, during RV infection is presented. Understanding these mechanisms is imperative for developing efficacious strategies to control RV infections, including development of antiviral therapies and vaccines that target specific immune system antagonists within IECs.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

NLRP9 in innate immunity and inflammation.

The nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) are a family of evolutionarily conserved proteins. Several members of NLRs, notably NLRP1, NLRP3 and NLRC4, are able to form cytosolic oligomeric signalling platforms termed inflammasomes to mediate immune response towards pathogens, damage and stress. However, the functions of many NLRs still remain elusive. In the past few years, a couple of less-characterized NLR members are emerging as important signalling molecules with fundamental functions in host defence and inflammation. Among them, NLRP9 is an NLR originally proposed to be expressed and function solely in the reproductive system. Recent evidence has suggested that NLRP9 is also capable of initiating inflammasome formation in the intestine to restrict replication and damage brought by rotavirus infection. Here, we highlight the latest progress in characterization of the role of NLRP9 in infectious and inflammatory diseases, as well as the newest crystallographic and biochemical studies on NLRP9. Finally, we discuss some important questions remained to be answered regarding the molecular and cellular mechanisms governing NLRP9's function in innate immunity and inflammation.