Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide1. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling2–5, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Type I (IFN-α/β) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets of target cells coordinately regulate antiviral defenses against heterologous or homologous rotaviruses with substantially different effectiveness.

"Bulk" measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. −/− mice revealed that murine but not simian RRV mediated accumulation of IkB-α protein and decreased transcription of NF-κB-dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of "averaged" innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.innate immunity | interferon and antiviral response | single-cell analysis | NF-κB signaling | IRF3 signaling F ollowing virus infection, eukaryotic cells respond by the activation of innate immunity in a cell type-and strain-specific manner. The early host innate response includes activation of the transcription factors IRF3 and NF-κB, which induce transcription of discrete and overlapping sets of genes, including those encoding type I IFNs and viral stress-induced genes (vSIGs) (1, 2). Following IFN secretion and binding to cognate receptors, there is feed-forward amplification of expression of IFN and several hundred IFN-stimulated genes (ISGs), resulting in the establishment of an antiviral state. Such early responses to the presence of viral particles are spatially distinct from those that occur in adjacent bystander cells lacking direct exposure to virus. The early host innate immune responses to virus generally have been measured using averaged signals from "bulk" populations of cells or whole tissues. Such analyses cannot reveal hierarchical responses in individual infected or bystander cells. Here, we used a single-cell analytic strategy to examine the diversity in the early host antiviral innate immune response to rotavirus (RV) in suckling mice.RVs cause severe dehydrating diarrhea in the young of many mammalian species, resulting in more than 400,000 deaths of children annually (3). Homologous RV (RV derived from the infected host species) replicates efficiently within mature absorptive villous enterocytes of the small intestine but in the immune competent host, does not replicate efficiently in crypts or colon or at extraintestinal systemic locations (4). RV has evolved mechanisms to effectively block IRF3 and NF-κB-dependent IFN induction, as well as feedback-dependent amplification stages of the innate antiviral response (5). Specifically, the RV nonstructural protein NSP1 mediates degradation of IRF3 and/or NF-κB regulatory factor β-TrCP, depending on the viral strain and host cell involved (6, 7). In addi...

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-␣/␤ and IFN-␥ receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B-or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-␣/␤ receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-␥ receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.

Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb's directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.

Although rotavirus infection has generally been felt to be restricted to the gastrointestinal tract, over the last two decades there have been sporadic reports of children with acute or fatal cases of rotavirus gastroenteritis testing positive for rotavirus antigen and/or nucleic acid in various extraintestinal locations such as serum, liver, kidney, bladder, testes, nasal secretions, cerebrospinal fluid, and the central nervous system. Recently, studies in animals and people have demonstrated that rotavirus antigenemia is a common event during natural infection. In this study, we extend these observations and compare the intestinal and extraintestinal spread of wild-type homologous murine rotavirus EC and a heterologous strain, rhesus rotavirus (RRV), in newborn mice. A strand-specific quantitative reverse transcription-PCR (ssQRT-PCR) assay was used to quantify the ability of different rotavirus strains to spread and replicate extraintestinally. Both strain EC and RRV were detected extraintestinally in the mesenteric lymph nodes (MLN), livers, lungs, blood, and kidneys. Extraintestinal replication, as measured by ssQRT-PCR, was most prominent in the MLN and occurred to a lesser degree in the livers, kidneys, and lungs. In the MLN, strain EC and RRV had similar (P < 0.05) RNA copy numbers, although EC was present at a 10,000-fold excess over RRV in the small intestine. Rotavirus nonstructural protein 4 (NSP4) and/or assembled triple-layered particles, indicated by immunostaining with the VP7 conformation-dependent monoclonal antibody 159, were detected in the MLN, lungs, and livers of ECand RRV-inoculated mice, confirming the ssQRT-PCR findings. Infectious RRV was detected in the MLN in quantities exceeding the amount present in the small intestines or blood. The cells in the MLN that supported rotavirus replication included dendritic cells and potentially B cells and macrophages. These data indicate that extraintestinal spread and replication occurs commonly during homologous and some heterologous rotaviral infections; that the substantial host range restrictions for rhesus rotavirus, a heterologous strain present in the intestine, are not necessarily apparent at systemic sites; that the level and location of extraintestinal replication varies between strains; that replication can occur in several leukocytes subsets; and that extraintestinal replication is likely a part of the normal pathogenic sequence of homologous rotavirus infection.Rotavirus-associated gastroenteritis affects an estimated 111 million children worldwide and is responsible for at least 440,000 deaths annually (25). Although in some reports rotavirus antigen and/or RNA was detected in the central nervous systems, lungs, kidneys, spleens, heart, testes, bladders, and pancreases of selected severely ill children (19,20), homologous rotavirus replication has generally been considered to be restricted to the terminally differentiated epithelial cells of the small intestinal villi. The sporadically reported findings of extraintestinal virus have ...

Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1's ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation.Rotavirus (RV) causes severe dehydrating diarrhea in the young of many mammalian species, and in humans the virus is responsible for approximately 600,000 deaths annually. RV is a member of the Reoviridae family, and virus particles are nonenveloped and icosahedral and contain an 11-segmented double-stranded RNA (dsRNA) genome (13). As a consequence of high error rates of the viral RNA-dependent RNA polymerase and frequent gene reassortment, a large diversity of RV strains constantly circulates in nature. However, despite this diversity RVs are naturally replication restricted in heterologous host species, generally resulting in poor replication (compared to homologous species RV replication) and the inability to cause diarrhea at low dosage in heterologous hosts (8,14,15,23). This natural phenomenon of host range restriction formed the basis for development of several attenuated RV vaccine strains (1, 3, 18). Notably, the mechanistic basis for host range res...

Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell-sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.