Commensal viruses maintain intestinal intraepithelial lymphocytes via noncanonical RIG-I signaling.

Much attention has focused on commensal bacteria in health and disease, but the role of commensal viruses is understudied. Although metagenomic analysis shows that the intestine of healthy humans and animals harbors various commensal viruses and the dysbiosis of these viruses can be associated with inflammatory diseases, there is still a lack of causal data and underlying mechanisms to understand the physiological role of commensal viruses in intestinal homeostasis. In the present study, we show that commensal viruses are essential for the homeostasis of intestinal intraepithelial lymphocytes (IELs). Mechanistically, the cytosolic viral RNA-sensing receptor RIG-I in antigen-presenting cells can recognize commensal viruses and maintain IELs via a type I interferon-independent, but MAVS-IRF1-IL-15 axis-dependent, manner. The recovery of IELs by interleukin-15 administration reverses the susceptibility of commensal virus-depleted mice to dextran sulfate sodium-induced colitis. Collectively, our results indicate that commensal viruses maintain the IELs and consequently sustain intestinal homeostasis via noncanonical RIG-I signaling.

Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination.

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.

Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Differentiation and Protective Capacity of Virus-Specific CD8+ T Cells Suggest Murine Norovirus Persistence in an Immune-Privileged Enteric Niche.

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

Analysis of Archival Sera From Norovirus-Infected Individuals Demonstrates That Cross-Blocking of Emerging Viruses Is Genotype-Specific.

BACKGROUND: Rapidly evolving RNA viruses, such as human norovirus, generate extraordinary sequence diversity, posing a significant challenge to vaccine design. This diversity, coupled with short-lasting natural immunity, leads to reinfection throughout one's lifetime. How reexposure shapes humoral immunity to future norovirus strains remains incompletely understood. METHODS: We profiled the antibody responses following 2 community gastroenteritis outbreaks with GII.2 and GII.6 noroviruses in 1971. Using diverse virus-like particles (VLPs), enzyme-linked immunosorbent assay (ELISA), and carbohydrate-blocking assays (surrogate for neutralization), we examined the antibody response at acute and convalescent timepoints following GII.6 infection. RESULTS: Convalescent sera displayed strong homologous blocking, demonstrating a 5-fold increase in GII.6 carbohydrate blockade over acute samples, and broad blocking of diverse archival and modern GII.6 noroviruses. Convalescent sera displayed limited carbohydrate blocking of heterotypic VLPs, despite high ELISA binding titers. Select individuals developed broad cross-genotype blockade, but this response was established before the second outbreak. Finally, we applied a novel competitive carbohydrate-blocking assay to demonstrate the epitope specificity and discrete compartments of the neutralizing response. CONCLUSIONS: Our data show that infection generates narrow, focused immunity directed toward the infecting genotype. We did detect broad cross-blocking in specific individuals, but these responses could be attributed to diverse, genotype-specific antibodies predating GII.6 infection.

N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

Novel stress granule-like structures are induced via a paracrine mechanism during viral infection.

To rapidly adapt to stresses such as infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs with RNA-binding proteins and signalling components into cytoplasmic biocondensates called stress granules (SGs). Increasing evidence suggests that SGs contribute to antiviral defence, and thus viruses need to evade these responses to propagate. We previously showed that feline calicivirus (FCV) impairs SG assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-positive granules, suggesting a paracrine response triggered by infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of SG-like foci, which we name paracrine granules. They are linked to antiviral activity and exhibit specific kinetics of assembly-disassembly, and protein and RNA composition that are different from canonical SGs. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.

Chimeric virus-like particles of human norovirus constructed by structure-guided epitope grafting elicit cross-reactive immunity against both GI.1 and GII.4 genotypes.

IMPORTANCE: Human norovirus (HuNoV) is highly infectious and can result in severe illnesses in the elderly and children. So far, there is no effective antiviral drug to treat HuNoV infection, and thus, the development of HuNoV vaccines is urgent. However, NoV evolves rapidly, and currently, at least 10 genogroups with numerous genotypes have been found. The genetic diversity of NoV and the lack of cross-protection between different genotypes pose challenges to the development of broadly protective vaccines. In this study, guided by structural alignment between GI.1 and GII.4 HuNoV VP1 proteins, several chimeric-type virus-like particles (VLPs) were designed through surface-exposed loop grafting. Mouse immunization studies show that two of the designed chimeric VLPs induced cross-immunity against both GI.1 and GII.4 HuNoVs. To our knowledge, this is the first designed chimeric VLPs that can induce cross-immune activities across different genogroups of HuNoV, which provides valuable strategies for the development of cross-reactive HuNoV vaccines.

Identification of a norovirus GII-specific antigenic epitope.

Noroviruses (NoVs) are the chief cause of acute viral gastroenteritis worldwide. By employing the major capsid protein VP1 of a GII.6 NoV strain as an immunogen, we generated two monoclonal antibodies (mAbs) with wide-spectrum binding activities against NoV genogroup II (GII) VP1 proteins. One mAb (10G7) could bind to native and denatured GII-specific VP1 proteins. The other mAb (10F2) could bind to all tested native GII VP1 proteins, but not to denatured GII.3, GII.4, GII.7, or GII.17 VP1 proteins. Using GII.6/GII.4 fusion proteins, the mAb 10F2 binding region was confirmed to be located in the C-terminal P1 domain. An enzyme-linked immunosorbent assay based on peptides covering the P domain did not detect any binding. Using a panel of VP1 proteins with swapped regions, deletions, and mutations, the mAb 10F2 binding region was determined to be located between residues 496 and 513. However, the residue(s) responsible for its varied binding affinity for different denatured GII VP1 proteins remain to be identified. In summary, two NoV GII-specific cross-reactive mAbs were generated, and their binding regions were determined. Our results might facilitate the detection and immunogenic study of NoVs.

Development of genogroup-specific ELISAs based on the VP1 protein to detect antibodies to GIV and GVI feline norovirus.

Feline norovirus (FNoV) is a potential pathogen of feline gastroenteritis and has two genogroups (GIV and GVI). Few epidemiological studies have been conducted on FNoV. We designed two enzyme-linked immunosorbent assays (ELISAs) to identify genogroup-specific FNoV antibodies for serological surveillance. Analysis of sera from cats experimentally infected with FNoV GIV or GVI and from specific-pathogen-free (SPF) cats confirmed that the two recombinant proteins used in the assay react in a genogroup-specific manner. Of the 183 samples tested, 6.6% were positive for GIV and 26.2% were positive for GVI. Antibodies to both FNoV genogroups were detected in sera collected in 2005, seven years before FNoV was first reported.

Characterisation of Lagovirus europaeus GI-RHDVs (Rabbit Haemorrhagic Disease Viruses) in Terms of Their Pathogenicity and Immunogenicity.

Rabbit haemorrhagic disease viruses (RHDV) belong to the family Caliciviridae, genus Lagovirus europaeus, genogroup GI, comprising four genotypes GI.1-GI.4, of which the genotypes GI.1 and GI.2 are pathogenic RHD viruses, while the genotypes GI.3 and GI.4 are non-pathogenic RCV (Rabbit calicivirus) viruses. Among the pathogenic genotypes GI.1 and GI.2 of RHD viruses, an antigenic variant of RHDV, named RHDVa-now GI.1a-RHDVa, was distinguished in 1996; and in 2010, a variant of RHDV-named RHDVb, later RHDV2 and now GI.2-RHDV2/b-was described; and recombinants of these viruses were registered. Pathogenic viruses of the genotype GI.1 were the cause of a disease described in 1984 in China in domestic (Oryctolagus (O.) cuniculus domesticus) and wild (O. cuniculus) rabbits, characterised by a very rapid course and a mortality rate of 90-100%, which spread in countries all over the world and which has been defined since 1989 as rabbit haemorrhagic disease. It is now accepted that GI.1-RHDV, including GI.1a-RHDVa, cause the predetermined primary haemorrhagic disease in domestic and wild rabbits, while GI.2-RHDV2/b cause it not only in rabbits, including domestic rabbits' young up to 4 weeks and rabbits immunised with rabbit haemorrhagic disease vaccine, but also in five various species of wild rabbits and seven different species of hares, as well as wild ruminants: mountain muskoxen and European badger. Among these viruses, haemagglutination-positive, doubtful and harmful viruses have been recorded and described and have been shown to form phylogenogroups, immunotypes, haematotypes and pathotypes, which, together with traits that alter and expand their infectious spectrum (rabbit, hare, wild ruminant, badger and various rabbit and hare species), are the determinants of their pathogenicity (infectivity) and immunogenicity and thus shape their virulence. These relationships are the aim of our consideration in this article.

MicroRNAs Regulate the Expression of Genes Related to the Innate Immune and Inflammatory Response in Rabbits Infected with Lagovirus europaeus GI.1 and GI.2 Genotypes.

MicroRNAs (miR) are a group of small, non-coding RNAs of 17-25 nucleotides that regulate gene expression at the post-transcriptional level. Dysregulation of miRNA expression or function may contribute to abnormal gene expression and signaling pathways, leading to disease pathology. Lagovirus europaeus (L. europaeus) causes severe disease in rabbits called rabbit hemorrhagic disease (RHD). The symptoms of liver, lung, kidney, and spleen degeneration observed during RHD are similar to those of acute liver failure (ALF) and multi-organ failure (MOF) in humans. In this study, we assessed the expression of miRs and their target genes involved in the innate immune and inflammatory response. Also, we assessed their potential impact on pathways in L. europaeus infection-two genotypes (GI.1 and GI.2)-in the liver, lungs, kidneys, and spleen. The expression of miRs and target genes was determined using quantitative real-time PCR (qPCR). We assessed the expression of miR-155 (MyD88, TAB2, p65, NLRP3), miR-146a (IRAK1, TRAF6), miR-223 (TLR4, IKKα, NLRP3), and miR-125b (MyD88). We also examined biomarkers of inflammation: IL-1ß, IL-6, TNF-α, and IL-18 in four tissues at the mRNA level. Our study shows that the main regulators of the innate immune and inflammatory response in L. europaeus/GI.1 and GI.2 infection, as well as RHD, are miR-155, miR-223, and miR-146a. During infection with L. europaeus/RHD, miR-155 has both pro- and anti-inflammatory effects in the liver and anti-inflammatory effects in the kidneys and spleen; miR-146a has anti-inflammatory effects in the liver, lungs and kidneys; miR-223 has anti-inflammatory effects in all tissues; however, miR-125b has anti-inflammatory effects only in the liver. In each case, such an effect may be a determinant of the pathogenesis of RHD. Our research shows that miRs may regulate three innate immune and inflammatory response pathways in L. europaeus infection. However, the result of this regulation may be influenced by the tissue microenvironment. Our research shows that infection of rabbits with L. europaeus/GI.1 and GI.2 genotypes causes an overexpression of two critical acute phase cytokines: IL-6 in all examined tissues and TNF-α (in the liver, lungs, and spleen). IL-1ß was highly expressed only in the lungs after L. europaeus infection. These facts indicate a strong and rapid involvement of the local innate immune and inflammatory response in L. europaeus infection-two genotypes (GI.1 and GI.2)-and in the pathogenesis of RHD. Profile of biomarkers of inflammation in rabbits infected with L. europaeus/GI.1 and GI.2 genotypes are similar regarding the nature of changes but are different for individual tissues. Therefore, we propose three inflammation profiles for L. europaeus infection for both GI.1 and GI.2 genotypes (pulmonary, renal, liver, and spleen).

Interferon-Driven Immune Dysregulation in Common Variable Immunodeficiency-Associated Villous Atrophy and Norovirus Infection.

PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.

Akt Plays Differential Roles during the Life Cycles of Acute and Persistent Murine Norovirus Strains in Macrophages.

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.

Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids.

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2 An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.

Advances in understanding of the innate immune response to human norovirus infection using organoid models.

Norovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and the most frequent cause of foodborne illness in the United States. There is no specific treatment for norovirus infections and therapeutic interventions are based on alleviating symptoms and limiting viral transmission. The immune response to norovirus is not completely understood and mechanistic studies have been hindered by lack of a robust cell culture system. In recent years, the human intestinal enteroid/human intestinal organoid system (HIE/HIO) has enabled successful human norovirus replication. Cells derived from HIE have also successfully been subjected to genetic manipulation using viral vectors as well as CRISPR/Cas9 technology, thereby allowing studies to identify antiviral signaling pathways important in controlling norovirus infection. RNA sequencing using HIE cells has been used to investigate the transcriptional landscape during norovirus infection and to identify antiviral genes important in infection. Other cell culture platforms such as the microfluidics-based gut-on-chip technology in combination with the HIE/HIO system also have the potential to address fundamental questions on innate immunity to human norovirus. In this review, we highlight the recent advances in understanding the innate immune response to human norovirus infections in the HIE system, including the application of advanced molecular technologies that have become available in recent years such as the CRISPR/Cas9 and RNA sequencing, as well as the potential application of single cell transcriptomics, viral proteomics, and gut-on-a-chip technology to further elucidate innate immunity to norovirus.