RESUMEN
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.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Infecciones por Caliciviridae/inmunología , Colitis/inmunología , Proteína 58 DEAD Box/metabolismo , Intestinos/inmunología , Linfocitos Intraepiteliales/inmunología , Norovirus/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Infecciones por Caliciviridae/virología , Células Cultivadas , Colitis/inducido químicamente , Colitis/virología , Proteína 58 DEAD Box/genética , Sulfato de Dextran , Susceptibilidad a Enfermedades , Homeostasis , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Interleucina-15/metabolismo , Intestinos/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Simbiosis/inmunologíaRESUMEN
Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis1-3. Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction.
Asunto(s)
Muerte Celular , Norovirus , Nucleósido-Trifosfatasa , Proteínas Quinasas , Proteínas Virales , Animales , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Norovirus/enzimología , Norovirus/crecimiento & desarrollo , Norovirus/patogenicidad , Norovirus/fisiología , Proteínas Quinasas/química , Replicación Viral , Proteínas Virales/química , Proteínas Virales/metabolismo , Nucleósido-Trifosfatasa/química , Nucleósido-Trifosfatasa/metabolismo , Señales de Clasificación de Proteína , Cardiolipinas/metabolismo , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/prevención & control , Norovirus/inmunología , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/química , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , Línea Celular , Secuencia Conservada , Epítopos/química , Epítopos/inmunología , Humanos , Inmunoglobulina G/inmunología , Modelos Moleculares , Norovirus/clasificación , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/inmunología , VacunaciónRESUMEN
Enteric viruses like norovirus, rotavirus and astrovirus have long been accepted as spreading in the population through fecal-oral transmission: viruses are shed into feces from one host and enter the oral cavity of another, bypassing salivary glands (SGs) and reaching the intestines to replicate, be shed in feces and repeat the transmission cycle1. Yet there are viruses (for example, rabies) that infect the SGs2,3, making the oral cavity one site of replication and saliva one conduit of transmission. Here we report that enteric viruses productively and persistently infect SGs, reaching titres comparable to those in the intestines. We demonstrate that enteric viruses get released into the saliva, identifying a second route of viral transmission. This is particularly significant for infected infants, whose saliva directly transmits enteric viruses to their mothers' mammary glands through backflow during suckling. This sidesteps the conventional gut-mammary axis route4 and leads to a rapid surge in maternal milk secretory IgA antibodies5,6. Lastly, we show that SG-derived spheroids7 and cell lines8 can replicate and propagate enteric viruses, generating a scalable and manageable system of production. Collectively, our research uncovers a new transmission route for enteric viruses with implications for therapeutics, diagnostics and importantly sanitation measures to prevent spread through saliva.
Asunto(s)
Saliva , Glándulas Salivales , Virosis , Virus , Astroviridae , Lactancia Materna , Células Cultivadas , Heces/virología , Femenino , Humanos , Inmunoglobulina A/inmunología , Lactante , Norovirus , Rotavirus , Saliva/virología , Glándulas Salivales/virología , Esferoides Celulares/virología , Virosis/transmisión , Virosis/virología , Virus/crecimiento & desarrolloAsunto(s)
Norovirus , Organoides , Betacoronavirus , COVID-19 , Infecciones por Coronavirus , Humanos , Pandemias , Neumonía Viral , SARS-CoV-2 , Células MadreRESUMEN
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.
Asunto(s)
Infecciones por Caliciviridae , Interferones , Norovirus , Animales , Norovirus/fisiología , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/inmunología , Ratones , Interferones/metabolismo , Infección Persistente/virología , Infección Persistente/inmunología , Ratones Endogámicos C57BL , Mucosa Intestinal/virología , Mucosa Intestinal/inmunología , Gastroenteritis/virología , Replicación Viral , Ratones Noqueados , Inmunidad Innata , Esparcimiento de VirusRESUMEN
Viruses are obligate intracellular parasites that rely on host cell metabolism for successful replication. Thus, viruses rewire host cell pathways involved in central carbon metabolism to increase the availability of building blocks for successful propagation. However, the underlying mechanisms of virus-induced alterations to host metabolism are largely unknown. Noroviruses (NoVs) are highly prevalent pathogens that cause sporadic and epidemic viral gastroenteritis. In the present study, we uncovered several strain-specific and shared host cell metabolic requirements of three murine norovirus (MNV) strains, MNV-1, CR3, and CR6. While all three strains required glycolysis, glutaminolysis, and the pentose phosphate pathway for optimal infection of macrophages, only MNV-1 relied on host oxidative phosphorylation. Furthermore, the first metabolic flux analysis of NoV-infected cells revealed that both glycolysis and glutaminolysis are upregulated during MNV-1 infection of macrophages. Glutamine deprivation affected the viral lifecycle at the stage of genome replication, resulting in decreased non-structural and structural protein synthesis, viral assembly, and egress. Mechanistic studies further showed that MNV infection and overexpression of the non-structural protein NS1/2 increased the enzymatic activity of the rate-limiting enzyme glutaminase. In conclusion, the inaugural investigation of NoV-induced alterations to host glutaminolysis identified NS1/2 as the first viral molecule for RNA viruses that regulates glutaminolysis either directly or indirectly. This increases our fundamental understanding of virus-induced metabolic alterations and may lead to improvements in the cultivation of human NoVs.
Asunto(s)
Infecciones por Caliciviridae , Glutamina , Norovirus , Proteínas no Estructurales Virales , Replicación Viral , Norovirus/fisiología , Replicación Viral/fisiología , Ratones , Animales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Glutamina/metabolismo , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/metabolismo , Macrófagos/virología , Macrófagos/metabolismo , Humanos , Glutaminasa/metabolismo , Glucólisis/fisiología , Células RAW 264.7RESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Caliciviridae/inmunología , Diferenciación Celular/inmunología , Gastroenteritis/inmunología , Norovirus/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Infecciones por Caliciviridae/genética , Infecciones por Caliciviridae/virología , Diferenciación Celular/genética , Línea Celular , Microambiente Celular/genética , Microambiente Celular/inmunología , Gastroenteritis/genética , Gastroenteritis/virología , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Ratones Endogámicos C57BL , Norovirus/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructural protein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells in vitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.
Asunto(s)
Norovirus , Infecciones por Rotavirus , Rotavirus , Niño , Lactante , Humanos , Animales , Ratones , Preescolar , Rotavirus/genética , Anticuerpos Neutralizantes , Membrana Mucosa , Anticuerpos AntiviralesRESUMEN
Since the discovery of norovirus in 1972 as a cause of what was contemporarily known as acute infectious non-bacterial gastroenteritis, scientific understanding of the viral gastroenteritides has continued to evolve. It is now recognised that a small number of viruses are the predominant cause of acute gastroenteritis worldwide, in both high-income and low-income settings. Although treatment is still largely restricted to the replacement of fluid and electrolytes, improved diagnostics have allowed attribution of illness, enabling both targeted treatment of individual patients and prioritisation of interventions for populations worldwide. Questions remain regarding specific genetic and immunological factors underlying host susceptibility, and the optimal clinical management of patients who are susceptible to severe or prolonged manifestations of disease. Meanwhile, the worldwide implementation of rotavirus vaccines has led to substantial reductions in morbidity and mortality, and spurred interest in vaccine development to diminish the impact of the most prevalent viruses that are implicated in this syndrome.
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Infecciones por Enterovirus , Gastroenteritis , Norovirus , Procedimientos de Cirugía Plástica , Humanos , Gastroenteritis/terapia , RentaRESUMEN
Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics.IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans, indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.
Asunto(s)
Proteínas de la Cápside , Norovirus , Animales , Humanos , Ratones , ADN Complementario/genética , Genes Reporteros , Norovirus/genética , Plásmidos/genética , Vacunas Virales/metabolismo , Proteínas de la Cápside/metabolismoRESUMEN
RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.
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Norovirus , Tropismo Viral , Internalización del Virus , Animales , Humanos , Ratones , Infecciones por Caliciviridae/virología , Genoma Viral , Células HeLa , Especificidad del Huésped , Mutación , Norovirus/genética , Norovirus/fisiología , Receptores Virales/metabolismo , Receptores Virales/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Acoplamiento Viral , Replicación ViralRESUMEN
Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.
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Calcio , Magnesio , Norovirus , Animales , Ratones , Ácidos y Sales Biliares , Cápside/ultraestructura , Proteínas de la Cápside/química , Microscopía por Crioelectrón , Norovirus/química , Norovirus/ultraestructura , Calcio/química , Magnesio/químicaRESUMEN
Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.
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Norovirus , Humanos , Antivirales/farmacología , Infecciones por Caliciviridae/tratamiento farmacológico , Infecciones por Caliciviridae/virología , Gastroenteritis/tratamiento farmacológico , Gastroenteritis/virología , Intestinos/virología , Norovirus/efectos de los fármacos , Norovirus/fisiología , Animales , Organoides/efectos de los fármacos , Organoides/virología , Cultivo de VirusRESUMEN
Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.
Asunto(s)
Ácidos y Sales Biliares , Norovirus , Receptores de Esfingosina-1-Fosfato , Replicación Viral , Humanos , Norovirus/efectos de los fármacos , Norovirus/fisiología , Norovirus/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Ácidos y Sales Biliares/metabolismo , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/metabolismo , Piridinas/farmacología , Gastroenteritis/virología , Yeyuno/virología , Yeyuno/metabolismo , Organoides/virología , Organoides/metabolismo , PirazolesRESUMEN
Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Norovirus , Humanos , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X , Modelos Moleculares , Norovirus/inmunologíaRESUMEN
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.
Asunto(s)
Infecciones por Caliciviridae , Proteínas de la Cápside , Norovirus , Anticuerpos de Dominio Único , Norovirus/genética , Norovirus/efectos de los fármacos , Norovirus/inmunología , Humanos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/terapia , Antivirales/farmacología , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/química , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Cápside/metabolismo , Cápside/inmunología , Antígenos de Grupos Sanguíneos/metabolismo , Replicación Viral/efectos de los fármacos , Gastroenteritis/virología , Inmunoglobulina G/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunologíaRESUMEN
Recently, we identified the coxsackie and adenovirus receptor (CAR) as the entry receptor for rhesus enteric calicivirus (ReCV) isolate FT285 and demonstrated that co-expression of the CAR and the type B histo-blood group antigen (HBGA) is required to convert the resistant CHO cell line susceptible to infection. To address whether the CAR is also the functional entry receptor for other ReCV isolates and the requirement for specific HBGAs or other glycans, here we used a panel of recombinant CHO cell lines expressing the CAR and the type A, B, or H HBGAs alone or in combination. Infection studies with three diverse ReCV strains, the prototype GI.1 Tulane virus (TV), GI.2 ReCV-FT285, and GI.3 ReCV-FT7, identified that cell surface expression of the CAR is an absolute requirement for all three strains to promote susceptibility to infection, while the requirement for HBGAs varies among the strains. In addition to the CAR, ReCV-FT285 and TV require type A or B HBGAs for infection. In the absence of HBGAs, TV, but not Re-CV FT285, can also utilize sialic acids, while ReCV-FT7 infection is HBGA-independent and relies on CAR and sialic acid expression. In summary, we demonstrated strain-specific diversity of susceptibility requirements for ReCV infections and that CAR, type A and B HBGA, and sialic acid expression control susceptibility to infection with the three ReCV isolates studied. Our study also indicates that the correlation between in vitro HBGA binding and HBGAs required for infection is relatively high, but not absolute. This has direct implications for human noroviruses.IMPORTANCEHuman noroviruses (HuNoVs) are important enteric pathogens. The lack of a robust HuNoV cell culture system is a bottleneck for HuNoV cell culture-based studies. Often, cell culture-adapted caliciviruses that rapidly replicate in conventional cell lines and recapitulate biological features of HuNoVs are utilized as surrogates. Particularly, rhesus enteric caliciviruses (ReCVs) display remarkable similarities, including the primate host, clinical manifestation of gastroenteritis, genetic/antigenic diversity, and reliance on histo-blood group antigens (HBGAs) for attachment. While the HuNoV entry receptor(s) is unknown, the coxsackie and adenovirus receptor (CAR) has recently been identified as the ReCV entry receptor. Here, we identified the CAR, the type A and B HBGAs, and sialic acids as critical cell surface molecules controlling susceptibility to ReCV infections. The CAR is required for all ReCV isolates studied. However, the requirement for the different carbohydrate molecules varies among different ReCV strains. Our findings have direct implications for HuNoVs.
Asunto(s)
Infecciones por Caliciviridae , Caliciviridae , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Animales , Cricetinae , Humanos , Antígenos de Grupos Sanguíneos/metabolismo , Caliciviridae/fisiología , Infecciones por Caliciviridae/virología , Células CHO , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Intestino Delgado/virología , Ácido N-Acetilneuramínico/metabolismo , Norovirus/fisiologíaRESUMEN
It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFalpha and IFNgamma and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.
Asunto(s)
Proteínas Portadoras/genética , Enfermedad de Crohn/genética , Enfermedad de Crohn/virología , Predisposición Genética a la Enfermedad , Íleon/patología , Norovirus , Animales , Proteínas Relacionadas con la Autofagia , Enfermedad de Crohn/patología , Perfilación de la Expresión Génica , Humanos , Interferón gamma/metabolismo , Ratones , Células de Paneth/metabolismo , Células de Paneth/virología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: This study compared trends in norovirus cases to determine whether chief complaint-based emergency department (ED) visit data could reflect trends of norovirus in Korea. METHODS: The ED visits from the National Emergency Department Information System database and the weekly reported number of noroviruses from the sentinel surveillance system were collected between August 2017 and December 2020. The correlation between weekly norovirus cases and weekly ED visits considering the chief complaint and discharge diagnosis code was estimated using a 3-week moving average. RESULTS: In total, 6 399 774 patients with chief complaints related to digestive system disease visited an ED. A higher correlation between reported norovirus cases and ED visit with chief complaint of vomiting and discharge diagnosis code of gastroenteritis and colitis of unspecified origin or other and unspecified gastroenteritis and colitis of infectious origin was observed (R = 0.88, P < .0001). The correlation was highest for the age group 0-4 years (R = 0.89, P < .0001). However, no correlation was observed between the reported norovirus cases and the number of ED visits with norovirus identified as a discharge diagnosis code. CONCLUSIONS: ED visit data considering a combination of chief complaints and discharged diagnosis code would be useful for early detection of infectious disease trends.