Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 460
Filtrar
1.
Arch Virol ; 165(11): 2605-2613, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32770483

RESUMO

Noroviruses are the main causative agents of acute viral gastroenteritis worldwide. However, no vaccine or specific antiviral treatment is available, imposing a heavy global health burden. The nucleoside analogue 2'-fluoro-2'-deoxycytidine (2'-FdC) has been reported to have broad antiviral activity. Here, we report that 2'-FdC significantly inhibits murine norovirus replication in macrophages. This effect was partially reversed by exogenous supplementation of cytidine triphosphate. The combination of 2'-FdC with mycophenolic acid, ribavirin or favipiravir (T705) exerts synergistic antiviral effects. These results indicate that 2'-FdC is a potential candidate for antiviral drug development against norovirus infection.


Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Norovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Amidas/farmacologia , Animais , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/virologia , Linhagem Celular , Desoxicitidina/farmacologia , Humanos , Camundongos , Ácido Micofenólico/farmacologia , Norovirus/fisiologia , Pirazinas/farmacologia , Células RAW 264.7 , Ribavirina/farmacologia , Replicação Viral/fisiologia
2.
Int J Food Microbiol ; 334: 108809, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32799118

RESUMO

The current popularity of minimally processed foods is an opportunity for natural antimicrobial agents to be combined with mild heat treatments to act synergistically in reducing viral foodborne pathogens. Viral inactivation by heat-treatments (at 25, 40, 50 and 63 °C for 30 min) combined with aged green tea extract (aged-GTE) was initially evaluated in phosphate buffered saline (PBS) against murine norovirus (MNV-1) and hepatitis A virus (HAV) by cell culture, and against human norovirus by in situ capture RT-qPCR. The combination of aged-GTE and heat treatment at 50 °C for 30 min exerted strong antiviral activity, reducing by more than 5 log MNV-1 infectivity in PBS. Heating at 40 °C for 30 min reduced the binding of norovirus to porcine gastric mucine (PGM) to 41.5% and the addition of aged-GTE further decreased the binding to 4.7%. Additionally, the reduction of MNV-1 and HAV infectivity was investigated in two different types of juices exposed to mild heat treatments alone, and combined with aged-GTE. The addition of aged-GTE increased to more than 4 log the inactivation of MNV-1 in juices exposed to 50 °C for 30 min. However, this synergistic effect of aged-GTE combined with heat treatments was not observed for HAV in any of the juices. Aged-GTE, then, could be considered as an additional control measure to improve the food safety of mild heat pasteurized juices.


Assuntos
Sucos de Frutas e Vegetais/virologia , Temperatura Alta , Pasteurização/métodos , Chá/química , Inativação de Vírus , Animais , Antivirais/farmacologia , Linhagem Celular , Doenças Transmitidas por Alimentos/prevenção & controle , Doenças Transmitidas por Alimentos/virologia , Vírus da Hepatite A/fisiologia , Humanos , Camundongos , Norovirus/fisiologia , Extratos Vegetais/farmacologia , Especificidade da Espécie , Suínos , Inativação de Vírus/efeitos dos fármacos
3.
PLoS One ; 15(8): e0237888, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813716

RESUMO

Norovirus, the leading cause of non-bacterial food poisoning, is responsible for several outbreaks associated with bivalves and ready-to-eat food products worldwide. As norovirus is resistant to alcohol, which is commonly used in food manufacturing processes, sodium hypochlorite is used for its inactivation. However, sodium hypochlorite has two disadvantages: it cannot be added to foods, and its effect is significantly reduced in the presence of organic compounds. Thus, a novel disinfectant against norovirus is urgently required for food hygiene. Thermally denatured egg white lysozyme inactivates norovirus; however, the optimal inactivating conditions and the underlying mechanism are unclear. In the present study, the inactivating mechanism of heat-denatured lysozyme against norovirus was analyzed using murine norovirus strain 1 (MNV-1). We found that the inactivating effect was enhanced by adjusting the pH of the lysozyme solution before thermal denaturation to 6.5 or higher. The reaction of heat-denatured lysozyme and MNV-1 was irreversible, and norovirus was completely inactivated after exposure to heat-denatured lysozyme. Furthermore, it was found that lysozyme residues 5-39 contributed to the norovirus-inactivating effect. Notably, the hydrophobicity and positive charges in this region contributed to the norovirus-inactivating effect, as evidenced by the norovirus inactivation test using mutated residues 5-39. These findings are novel and highlight the possible application of heat-denatured lysozyme as a disinfectant against norovirus in a wide range of food processes.


Assuntos
Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Muramidase/metabolismo , Norovirus/fisiologia , Desnaturação Proteica , Inativação de Vírus , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Macrófagos/virologia , Camundongos , Muramidase/química , Peptídeos/química , Domínios Proteicos , Células RAW 264.7
4.
Int J Food Microbiol ; 330: 108695, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-32502761

RESUMO

Norovirus (NoV) is the leading cause of epidemic and sporadic gastroenteritis worldwide; a high number of those cases are attributed to the consumption of contaminated food. Crop producers have used several strategies to inactivate the virus present in these products and thus stop the NoV transmission chain. Physical methods such as gamma radiation show excellent results in the inactivation of bacteria, but its effect on NoV has been little studied. This study aimed to evaluate the effect of gamma radiation for NoV inactivation, and over the surface topographic characteristics of strawberry cells, as a prototype of soft fruit. A 10% suspension of GII norovirus-positive stool samples were treated with either 200 mg/L of sodium hypochlorite (NaClO) or gamma-irradiated at doses of 5, 10, 15 and 20 kilograys (kGy). Viral inactivation was determined by measuring the integrity of viral capsid using RNase A alone or in combination with proteinase K followed by RT-qPCR. The effect over cellular surface topology characteristics of the fruit was measured by atomic force microscopy (AFM) and confocal microscopy. High doses of radiation (20 kGy) were necessary to detect a significant (p < 0.05) decrease of up to 1.26 log10 viral copy number. This dose significantly (p < 0.05) raises the root means square roughness (Rq), which affects directly the quality and texture of the product. The gamma irradiation doses tested in this study were not enough to inactivate NoV. The allowed gamma irradiation doses for fresh produce does not alter the surface topology of the fruit, but they affect the content of fluorescent compounds, responsible for the antioxidant activity of the fruit.


Assuntos
Fragaria/efeitos da radiação , Fragaria/virologia , Raios gama , Norovirus/efeitos da radiação , Inativação de Vírus/efeitos da radiação , Relação Dose-Resposta à Radiação , Microbiologia de Alimentos , Fragaria/efeitos dos fármacos , Frutas/efeitos dos fármacos , Frutas/efeitos da radiação , Frutas/virologia , Norovirus/fisiologia , Hipoclorito de Sódio/farmacologia , Inativação de Vírus/efeitos dos fármacos
5.
Nat Commun ; 11(1): 2759, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488028

RESUMO

Human noroviruses are a major cause of diarrheal illness, but pathogenesis is poorly understood. Here, we investigate the cellular tropism of norovirus in specimens from four immunocompromised patients. Abundant norovirus antigen and RNA are detected throughout the small intestinal tract in jejunal and ileal tissue from one pediatric intestinal transplant recipient with severe gastroenteritis. Negative-sense viral RNA, a marker of active viral replication, is found predominantly in intestinal epithelial cells, with chromogranin A-positive enteroendocrine cells (EECs) identified as a permissive cell type in this patient. These findings are consistent with the detection of norovirus-positive EECs in the other three immunocompromised patients. Investigation of the signaling pathways induced in EECs that mediate communication between the gut and brain may clarify mechanisms of pathogenesis and lead to the development of in vitro model systems in which to evaluate norovirus vaccines and treatment.


Assuntos
Células Enteroendócrinas/imunologia , Células Epiteliais/imunologia , Norovirus/fisiologia , Doença Aguda , District of Columbia , Células Enteroendócrinas/metabolismo , Gastroenterite/virologia , Genótipo , Humanos , Intestino Delgado/patologia , Intestino Delgado/virologia , Norovirus/genética , RNA Viral , Replicação Viral
6.
J Vis Exp ; (158)2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32420984

RESUMO

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.


Assuntos
Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Interações Microbianas , Norovirus/fisiologia , Ligação Viral , Anticorpos Antivirais/fisiologia , Citometria de Fluxo
7.
J Biotechnol ; 318: 31-38, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32387450

RESUMO

Norovirus infections cause severe gastroenteritis in millions of people every year. Infection requires the recognition of histo-blood group antigens (HBGAs), but such interactions can be inhibited by human milk oligosaccharides (HMOs), which act as structurally-similar decoys. HMO supplements could help to prevent norovirus infections, but the industrial production of complex HMOs is challenging. Here we describe a large-scale fermentation process that yields several kilograms of lacto-N-fucopentaose I (LNFP I). The product was synthesized in Escherichia coli BL21(DE3) cells expressing a recombinant N-acetylglucosaminyltransferase, ß(1,3)galactosyltransferase and α(1,2)fucosyltransferase. Subsequent in vitro enzymatic conversion produced HBGA types A1 and B1 for norovirus inhibition assays. These carbohydrates inhibited the binding of GII.17 virus-like particles (VLPs) to type A1 and B1 trisaccharides more efficiently than simpler fucosylated HMOs, which were in turn more effective than any non-fucosylated structures. However, we found that the simpler fucosylated HMOs were more effective than complex molecules such as LNFP I when inhibiting the binding of GII.17 and GII.4 VLPs to human gastric mucins and mucins from human amniotic fluid. Our results show that complex fucosylated HMOs can be produced by large-scale fermentation and that a combination of simple and complex fucosylated structures is more likely to prevent norovirus infections.


Assuntos
Norovirus/efeitos dos fármacos , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Receptores Virais/metabolismo , Biotecnologia , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Antígenos de Grupos Sanguíneos/farmacologia , Fermentação , Humanos , Concentração Inibidora 50 , Leite Humano/química , Mucinas/metabolismo , Norovirus/fisiologia , Oligossacarídeos/química , Trissacarídeos/metabolismo
8.
Virology ; 546: 109-121, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452409

RESUMO

The inflammasome machinery has recently been recognized as an emerging pillar of innate immunity. However, little is known regarding the interaction between the classical interferon (IFN) response and inflammasome activation in response to norovirus infection. We found that murine norovirus (MNV-1) infection induces the transcription of IL-1ß, a hallmark of inflammasome activation, which is further increased by inhibition of IFN response, but fails to trigger the release of mature IL-1ß. Interestingly, pharmacological inflammasome inhibitors do not affect viral replication, but slightly reverse the inflammasome activator lipopolysaccharide (LPS)-mediated inhibition of MNV replication. LPS efficiently stimulates the transcription of IFN-ß through NF-ĸB, which requires the transcription factors IRF3 and IRF7. This activates downstream antiviral IFN-stimulated genes (ISGs) via the JAK-STAT pathway. Moreover, inhibition of NF-ĸB and JAK-STAT signaling partially reverse LPS-mediated anti-MNV activity, suggesting additional antiviral mechanisms activated by NF-ĸB. This study reveals additional insight in host defense against MNV infection.


Assuntos
Antivirais/farmacologia , Infecções por Caliciviridae/imunologia , Janus Quinases/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , NF-kappa B/imunologia , Norovirus/efeitos dos fármacos , Fatores de Transcrição STAT/imunologia , Animais , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/virologia , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Janus Quinases/genética , Macrófagos/virologia , Camundongos , NF-kappa B/genética , Norovirus/genética , Norovirus/fisiologia , Fatores de Transcrição STAT/genética , Transdução de Sinais/efeitos dos fármacos
9.
Food Microbiol ; 90: 103461, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32336357

RESUMO

Human noroviruses (HNoV) and hepatitis A virus (HAV) are predominantly linked to foodborne outbreaks worldwide. As cell-culture systems to propagate HNoV in laboratories are not easily available, Tulane virus (TV) is used as a cultivable HNoV surrogate to determine inactivation. Heat-sensitization of HAV and TV by "generally recognized as safe'' (GRAS) substances can potentially reduce their time-temperature inactivation parameters during processing to ensure food safety. Curcumin, gingerol (from ginger), and grape seed extract (GSE) reportedly have anti-inflammatory, immune-modulating and antiviral properties. The objective of this study was to determine and compare the D-values and z-values of HAV and TV at 52-68 °C with or without curcumin (0.015 mg/ml), gingerol (0.1 mg/ml), or GSE (1 mg/ml) in 2-ml glass vials. HAV at ~7 log PFU/ml and TV at ~6 log PFU/ml were diluted in phosphate buffered saline (PBS) and added to two sets of six 2-mL sterile glass vials. One set served as the control and the second set had the three extracts individually added for thermal treatments in a circulating water bath for 0-10 min. The D-values for TV in PBS ranged from 4.55 ± 0.28 to 1.08 ± 0.16 min, and for HAV in PBS ranged from to 9.21 ± 0.24 to 0.67 ± 0.19 min at 52-68 °C. Decreased D-values (52-58 °C) for TV with curcumin ranging from 4.32 ± 0.25 to 0.62 ± 0.17 min, gingerol from 4.09 ± 0.18 to 0.72 ± 0.09 min and GSE from 3.82 ± 0.18 to 0.80 ± 0.07 min, with similar trends for HAV were observed. The linear model showed significant differences (p < 0.05) between the D-values of HAV and TV with and without plant extracts for most tested temperatures. This suggests that GRAS substances can potentially lower temperature and time regimens needed to inactivate HAV and TV.


Assuntos
Antivirais/farmacologia , Microbiologia de Alimentos/métodos , Vírus da Hepatite A/efeitos dos fármacos , Temperatura Alta , Norovirus/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Catecóis/farmacologia , Curcumina/farmacologia , Álcoois Graxos/farmacologia , Extrato de Sementes de Uva/farmacologia , Vírus da Hepatite A/fisiologia , Norovirus/fisiologia
10.
Appl Environ Microbiol ; 86(12)2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32303551

RESUMO

Noroviruses (NoV) are responsible for many shellfish outbreaks. Purification processes may be applied to oysters before marketing to decrease potential fecal pollution. This step is rapidly highly effective in reducing Escherichia coli; nevertheless, the elimination of virus genomes has been described to be much slower. It is therefore important to identify (i) the purification conditions that optimize virus removal and (ii) the mechanism involved. To this end, the effects of oyster stress, nutrients, and the presence of a potential competitor to NoV adhesion during purification were investigated using naturally contaminated oysters. Concentrations of NoV (genomes) and of the viral indicator F-specific RNA bacteriophage (FRNAPH; genomes and infectious particles) were regularly monitored. No significant differences were observed under the test conditions. The decrease kinetics of both virus genomes were similar, again showing the potential of FRNAPH as an indicator of NoV behavior during purification. The T90 (time to reduce 90% of the initial titer) values were 47.8 days for the genogroup I NoV genome, 26.7 days for the genogroup II NoV genome, and 43.9 days for the FRNAPH-II genome. Conversely, monitoring of the viral genomes could not be used to determine the behavior of infectious viruses because the T90 values were more than two times lower for infectious FRNAPH (20.6 days) compared to their genomes (43.9 days). Finally, this study highlighted that viruses are primarily inactivated in oysters rather than released in the water during purification processes.IMPORTANCE This study provides new data about the behavior of viruses in oysters under purification processes and about their elimination mechanism. First, a high correlation has been observed between F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and norovirus (NoV) in oysters impacted by fecal contamination when both are detected using molecular approaches. Second, when using reverse transcription-quantitative PCR and culture to detect FRNAPH-II genomes and infectious FRNAPH in oysters, respectively, it appears that genome detection provides limited information about the presence of infectious particles. The comparison of both genomes and infectious particles highlights that the main mechanism of virus elimination in oysters is inactivation. Finally, this study shows that none of the conditions tested modify virus removal.


Assuntos
Crassostrea/virologia , Fagos RNA/fisiologia , Inativação de Vírus , Eliminação de Partículas Virais , Animais , Ácido Cítrico/análise , Norovirus/fisiologia , Nutrientes/análise , Estresse Fisiológico
11.
J Food Prot ; 83(4): 661-667, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32221571

RESUMO

Noroviruses encounter numerous and diverse bacterial populations in the host and environment, but the impact of bacteria on norovirus transmission, infection, detection, and inactivation are not well understood. Tulane virus (TV), a human norovirus surrogate, was exposed to viable bacteria, bacterial metabolic products, and bacterial cell constituents and was evaluated for impact on viral recovery, propagation, and inactivation resistance, respectively. TV was incubated with common soil, intestinal, skin, and phyllosphere bacteria, and unbound viruses were recovered by centrifugation and filtration. TV recovery from various bacterial suspensions was not impeded, which suggests a lack of direct, stable binding between viruses and bacteria. The cell-free supernatant (CFS) of Bifidobacterium bifidum 35914, a bacterium that produces glycan-modifying enzymes, was evaluated for effect on the propagation of TV in LLC-MK2 cells. CFS did not limit TV propagation relative to TV absent of CFS. The impact of Escherichia coli O111:B4 lipopolysaccharide (LPS) and Bacillus subtilis peptidoglycan (PEP) on TV thermal and chlorine inactivation resistance was evaluated. PEP increased TV thermal and chlorine inactivation resistance compared with control TV in phosphate-buffered saline (PBS). TV suspended in PBS and LPS was reduced by more than 3.7 log at 60°C, whereas in PEP, TV reduction was approximately 2 log. Chlorine treatment (200 ppm) rendered TV undetectable (>3-log reduction) in PBS and LPS; however, TV was still detected in PEP, reduced by 2.9 log. Virus inactivation studies and food processing practices should account for potential impact of bacteria on viral resistance.


Assuntos
Norovirus , Fenômenos Fisiológicos Bacterianos , Microbiologia de Alimentos , Humanos , Norovirus/patogenicidade , Norovirus/fisiologia , Inativação de Vírus , Vírus
12.
Rev Epidemiol Sante Publique ; 68(2): 99-107, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32037129

RESUMO

BACKGROUND: Concern about health misinformation is longstanding, especially on the Internet. METHODS: Using agent-based models, we considered the effects of such misinformation on a norovirus outbreak, and some methods for countering the possible impacts of "good" and "bad" health advice. The work explicitly models spread of physical disease and information (both online and offline) as two separate but interacting processes. The models have multiple stochastic elements; repeat model runs were made to identify parameter values that most consistently produced the desired target baseline scenario. Next, parameters were found that most consistently led to a scenario when outbreak severity was clearly made worse by circulating poor quality disease prevention advice. Strategies to counter "fake" health news were tested. RESULTS: Reducing bad advice to 30% of total information or making at least 30% of people fully resistant to believing in and sharing bad health advice were effective thresholds to counteract the negative impacts of bad advice during a norovirus outbreak. CONCLUSION: How feasible it is to achieve these targets within communication networks (online and offline) should be explored.


Assuntos
Infecções por Caliciviridae/epidemiologia , Comunicação , Surtos de Doenças , Letramento em Saúde , Internet , Norovirus/fisiologia , Análise de Sistemas , Acesso à Informação , Infecções por Caliciviridae/transmissão , Infecções por Caliciviridae/virologia , Informação de Saúde ao Consumidor/organização & administração , Informação de Saúde ao Consumidor/normas , Informação de Saúde ao Consumidor/estatística & dados numéricos , Letramento em Saúde/organização & administração , Letramento em Saúde/normas , Letramento em Saúde/estatística & dados numéricos , Humanos , Disseminação de Informação , Serviços de Informação/organização & administração , Serviços de Informação/normas , Registros Públicos de Dados de Cuidados de Saúde
13.
PLoS Pathog ; 16(1): e1008250, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905230

RESUMO

Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2α signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2α signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome.


Assuntos
Infecções por Caliciviridae/virologia , DNA Helicases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Norovirus/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Animais , Infecções por Caliciviridae/genética , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , RNA/metabolismo , Transdução de Sinais , Replicação Viral
14.
J Agric Food Chem ; 68(5): 1207-1212, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31755264

RESUMO

Human noroviruses (HuNoVs) are among the main pathogens causing acute nonbacterial gastroenteritis. Histo-blood group antigens (HBGAs) are widely accepted receptors for HuNoV specific binding. HBGA-like substances in produce are also considered as the critical ligands for capture of HuNoVs. However, the composition of viral ligands from food substrates remains unknown. In this study, an oligosaccharide (H2N2F2) was captured and isolated from romaine lettuce extract by a bacterial surface display system. Using electrospray ionization mass spectrometry and tandem mass spectrometry, it was shown that H2N2F2 was most likely to be a chimera of type A, H, and Lewis a HBGAs. The composition was consistent with our ELISA results using a panel of monoclonal antibodies against HBGAs. Our results revealed a possible interaction mechanism between HuNoVs and romaine lettuce. Better understanding of the interaction of HuNoVs with easily contaminated produce will ultimately aid in the control of and reduction in disease outbreaks.


Assuntos
Antígenos de Plantas/metabolismo , Antígenos de Grupos Sanguíneos/metabolismo , Alface/virologia , Norovirus/fisiologia , Receptores Virais/metabolismo , Ligação Viral , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/genética , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Humanos , Alface/química , Alface/genética , Alface/metabolismo , Espectrometria de Massas , Norovirus/genética , Oligossacarídeos/química , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Ligação Proteica , Receptores Virais/química , Receptores Virais/genética
15.
Nat Immunol ; 20(12): 1681-1691, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31636462

RESUMO

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.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Infecções por Caliciviridae/imunologia , Colite/imunologia , Proteína DEAD-box 58/metabolismo , Intestinos/imunologia , Linfócitos Intraepiteliais/imunologia , Norovirus/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Infecções por Caliciviridae/virologia , Células Cultivadas , Colite/induzido quimicamente , Colite/virologia , Proteína DEAD-box 58/genética , Sulfato de Dextrana , Suscetibilidade a Doenças , Homeostase , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Interleucina-15/metabolismo , Intestinos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Simbiose/imunologia
16.
mBio ; 10(5)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575769

RESUMO

Human norovirus (HuNoV) is the main cause of gastroenteritis worldwide, yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an interferon (IFN)-cured population of replicon-harboring HGT cells (HGT-Cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-Cured cells created an environment favoring norovirus replication. Microarrays were used to identify genes differentially regulated in HGT-NV and HGT-Cured compared to parental cells. We found that IFN lambda receptor (IFNLR1) expression was highly reduced in HGT-NV and HGT-Cured cells. While all three cell lines responded to exogenous IFN-ß by inducing interferon-stimulated genes, HGT-NV and HGT-Cured cells failed to respond to exogenous IFN-λ. Methylation-sensitive PCR showed that an increased methylation of the IFNLR1 promoter and inhibition of DNA methyltransferase activity partially reactivated IFNLR1 expression in HGT-NV and HGT-Cured cells, indicating that host adaptation occurred via epigenetic reprogramming. Moreover, IFNLR1 ectopic expression rescued response to IFN-λ and restricted HuNoV replication in HGT-NV cells. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. This study unravels for the first time epigenetic reprogramming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signaling is able to control human norovirus replication.IMPORTANCE Noroviruses are one of the most widespread causes of gastroenteritis, yet no suitable therapeutics are available for their control. Moreover, to date, knowledge of the precise cellular processes that control the replication of the human norovirus remains ill defined. Recent work has highlighted the importance of type III interferon (IFN) responses in the restriction of viruses that infect the intestine. Here, we analyzed the adaptive changes required to support long-term replication of noroviruses in cell culture and found that the receptor for type III IFN is decreased in its expression. We confirmed that this decreased expression was driven by epigenetic modifications and that cells lacking the type III IFN receptor are more permissive for norovirus replication. This work provides new insights into key host-virus interactions required for the control of noroviruses and opens potential novel avenues for their therapeutic control.


Assuntos
Epigênese Genética , Norovirus/fisiologia , Receptores de Interferon/metabolismo , Replicação Viral , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Interferons/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interferon/genética , Análise Serial de Tecidos
17.
PLoS Pathog ; 15(9): e1008009, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31536612

RESUMO

Human noroviruses (HuNoVs) are the most common cause of foodborne illness, with a societal cost of $60 billion and 219,000 deaths/year. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. The virus (HuNoV GII.4) could be passaged from larva to larva two consecutive times. HuNoV is detected in cells of the hematopoietic lineage and the intestine, supporting the notion of a dual tropism. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.


Assuntos
Norovirus/fisiologia , Norovirus/patogenicidade , Replicação Viral/fisiologia , Peixe-Zebra/virologia , Animais , Antivirais/administração & dosagem , Infecções por Caliciviridae/virologia , Doenças Transmitidas por Alimentos/virologia , Gastroenterite/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Larva/virologia , Metagenômica , Modelos Animais , Norovirus/genética , Cultura de Vírus/métodos , Replicação Viral/efeitos dos fármacos
18.
J Virol ; 93(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31484750

RESUMO

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCV strains can be propagated to high titers in standard nonhuman primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells, which identified the coxsackievirus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that short interfering RNA, anti-human CAR (hCAR) monoclonal antibody RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9-targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection, and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate, caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays, we identified and validated the coxsackievirus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs both are necessary to convert a nonsusceptible cell line to being susceptible to ReCV infection. Follow-up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.


Assuntos
Infecções por Caliciviridae/metabolismo , Receptores Virais/metabolismo , Replicação Viral/fisiologia , Infecções por Adenoviridae/metabolismo , Animais , Células CHO , Caliciviridae/metabolismo , Chlorocebus aethiops , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Infecções por Coxsackievirus/metabolismo , Cricetulus , Gastroenterite/virologia , Intestino Delgado/imunologia , Macaca mulatta/imunologia , Modelos Biológicos , Norovirus/fisiologia , Vírus de RNA/metabolismo , Receptores Virais/genética , Receptores Virais/fisiologia , Células Vero , Ligação Viral
19.
Am J Clin Nutr ; 110(6): 1456-1464, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504095

RESUMO

BACKGROUND: To accurately assess micronutrient status, it is necessary to characterize the effects of inflammation and the acute-phase response on nutrient biomarkers. OBJECTIVE: Within a norovirus human challenge study, we aimed to model the inflammatory response of C-reactive protein (CRP) and α-1-acid glycoprotein (AGP) by infection status, model kinetics of micronutrient biomarkers by inflammation status, and evaluate associations between inflammation and micronutrient biomarkers from 0 to 35 d post-norovirus exposure. METHODS: Fifty-two healthy adults were enrolled into challenge studies in a hospital setting and followed longitudinally; all were exposed to norovirus, half were infected. Post hoc analysis of inflammatory and nutritional biomarkers was performed. Subjects were stratified by inflammation resulting from norovirus exposure. Smoothed regression models analyzed the kinetics of CRP and AGP by infection status, and nutritional biomarkers by inflammation. Linear mixed-effects models were used to analyze the independent relations between CRP, AGP, and biomarkers for iron, vitamin A, vitamin D, vitamin B-12, and folate from 0 to 35 d post-norovirus exposure. RESULTS: Norovirus-infected subjects had median (IQR) peak concentrations for CRP [16.0 (7.9-29.5) mg/L] and AGP [0.9 (0.8-1.2) g/L] on day 3 and day 4 postexposure, respectively. Nutritional biomarkers that differed (P < 0.05) from baseline within the inflamed group were ferritin (elevated day 3), hepcidin (elevated days 2, 3), serum iron (depressed days 2-4), transferrin saturation (depressed days 2-4), and retinol (depressed days 3, 4, and 7). Nutritional biomarker concentrations did not differ over time within the uninflamed group. In mixed models, CRP was associated with ferritin (positive) and serum iron and retinol (negative, P < 0.05). CONCLUSION: Using an experimental infectious challenge model in healthy adults, norovirus infection elicited a time-limited inflammatory response associated with altered serum concentrations of certain iron and vitamin A biomarkers, confirming the need to consider adjustments of these biomarkers to account for inflammation when assessing nutritional status. These trials were registered at clinicaltrials.gov as NCT00313404 and NCT00674336.


Assuntos
Biomarcadores/sangue , Infecções por Caliciviridae/sangue , Micronutrientes/sangue , Adulto , Proteína C-Reativa/análise , Proteína C-Reativa/imunologia , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Feminino , Ferritinas/sangue , Ácido Fólico/sangue , Humanos , Ferro/sangue , Estudos Longitudinais , Masculino , Norovirus/fisiologia , Vitamina A/sangue , Vitamina D/sangue
20.
J Am Chem Soc ; 141(41): 16303-16311, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31533424

RESUMO

Virus internalization into the host cells occurs via multivalent interactions, in which a single virus binds to multiple receptors in parallel. Because of analytical and experimental limitations this complex type of interaction is still poorly understood and quantified. Herein, the multivalent interaction of norovirus-like particles (noroVLPs) with H or B type 1 glycosphingolipids (GSLs), embedded in a supported phospholipid bilayer, is investigated by following the competition between noroVLPs and a lectin (from Ralstonia solanacearum) upon binding to these GSLs. Changes in noroVLP and lectin coverage, caused by competition, were monitored for both GSLs and at different GSL concentrations using quartz crystal microbalance with dissipation monitoring. The study yields information about the minimum GSL concentration needed for (i) noroVLPs to achieve firm attachment to the bilayer prior to competition and to (ii) remain firmly attached to the bilayer during competition. We show that these two concentrations are almost identical for the H type 1-noroVLP interaction but differ for B type 1, indicating an accumulation of B type 1 GSLs in the noroVLP-bilayer interaction area. Furthermore, the GSL concentration required for firm attachment is significantly larger for H type 1 than for B type 1, indicating a higher affinity of noroVLP toward B type 1. This finding is supported by extracting the energy of single noroVLP-H type 1 and noroVLP-B type 1 bonds from the competition kinetics, which were estimated to be 5 and 6 kcal/mol, respectively. This demonstrates the potential of utilizing competitive binding kinetics to analyze multivalent interactions, which has remained difficult to quantify using conventional approaches.


Assuntos
Lectinas/farmacologia , Norovirus/fisiologia , Receptores de Superfície Celular/fisiologia , Ligação Viral/efeitos dos fármacos , Membrana Celular , Bicamadas Lipídicas , Fosfolipídeos , Técnicas de Microbalança de Cristal de Quartzo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA