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1.
Arch Virol ; 165(12): 2767-2776, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32949263

RESUMO

Human norovirus is the leading cause of viral gastroenteritis worldwide. Rapid detection facilitates management of disease outbreaks, but field diagnosis is difficult to achieve due to the lack of reliable and portable methods. Recombinase polymerase amplification (RPA) is a robust isothermal amplification method that is capable of rapidly amplifying and detecting nucleic acids using simple equipment. In this study, RPA combined with lateral flow (LF) strips specific for human genogroup II (GII) noroviruses was established and evaluated. The assay specifically detects purified GII noroviruses as well as RNA in boiled human stool samples, with a sensitivity of 50 norovirus genome copies per reaction. The whole detection procedure of the one-step RT-RPA-LF is completed within 20 min, which is eight times faster than that of the standard real-time RT-PCR. The RT-RPA-LF method described here is suitable for rapid field diagnosis of all GII noroviruses in human stool samples.


Assuntos
Infecções por Caliciviridae/diagnóstico , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Caliciviridae/genética , Fezes/virologia , Humanos , Norovirus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/química , Sensibilidade e Especificidade
2.
PLoS One ; 15(8): e0237044, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745122

RESUMO

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis. Outbreaks normally occur via the fecal-oral route. HuNoV infection is thought to occur by viral particle transmission, but increasing evidence suggests a function for exosomes in HuNoV infection. HuNoV is contained within stool-derived exosomes, and exosome-associated HuNoV has been shown to replicate in human intestinal enteroids. In this study, we examine exosome-associated HuNoV infection of Vero cells and show that exosomes containing HuNoV may attach, infect, and be passaged in Vero cells. These findings support earlier findings and have implications for developing HuNoV disease intervention strategies.


Assuntos
Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/transmissão , Exossomos/metabolismo , Animais , Infecções por Caliciviridae/genética , Criança , Pré-Escolar , Chlorocebus aethiops , Enterocolite/virologia , Exossomos/genética , Fezes/virologia , Feminino , Gastroenterite/virologia , Humanos , Masculino , Norovirus/patogenicidade , Células Vero , Vírion
3.
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
4.
Biomed Res Int ; 2020: 4707538, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104692

RESUMO

Norovirus is the leading cause of food-borne disease outbreaks. We conducted this study to examine the incidence and molecular characteristics of norovirus genogroup I infections from acute gastroenteritis outbreaks in Taiwan. Between January 2015 and June 2019, 2121 acute gastroenteritis clusters were reported to Taiwan CDC, of which 351 (16.5%) clusters were positive for NoV GI, and GI.3 was the most prevalent (36.8%) during the study period. The GI.3 infections were significantly higher than non-GI.3 infections in the age groups of 0-5 and 6-18 years. The phylogenetic analysis of the MCC tree revealed that VP1 genes were divided into 3 groups: the GI.P3-GI.3 strains in Taiwan were genetically close to Japan and the GI.Pd-GI.3 strains were segregated into 2 other groups which were genetically closely related to China. In addition, 7 GI.Pd-GI.3 recombinants were identified circulating in Taiwan between 2018 and 2019, and the prevalence of GI.Pd-GI.3 should be monitored to assess whether this could become the new predominant strains in neighboring Asian countries or other parts of the world. Both GI.P3-GI.3 and GI.Pd-GI.3 strains cocirculate, the recombination among these two lineages occurs frequently, contributing to the genetic diversity and multiple occurrences of different norovirus lineages, and their rapid evolution makes future control more difficult. Continued surveillance and timely interventions are critical to understand the complexity of norovirus gene variation and to monitor the new emerging norovirus strains.


Assuntos
Infecções por Caliciviridae , Gastroenterite , Norovirus/genética , Filogenia , Doença Aguda , Adolescente , Adulto , Idoso , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/genética , Criança , Pré-Escolar , Feminino , Gastroenterite/epidemiologia , Gastroenterite/genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Taiwan
5.
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
6.
Virus Genes ; 56(2): 174-181, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31912284

RESUMO

Genogroup II, genotype 4 noroviruses (GII.4 NoVs) are a leading cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. In this study, we isolated a GII.4 NoV strain (designated 2015HN08) from a kid presenting with acute gastroenteritis and determined its near-complete genome sequence. We then performed sequence analysis by comparing this strain with the prototypical GII.4 strain. Virus-like particles (VLPs) derived from the major capsid protein (VP1) were expressed by using a recombinant-baculovirus expression system, and monoclonal antibodies (mAbs) were produced to compare changes in antigenic or histo-blood group antigens (HBGAs) binding sites with the previously characterized GII.4 NoV strain (JZ403). The genome of 2015HN08 was 7559 nucleotides (nt) long, excluding the poly(A) tail. Genotyping analysis indicated that this strain was a Sydney 2012 variant. In comparison with the prototype Sydney 2012 strain, there were 74, 35, and 16 differences in nucleotide sequences in ORF1, OFR2, and OFR3, causing 7, 10, and 6 amino acid (aa) changes, respectively. Expression of VP1 led to successful assembly of VLPs, as demonstrated by electron microscopy. Screening of hybridoma cell supernatants with an in vitro VLP-HBGAs binding blockade assay led to the identification of a cell clone 3G10 that exhibited HBGA-blocking effects. This mAb also exhibited blocking effects against JZ403 strain, suggesting maintenance of the antigenic site and/or HBGAs binding sites between the two strains. In summary, we determined the near-complete genome sequence of a GII.4 Sydney 2012 variant and produced an mAb with blocking effects that might be useful in evaluating the evolution of current Sydney 2012 NoV strains.


Assuntos
Infecções por Caliciviridae/genética , Proteínas do Capsídeo/genética , Gastroenterite/genética , Norovirus/genética , Sítios de Ligação , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genoma Viral/genética , Genômica , Genótipo , Humanos , Norovirus/patogenicidade , Pandemias , Ligação Proteica
7.
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
8.
Emerg Microbes Infect ; 8(1): 1642-1657, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31711377

RESUMO

Diverse noroviruses infect humans and animals via the recognition of host-specific glycan ligands. Genogroup II (GII) noroviruses consist of human noroviruses (huNoVs) that generally bind histo-blood group antigens (HBGAs) as host factors and three porcine norovirus (porNoV) genotypes (GII.11/18/19) that form a genetic lineage lacking HBGA-binding ability. Thus, these GII porNoVs provide an excellent model to study norovirus evolution with host ligand specificity changes. Here we solved the crystal structures of a native GII.11 porNoV P protein and a closely-related GII.3 huNoV P protein complexed with an HBGA, focusing on the HBGA-binding sites (HBSs) compared with the previously known ones to understand the structural basis of the host ligand specificity change. We found that the GII.3 huNoV binds HBGAs via a conventional GII HBS that uses an arginine instead of the conserved aromatic residue for the required Van der Waals interaction, while the GII.11 porNoV HBS loses its HBGA-binding function because of two mutations (Q355/V451). A mutant that reversed the two mutated residues back to the conventional A355/Y451 restored the HBGA-binding function of the GII.11 porNoV P protein, which validated our observations. Similar mutations are also found in GII.19 porNoVs and a GII.19 P protein mutant with double reverse mutations restored the HBS function. This is the first reconstruction of a functional HBS based on one with new host specificity back to its parental one. These data shed light on the molecular basis of structural adaptation of the GII porNoVs to the pig hosts through mutations at their HBSs.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Infecções por Caliciviridae/veterinária , Infecções por Caliciviridae/virologia , Norovirus/metabolismo , Doenças dos Suínos/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Antígenos de Grupos Sanguíneos/genética , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Genótipo , Humanos , Ligantes , Mutação , Norovirus/química , Norovirus/classificação , Norovirus/genética , Filogenia , Ligação Proteica , Alinhamento de Sequência , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
9.
Viruses ; 11(11)2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671627

RESUMO

It is known that levels of the anti-apoptotic protein survivin are reduced during Murine norovirus MNV-1 and Feline calicivirus (FCV) infection as part of the apoptosis establishment required for virus release and propagation in the host. Recently, our group has reported that overexpression of survivin causes a reduction of FCV protein synthesis and viral progeny production, suggesting that survivin may affect early steps of the replicative cycle. Using immunofluorescence assays, we observed that overexpression of survivin, resulted in the reduction of FCV infection not only in transfected but also in the neighboring nontransfected CrFK cells, thus suggesting autocrine and paracrine protective effects. Cells treated with the supernatants collected from CrFK cells overexpressing survivin showed a reduction in FCV but not MNV-1 protein production and viral yield, suggesting that FCV binding and/or entry were specifically altered. The reduced ability of FCV to bind to the surface of the cells overexpressing survivin, or treated with the supernatants collected from these cells, correlate with the reduction in the cell surface of the FCV receptor, the feline junctional adhesion molecule (fJAM) 1, while no effect was observed in the cells transfected with the pAm-Cyan vector or in cells treated with the corresponding supernatants. Moreover, the overexpression of survivin affects neither Vaccinia virus (VACV) production in CrFK cells nor MNV-1 virus production in RAW 267.4 cells, indicating that the effect is specific for FCV. All of these results taken together indicate that cells that overexpress survivin, or cell treatment with the conditioned medium from these cells, results in the reduction of the fJAM-1 molecule and, therefore, a specific reduction in FCV entry and infection.


Assuntos
Infecções por Caliciviridae/virologia , Calicivirus Felino/fisiologia , Survivina/metabolismo , Animais , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Calicivirus Felino/metabolismo , Gatos , Linhagem Celular , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Expressão Gênica , Interações Hospedeiro-Patógeno , Moléculas de Adesão Juncional/metabolismo , Receptores Virais/metabolismo , Especificidade da Espécie , Survivina/genética , Proteínas Virais/biossíntese , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
10.
Medicine (Baltimore) ; 98(40): e17269, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31577718

RESUMO

This study examined the characteristics of norovirus (NoV) gastroenteritis associated with convulsions in children and its molecular epidemiology. From July 2006 through December 2015, NoV infection was confirmed by the genome detection using reverse transcriptase polymerase chain reaction. Viral genotyping with strain validation was achieved using sequence analyses with Basic Local Alignment Search Tool genome identification. The patients' clinical features were assessed retrospectively, focusing on convulsive disorders. The diagnosis of encephalitis followed the International Encephalitis Consortium. Seizures occurred in 52 (20.9%) of 249 NoV infections. GII.4 Den_Haag_2006b (n = 22, 42.3%) and GII.4 Sydney 2012 (n = 10, 19.2%) were major variants correlated with convulsions. Patient with convulsions tend to have GII.4 genotype infection (P < .001), short vomiting (≤2 days) (P < .001), and no fever (P = .002). Compared to GII.4 Den_Haag_2006b, the GII.4 Sydney 2012-associated convulsions had similar manifestations except without significant winter preponderance (P = .049). The NoV infection with convulsions had less febrile course, specific genotype (GII.4) infections, and with shorter symptom of vomiting. Continuous surveillance is important for uncommon disease associated with emerging NoV strain infections. The prevention of NoV diseases requires the development of vaccines targeting highly virulent variants.


Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/genética , Gastroenterite/epidemiologia , Convulsões/epidemiologia , Adolescente , Criança , Pré-Escolar , Encefalite/epidemiologia , Feminino , Febre/epidemiologia , Gastroenterite/virologia , Genótipo , Humanos , Lactente , Masculino , Epidemiologia Molecular , RNA Viral/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estações do Ano , Análise de Sequência de DNA , Taiwan/epidemiologia
11.
mBio ; 10(5)2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551337

RESUMO

GII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially explained by the continuous emergence of antigenically distinct variants. To gain insights into the mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 noroviruses exhibited a periodic replacement of predominant variants with accumulation of amino acid substitutions. Genomic analyses revealed (i) a large proportion (87%) of conserved residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) variable residues that map outside the antigenic sites. Residues in the third pattern category formed motifs on the surface of VP1, which suggested extensions of previously predicted and new uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic makeup of the GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the circulation patterns of GII.4 variants, with three of them (A, C, and G) containing residues (352, 357, 368, and 378) linked with the diversifying selective pressure on the emergence of new GII.4 variants. Notably, the emergence of each variant was followed by stochastic diversification with minimal changes that did not progress toward the next variant. This report provides a methodological framework for antigenic characterization of viruses and expands our understanding of the dynamics of GII.4 noroviruses and could facilitate the design of cross-reactive vaccines.IMPORTANCE Noroviruses are an important cause of viral gastroenteritis around the world. An obstacle delaying the development of norovirus vaccines is inadequate understanding of the role of norovirus diversity in immunity. Using a population genomics approach, we identified new residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful monitoring of the substitutions in those residues involved in the diversification and emergence of new viruses could help in the early detection of future novel variants with pandemic potential. Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus vaccine design.


Assuntos
Anticorpos Antivirais/genética , Variação Antigênica/genética , Infecções por Caliciviridae/genética , Variação Genética , Metagenômica , Norovirus/genética , Pandemias , Infecções por Caliciviridae/epidemiologia , Humanos
12.
Epidemiol Infect ; 147: e218, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-31364546

RESUMO

Childhood morbidity and mortality of diarrhoeal diseases are high, particularly in low-income countries and noroviruses and sapoviruses are among the most frequent causes worldwide. Their epidemiology and diversity remain not well studied in many African countries. To assess the positivity rate and the diversity of sapoviruses and noroviruses in Northwest Ethiopia, during November 2015 and April 2016, a total of 450 faecal samples were collected from outpatient children aged <5 years who presented with diarrhoea. Samples were screened for noroviruses and sapoviruses by real-time RT-PCR. Partial VP1 genes were sequenced, genotyped and phylogenetically analysed. Norovirus and sapovirus stool positivity rate was 13.3% and 10.0%, respectively. Noroviruses included GII.4 (35%), GII.6 (20%), GII.17 (13.3%), GII.10 (10%), GII.2 (6.7%), GII.16 (5%), GII.7 (3.3%), GII.9, GII.13, GII.20 and GI.3 (1.7% each) strains. For sapoviruses, GI.1, GII.1 (20.0% each), GII.6 (13.3%), GI.2 (8.9%), GII.2 (11.1%), GV.1 (8.9%), GIV.1 (6.7%), GI.3 and GII.4 (2.2% each) genotypes were detected. This study demonstrates a high genetic diversity of noroviruses and sapoviruses in Northwest Ethiopia. The positivity rate in stool samples from young children with diarrhoea was high for both caliciviruses. Continued monitoring is recommended to identify trends in genetic diversity and seasonal variations.


Assuntos
Proteínas de Bactérias/genética , Infecções por Caliciviridae/epidemiologia , Gastroenterite/epidemiologia , Norovirus/genética , Sapovirus/genética , Infecções por Caliciviridae/genética , Criança , Pré-Escolar , Estudos de Coortes , Países em Desenvolvimento , Etiópia/epidemiologia , Fezes/virologia , Feminino , Gastroenterite/genética , Gastroenterite/microbiologia , Variação Genética , Genótipo , Humanos , Incidência , Lactente , Masculino , Norovirus/isolamento & purificação , Pacientes Ambulatoriais/estatística & dados numéricos , Filogenia , Saúde Pública , Estudos Retrospectivos , Medição de Risco , Sapovirus/isolamento & purificação , Estações do Ano , Análise de Sobrevida
13.
Zhonghua Yu Fang Yi Xue Za Zhi ; 53(6): 581-585, 2019 Jun 06.
Artigo em Chinês | MEDLINE | ID: mdl-31177754

RESUMO

Objective: To understand the infection status and genetic characteristics of Norovirus from foodborne diseases in sentinel hospital of Ma'anshan city. Methods: The 911 stool samples and epidemiological data of the patients with foodborne disease were collected from three hospitals of Ma'anshan city during January 2015 to June 2018. The G Ⅰ and G Ⅱ Norovirus were detected by real-time reverse transcription PCR. Some of the positive specimens were amplified by conventional reverse transcription PCR, and the PCR products were sequenced for sequence alignment and phylogenetic analysis. Results: The positive rate of Norovirus was 14.7% (134/911), in which 7 strains were G Ⅰ, 124 strains were G Ⅱ and 3 strains were mixed infection. Norovirus can be detected throughout the year, with high positive rate from December to April of the next year [24.4%(20/82)-45.3%(24/53)]. The 78 males (15.5%) and 55 females (13.7%) were positive for Norovirus (χ(2)=0.58,P=0.448). There was no significant difference in different age groups (χ(2)=9.55, P=0.089). A total of 79 strains were successfully sequenced, 4 strains were G Ⅰ group(5.1%), 75 strains were G Ⅱ group (94.9%). The predominant strains were GⅡ.17 and GⅡ.4, aud the number were 35 and 15 respectively. The predominant strains were different in different years. The main strain was GⅡ.17 in 2015 (30, 68.2%),GⅡ.4 in 2016 (5/9) and 2017 (8/16), but GⅡ.3 in 2018 (3/6). Conclusion: Norovirus diarrhea was popular in Ma'anshan city throughout the year,especially in winter and spring. The prevalent strain was GⅡ,genotypes were diversified distribution,the dominant strains were GⅡ.17 and GⅡ.4. The predominant strains were different in different years.


Assuntos
Infecções por Caliciviridae , Doenças Transmitidas por Alimentos , Gastroenterite , Norovirus , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/genética , China , Cidades , Fezes , Feminino , Doenças Transmitidas por Alimentos/virologia , Genótipo , Humanos , Masculino , Norovirus/genética , Norovirus/isolamento & purificação , Filogenia
14.
Immunol Lett ; 215: 40-44, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31154053

RESUMO

Norovirus (NoV) is now recognized as a major cause of gastroenteritis outbreaks, worldwide. Norovirus replication mechanisms are still poorly understood, mainly because a reliable cell culture system is still lacking. The present study aims at understanding some aspects of the immune response against norovirus, and particularly the capacity of virus like particles (VLPs) from an Italian strain, belonging to the GII.4 genotype predominating worldwide, to interact with target cells via Toll Like Receptors (TLRs). The capacity of GII.4 NoV VLPs to interact and cause the activation of TLR2, 4 and 5 was studied in recombinant HEK cells. The results obtained show the ability of GII.4 NoV VLPs to induce activation of TLR2 and 5. The results on TLRs activation confirm that GII.4 NoV VLPs interact with TLR2 and 5, that may represent putative receptors and play a role in NoV infection of intestinal cells.


Assuntos
Norovirus/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 5 Toll-Like/imunologia , Vírion/imunologia , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/patologia , Células HEK293 , Humanos , Norovirus/genética , Receptor 2 Toll-Like/genética , Receptor 5 Toll-Like/genética , Vírion/genética
15.
Viruses ; 11(3)2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845670

RESUMO

Noroviruses are the most common etiological agent of acute gastroenteritis worldwide. Despite their high infectivity, a subpopulation of individuals is resistant to infection and disease. This susceptibility is norovirus genotype-dependent and is largely mediated by the presence or absence of human histo-blood group antigens (HBGAs) on gut epithelial surfaces. The synthesis of these HBGAs is mediated by fucosyl- and glycosyltransferases under the genetic control of the FUT2 (secretor), FUT3 (Lewis) and ABO(H) genes. The so-called non-secretors, having an inactivated FUT2 enzyme, do not express blood group antigens and are resistant to several norovirus genotypes, including the predominant GII.4. Significant genotypic and phenotypic diversity of HBGA expression exists between different human populations. Here, we review previous in vivo studies on genetic susceptibility to norovirus infection. These are discussed in relation to population susceptibility, vaccines, norovirus epidemiology and the impact on public health.


Assuntos
Infecções por Caliciviridae/genética , Gastroenterite/virologia , Predisposição Genética para Doença , Antígenos de Grupos Sanguíneos/genética , Fucosiltransferases/genética , Genótipo , Glicosiltransferases , Humanos , Norovirus/fisiologia
16.
J Med Virol ; 91(6): 1014-1021, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30735247

RESUMO

Norovirus (NoV) and rotavirus group A (RVA) are major agents of acute gastroenteritis worldwide. This study aimed to investigate their epidemiological profile in Portuguese elderly living in long-term care facilities and to assess the host genetic factors mediating infection susceptibility. From November 2013 to June 2015, 636 faecal specimens from 169 elderly, mainly asymptomatic, living in nursing homes in Greater Lisbon and Faro district, Portugal, were collected. NoV and RVA were detected by real-time polymerase chain reaction and NoV genotyped by phylogenetic analysis. NoV detection rate was 7.1% (12 of 169). Three GI.3 and one GII.6 strains were genotyped. RVA detection rate was 3.6% (6 of 169), exclusively in asymptomatic individuals. Host genetic factors associated with infection susceptibility were described on 250 samples by saliva-based enzyme-linked immunosorbent assays. The Lewis-negative phenotype was 8.8% (22 of 250) and the rate of nonsecretors was 16.8% (42 of 250). Association to NoV and RVA infection was performed in the subgroup of individuals (n = 147) who delivered both faecal and saliva samples. The majority of NoV- and RVA-positive individuals (90.9% and 83.3%, respectively) were secretor-positive, with Lewis B phenotype. In a subset of individuals, FUT2 and FUT3 genes were genotyped to assess mutations and validate the secretor and Lewis phenotypes. All sequenced nonsecretors were homozygous for FUT2 nonsense mutation G428A. In this study, low detection rates of NoV and RVA infections were found during two winter seasons. However, even in the absence of any outbreak, the importance of finding these infections in a nonepidemic situation in long-term care facilities may have important implications for infection control.


Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/genética , Instituição de Longa Permanência para Idosos/estatística & dados numéricos , Norovirus/genética , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/genética , Rotavirus/genética , Idoso , Idoso de 80 Anos ou mais , Surtos de Doenças/estatística & dados numéricos , Fezes/virologia , Feminino , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genótipo , Humanos , Masculino , Norovirus/isolamento & purificação , Fenótipo , Filogenia , Portugal/epidemiologia , RNA Viral/genética , Rotavirus/isolamento & purificação
17.
Nucleic Acids Res ; 47(4): 1920-1934, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30668745

RESUMO

Caliciviruses use a termination/reinitiation mechanism for translation of their minor capsid protein VP2. A sequence element of about 80 nucleotides denoted 'termination upstream ribosomal binding site' (TURBS) is crucial for reinitiation. RNA secondary structure probing and computer aided secondary structure prediction revealed a rather low degree of secondary structure determinants for the TURBS of the rabbit hermorrhagic disease virus. Mutation analysis showed that prevention of duplex formation had major impact on the VP2 expression levels. Restoration of complementarity of the respective sequences by reciprocal mutation at least partially restored reinitiating rates. Synthetic TURBS structures preserving only the secondary structure forming sequences and the known short motifs important for TURBS function were found to drive reinitiation when the altered sequence could be predicted to allow establishment of the crucial secondary structures of the TURBS.


Assuntos
Infecções por Caliciviridae/genética , Proteínas do Capsídeo/genética , Vírus da Doença Hemorrágica de Coelhos/genética , Relação Estrutura-Atividade , Animais , Sítios de Ligação , Infecções por Caliciviridae/virologia , Regulação Viral da Expressão Gênica/genética , Vírus da Doença Hemorrágica de Coelhos/patogenicidade , Mutação , Biossíntese de Proteínas/genética , Coelhos , Ribossomos/genética
18.
J Virol ; 93(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30305350

RESUMO

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprisingly, the majority of the genes identified are neither associated with innate or adaptive immunity nor associated with any antiviral activity. Confirmatory studies of eight of the genes validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together, these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules.IMPORTANCE Norovirus is one of the leading causes of food-borne illness worldwide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed, we performed genome-wide CRISPR activation screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 49 genes that could block murine norovirus replication in human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data are a resource for those studying noroviruses, and we provide a robust approach to identify novel antiviral genes.


Assuntos
Antivirais/farmacologia , Infecções por Caliciviridae/genética , Proteínas de Transporte/farmacologia , Redes Reguladoras de Genes , Norovirus/fisiologia , Animais , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/virologia , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Norovirus/efeitos dos fármacos , Ativação Transcricional , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Regulação para Cima , Internalização do Vírus , Replicação Viral/efeitos dos fármacos
19.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30209176

RESUMO

The linear ubiquitin chain assembly complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1), HOIL1-interacting protein (HOIP), and SHANK-associated RH domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1 or HOIP deficiency is associated with an immune disorder involving autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I). Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor 3 (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, melanoma differentiation-associated protein 5 (MDA5), is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler's murine encephalomyelitis virus infection and poly(I·C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required selectively for MDA5 signaling. Moreover, Hoil1 - / - mice exhibited defective control of acute and persistent murine norovirus infection and defective regulation of MNoV persistence by the microbiome as also observed previously for mice deficient in interferon lambda (IFN-λ) receptor, signal transducer and activator of transcription factor 1 (STAT1), and IRF3. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world but are challenging to study in vivo and in vitro Murine norovirus (MNoV) provides a tractable genetic and small-animal model to study norovirus biology and immune responses. Interferons are critical mediators of antiviral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces, including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. LUBAC, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.


Assuntos
Infecções por Caliciviridae/virologia , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferons/metabolismo , Norovirus/patogenicidade , Ubiquitina-Proteína Ligases/fisiologia , Animais , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/microbiologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Fibroblastos/virologia , Genoma Viral , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Helicase IFIH1 Induzida por Interferon/genética , Interferons/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota , Norovirus/genética , Fosforilação
20.
Viruses ; 10(9)2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30235853

RESUMO

The rabbit caliciviruses Lagovirus europaeus GI.1 and GI.2 both cause acute necrotizing hepatitis in European rabbits (Oryctolagus cuniculus). Whilst GI.2 is highly virulent in both young and adult rabbits, rabbits younger than eight weeks of age are highly resistant to disease caused by GI.1, although they are still permissive to infection and viral replication. To investigate the underlying mechanism(s) of this age related resistance to GI.1, we compared liver transcriptomes of young rabbits infected with GI.1 to those of adult rabbits infected with GI.1 and young rabbits infected with GI.2. Our data suggest that kittens have constitutively heightened innate immune responses compared to adult rabbits, particularly associated with increased expression of major histocompatibility class II molecules and activity of natural killer cells, macrophages, and cholangiocytes. This enables them to respond more rapidly to GI.1 infection than adult rabbits and thus limit virus-induced pathology. In contrast, these responses were not fully developed during GI.2 infection. We speculate that the observed downregulation of multiple genes associated with innate immunity in kittens during GI.2 infection may be due to virally-mediated immunomodulation, permitting fatal disease to develop. Our study provides insight into the fundamental host⁻pathogen interactions responsible for the differences in age-related susceptibility, which likely plays a critical role in defining the success of GI.2 in outcompeting GI.1 in the field.


Assuntos
Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Resistência à Doença/imunologia , Vírus da Doença Hemorrágica de Coelhos/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Animais , Apresentação do Antígeno/imunologia , Antígenos Virais/imunologia , Biomarcadores , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Genoma Viral , Genômica/métodos , Vírus da Doença Hemorrágica de Coelhos/classificação , Interações Hospedeiro-Patógeno/genética , RNA Viral , Coelhos , Transdução de Sinais , Carga Viral
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