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1.
Nat Commun ; 12(1): 7288, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34911947

RESUMO

Identifying risk factors for impaired oral rotavirus vaccine (ORV) efficacy in low-income countries may lead to improvements in vaccine design and delivery. In this prospective cohort study, we measure maternal rotavirus antibodies, environmental enteric dysfunction (EED), and bacterial gut microbiota development among infants receiving two doses of Rotarix in India (n = 307), Malawi (n = 119), and the UK (n = 60), using standardised methods across cohorts. We observe ORV shedding and seroconversion rates to be significantly lower in Malawi and India than the UK. Maternal rotavirus-specific antibodies in serum and breastmilk are negatively correlated with ORV response in India and Malawi, mediated partly by a reduction in ORV shedding. In the UK, ORV shedding is not inhibited despite comparable maternal antibody levels to the other cohorts. In both India and Malawi, increased microbiota diversity is negatively correlated with ORV immunogenicity, suggesting that high early-life microbial exposure may contribute to impaired vaccine efficacy.


Assuntos
Microbioma Gastrointestinal , Doenças do Recém-Nascido/prevenção & controle , Infecções por Rotavirus/microbiologia , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/administração & dosagem , Rotavirus/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imunidade Materno-Adquirida , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Índia , Lactente , Recém-Nascido , Doenças do Recém-Nascido/sangue , Doenças do Recém-Nascido/microbiologia , Doenças do Recém-Nascido/virologia , Malaui , Masculino , Leite Humano/química , Leite Humano/imunologia , Gravidez , Estudos Prospectivos , Rotavirus/genética , Rotavirus/fisiologia , Infecções por Rotavirus/sangue , Infecções por Rotavirus/virologia , Vacinas contra Rotavirus/imunologia , Reino Unido , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Eliminação de Partículas Virais
2.
PLoS One ; 16(8): e0247738, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34383769

RESUMO

The commensal microbiota regulates susceptibility to enteric pathogens by fine-tuning mucosal innate immune responses, but how susceptibility to enteric viruses is shaped by the microbiota remains incompletely understood. Past reports have indicated that commensal bacteria may either promote or repress rotavirus replication in the small intestine of mice. We now report that rotavirus replicated more efficiently in the intestines of germ-free and antibiotic-treated mice compared to animals with an unmodified microbiota. Antibiotic treatment also facilitated rotavirus replication in type I and type III interferon (IFN) receptor-deficient mice, revealing IFN-independent proviral effects. Expression of interleukin-22 (IL-22) was strongly diminished in the intestine of antibiotic-treated mice. Treatment with exogenous IL-22 blocked rotavirus replication in microbiota-depleted wild-type and Stat1-/- mice, demonstrating that the antiviral effect of IL-22 in animals with altered microbiome is not dependent on IFN signaling. In antibiotic-treated animals, IL-22-induced a specific set of genes including Fut2, encoding fucosyl-transferase 2 that participates in the biosynthesis of fucosylated glycans which can mediate rotavirus binding. Interestingly, IL-22 also blocked rotavirus replication in antibiotic-treated Fut2-/- mice. Furthermore, IL-22 inhibited rotavirus replication in antibiotic-treated mice lacking key molecules of the necroptosis or pyroptosis pathways of programmed cell death. Taken together, our results demonstrate that IL-22 determines rotavirus susceptibility of antibiotic-treated mice, yet the IL-22-induced effector molecules conferring rotavirus resistance remain elusive.


Assuntos
Antibacterianos/efeitos adversos , Interleucinas/metabolismo , Infecções por Rotavirus/etiologia , Animais , Antibacterianos/farmacologia , Suscetibilidade a Doenças , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Perfilação da Expressão Gênica , Interleucinas/fisiologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Rotavirus/fisiologia
3.
Sci Rep ; 11(1): 13945, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230563

RESUMO

Acute gastroenteritis associated with diarrhea is considered a serious disease in Africa and South Asia. In this study, we examined the trends in the causative pathogens of diarrhea and the corresponding gut microbiota in Ghana using microbiome analysis performed on diarrheic stools via 16S rRNA sequencing. In total, 80 patients with diarrhea and 34 healthy adults as controls, from 2017 to 2018, were enrolled in the study. Among the patients with diarrhea, 39 were norovirus-positive and 18 were rotavirus-positive. The analysis of species richness (Chao1) was lower in patients with diarrhea than that in controls. Beta-diversity analysis revealed significant differences between the two groups. Several diarrhea-related pathogens (e.g., Escherichia-Shigella, Klebsiella and Campylobacter) were detected in patients with diarrhea. Furthermore, co-infection with these pathogens and enteroviruses (e.g., norovirus and rotavirus) was observed in several cases. Levels of both Erysipelotrichaceae and Staphylococcaceae family markedly differed between norovirus-positive and -negative diarrheic stools, and the 10 predicted metabolic pathways, including the carbohydrate metabolism pathway, showed significant differences between rotavirus-positive patients with diarrhea and controls. This comparative study of diarrheal pathogens in Ghana revealed specific trends in the gut microbiota signature associated with diarrhea and that pathogen-dependent dysbiosis occurred in viral gastroenteritis.


Assuntos
Disbiose/microbiologia , Disbiose/virologia , Gastroenterite/microbiologia , Gastroenterite/virologia , Microbioma Gastrointestinal , Adolescente , Adulto , Bactérias/classificação , Biodiversidade , Estudos de Casos e Controles , Criança , Pré-Escolar , Diarreia/microbiologia , Diarreia/virologia , Fezes/microbiologia , Feminino , Gana , Humanos , Masculino , Filogenia , Rotavirus/fisiologia
4.
Viruses ; 13(6)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070283

RESUMO

Every year, millions of children are infected with viruses that target the gastrointestinal tract, causing acute gastroenteritis and diarrheal illness. Indeed, approximately 700 million episodes of diarrhea occur in children under five annually, with RNA viruses norovirus, rotavirus, and astrovirus serving as major causative pathogens. Numerous methodological advancements in recent years, including the establishment of novel cultivation systems using enteroids as well as the development of murine and other animal models of infection, have helped provide insight into many features of viral pathogenesis. However, many aspects of enteric viral infections remain elusive, demanding further study. Here, we describe the different in vitro and in vivo tools available to explore different pathophysiological attributes of human enteric RNA viruses, highlighting their advantages and limitations depending upon the question being explored. In addition, we discuss key areas and opportunities that would benefit from further methodological progress.


Assuntos
Suscetibilidade a Doenças , Gastroenterite/virologia , Vírus de RNA/fisiologia , Doenças dos Animais/diagnóstico , Doenças dos Animais/virologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Gastroenterite/diagnóstico , Predisposição Genética para Doença , Humanos , Norovirus/fisiologia , Rotavirus/fisiologia
5.
J Virol ; 95(15): e0075121, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980599

RESUMO

Rotavirus infection is highly prevalent in children, and the most severe effects are diarrhea and vomiting. It is well accepted that the enteric nervous system (ENS) is activated and plays an important role, but knowledge of how rotavirus activates nerves within ENS and to the vomiting center is lacking. Serotonin is released during rotavirus infection, and antagonists to the serotonin receptor subtype 3 (5-HT3 receptor) can attenuate rotavirus-induced diarrhea. In this study, we used a 5-HT3 receptor knockout (KO) mouse model to investigate the role of this receptor in rotavirus-induced diarrhea, motility, electrolyte secretion, inflammatory response, and vomiting reflex. The number of diarrhea days (P = 0.03) and the number of mice with diarrhea were lower in infected 5-HT3 receptor KO than wild-type pups. In vivo investigation of fluorescein isothiocyanate (FITC)-dextran transit time showed that intestinal motility was lower in the infected 5-HT3 receptor KO compared to wild-type mice (P = 0.0023). Ex vivo Ussing chamber measurements of potential difference across the intestinal epithelia showed no significant difference in electrolyte secretion between the two groups. Immediate early gene cFos expression level showed no difference in activation of the vomiting center in the brain. Cytokine analysis of the intestine indicated a low effect of inflammatory response in rotavirus-infected mice lacking the 5-HT3 receptor. Our findings indicate that the 5-HT3 receptor is involved in rotavirus-induced diarrhea via its effect on intestinal motility and that the vagus nerve signaling to the vomiting center occurs also in the absence of the 5-HT3 receptor. IMPORTANCE The mechanisms underlying rotavirus-induced diarrhea and vomiting are not yet fully understood. To better understand rotavirus pathophysiology, characterization of nerve signaling within the ENS and through vagal efferent nerves to the brain, which have been shown to be of great importance to the disease, is necessary. Serotonin (5-HT), a mediator of both diarrhea and vomiting, has been shown to be released from enterochromaffin cells in response to rotavirus infection and the rotavirus enterotoxin NSP4. Here, we investigated the role of the serotonin receptor 5-HT3, which is known to be involved in the nerve signals that regulate gut motility, intestinal secretion, and signal transduction through the vagus nerve to the brain. We show that the 5-HT3 receptor is involved in rotavirus-induced diarrhea by promoting intestinal motility. The findings shed light on new treatment possibilities for rotavirus diarrhea.


Assuntos
Diarreia/fisiopatologia , Sistema Nervoso Entérico/fisiopatologia , Motilidade Gastrointestinal/fisiologia , Receptores 5-HT3 de Serotonina/metabolismo , Infecções por Rotavirus/patologia , Vômito/fisiopatologia , Animais , Células Enterocromafins/metabolismo , Motilidade Gastrointestinal/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores 5-HT3 de Serotonina/genética , Rotavirus/fisiologia , Serotonina/metabolismo , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia
6.
J Gen Virol ; 102(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33843576

RESUMO

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.


Assuntos
Genes Reporter , Vetores Genéticos , RNA Viral , Genética Reversa , Rotavirus/genética , Animais , Linhagem Celular , Cricetinae , Haplorrinos , Plasmídeos , Rotavirus/fisiologia , Replicação Viral
7.
Curr Opin Virol ; 48: 42-48, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33887683

RESUMO

Rotaviruses are important agents of severe gastroenteritis in young children, and show a very selective cell and tissue tropism, as well as significant age and host restriction. In the last few years, these properties have been associated with the initial interaction of the virus with histo-blood group antigens on the cell surface, although post-attachment interactions have also been found to define the susceptibility to infection of human enteroids. These initial interactions seem also to determine the virus entry pathway, as well as the induction of signaling cascades that influence the virus intracellular vesicular traffic and escape from endosomes. Here we review the current knowledge of the different stages of the virus entry journey.


Assuntos
Rotavirus/fisiologia , Internalização do Vírus , Animais , Sítios de Ligação , Membrana Celular , Endossomos , Gastroenterite/virologia , Humanos , Infecções por Rotavirus/virologia , Proteínas Virais de Fusão/química
8.
PLoS One ; 16(2): e0246025, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33539399

RESUMO

BACKGROUND: In Zambia, before rotavirus vaccine introduction, the virus accounted for about 10 million episodes of diarrhoea, 63 000 hospitalisations and 15 000 deaths in 2015, making diarrhoea the third leading cause of death after pneumonia and malaria. In Zambia, despite the introduction of the vaccine acute diarrhoea due to rotaviruses has continued to affect children aged five years and below. This study aimed to characterise the rotavirus genotypes which were responsible for diarrhoeal infections in vaccinated infants aged 2 to 12 months and to determine the relationship between rotavirus strains and the severity of diarrhoea in 2016. METHODS: Stool samples from infants aged 2 to 12 months who presented to the hospital with acute diarrhoea of three or more episodes in 24 hours were tested for group A rotavirus. All positive specimens that had enough sample were genotyped using reverse transcriptase Polymerase Chain Reaction (RT-PCR). A 20-point Vesikari clinical score between 1-5 was considered as mild, 6-10 as moderate and greater or equal to 11 as severe. RESULTS: A total of 424 stool specimens were tested of which 153 (36%, 95% CI 31.5% to 40.9%) were positive for VP6 rotavirus antigen. The age-specific rotavirus infections decreased significantly (p = 0.041) from 2-4 months, 32.0% (49/118) followed by a 38.8% (70/181) infection rate in the 5-8 months' category and subsequently dropped in the infants aged 9-12 months with a positivity rate of 27.2%. 38.5% of infants who received a single dose, 34.5% of those who received a complete dose and 45.2% (19/42) of the unvaccinated tested positive for rotavirus. The predominant rotavirus genotypes included G2P[6] 36%, G1P[8] 32%, mixed infections 19%, G2P[4] 6%, G1P[6] 4% and G9P[6] 3%. DISCUSSION AND CONCLUSION: Results suggest breakthrough infection of heterotypic strains (G2P[6] (36%), homotypic, G1P[8] (32%) and mixed infections (19%) raises concerns about the effects of the vaccination on the rotavirus diversity, considering the selective pressure that rotavirus vaccines could exert on viral populations. This data indicates that the rotavirus vaccine has generally reduced the severity of diarrhoea despite the detection of the virus strains.


Assuntos
Diarreia/virologia , Gastroenterite/virologia , Hospitais Universitários/estatística & dados numéricos , Infecções por Rotavirus/prevenção & controle , Rotavirus/fisiologia , Vacinação , Doença Aguda , Diarreia/complicações , Feminino , Gastroenterite/complicações , Humanos , Lactente , Masculino , Infecções por Rotavirus/complicações , Zâmbia
9.
PLoS One ; 16(2): e0246193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33592026

RESUMO

Human rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model.


Assuntos
Biofilmes , Dextranos/química , Escherichia coli/fisiologia , Fezes/microbiologia , Desnutrição/virologia , Microbiota , Rotavirus/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Modelos Animais de Doenças , Desnutrição/microbiologia , Microesferas , RNA Mensageiro/genética , Rotavirus/fisiologia , Fatores de Transcrição SOX9/genética , Suínos , Regulação para Cima
10.
Int J Infect Dis ; 105: 277-285, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33596479

RESUMO

OBJECTIVES: Rotavirus (RV) genotypes vary geographically, and this can affect vaccine effectiveness (VE). This study investigated the genotype distribution of RV and explored VE before introducing the RV vaccine to the national immunization programme in Vietnam. METHODS: This hospital-based surveillance study was conducted at Children's Hospital 1, Ho Chi Minh City in 2013-2018. Stool samples and relevant data, including vaccination history, were collected from children aged <5 years who were hospitalized with gastroenteritis. RV was detected using enzyme immunoassays and then genotyped. Children aged ≥6 months were included in the VE analysis. RESULTS: Overall, 5176 children were included in this study. RV was detected in 2421 children (46.8%). RV positivity decreased over the study period and was associated with age, seasonality, location and previous vaccination. Among 1105 RV-positive samples, G3P[8] was the most prevalent genotype (43.1%), followed by G8P[8] (19.7%), G1P[8] (12.9%) and G2P[4] (12.9%). Overall VE was 69.7% [95% confidence interval (CI) 53.3-80.6%] in fully vaccinated children and 58.6% (95% CI 44.1-69.4%) in children who had received at least one dose of RV vaccine. VE was highest for G3P[8] (95% CI 75.1-84.5%) and lowest for G2P[4] (95% CI 32.4-57.2%). CONCLUSIONS: RV remains a major cause of acute gastroenteritis requiring hospitalization in southern Vietnam. The RV vaccine is effective, but its effectiveness varies with RV genotype.


Assuntos
Genótipo , Vacinas contra Rotavirus/imunologia , Rotavirus/genética , Rotavirus/imunologia , Vacinação/estatística & dados numéricos , Criança , Pré-Escolar , Fezes/virologia , Feminino , Gastroenterite/epidemiologia , Gastroenterite/prevenção & controle , Hospitalização , Humanos , Lactente , Masculino , Avaliação de Resultados em Cuidados de Saúde , Rotavirus/fisiologia , Vietnã/epidemiologia
11.
Nature ; 590(7847): 666-670, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33442061

RESUMO

A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Redobramento de Proteína , Rotavirus/metabolismo , Rotavirus/ultraestrutura , Internalização do Vírus , Animais , Antígenos Virais/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dissulfetos/química , Dissulfetos/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestrutura , Mutação , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Rotavirus/química , Rotavirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Vírion/química , Vírion/metabolismo , Vírion/ultraestrutura
12.
PLoS Pathog ; 17(1): e1009237, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33513201

RESUMO

Rotaviruses (RVs) are a leading cause of acute viral gastroenteritis in young children and livestock worldwide. Growing evidence suggests that host cellular glycans, such as histo-blood group antigens (HBGAs) and sialic acids (SA), are recognized by the RV surface protein VP4. However, a mechanistic understanding of these interactions and their effects on RV infection and pathogenesis is lacking. Here, we established a porcine crypt-derived 3D intestinal enteroids (PIEs) culture system which contains all intestinal epithelial cells identified in vivo and represents a unique physiologically functional model to study RV-glycan interactions in vitro. PIEs expressing different HBGAs (A+, H+, and A+/H+) were established and isolation, propagation, differentiation and RV infection conditions were optimized. Differentiated PIEs were infected with human RV (HRV) G1P[8] Wa, porcine RV (PRV) G9P[13], PRV Gottfried G4P[6] or PRV OSU G5P[7] virulent and attenuated strains and virus replication was measured by qRT-PCR. Our results indicated that virulent HRV G1P[8] Wa replicated to the highest titers in A+ PIEs, while a distinct trend was observed for PRV G9P[13] or G5P[7] with highest titers in H+ PIEs. Attenuated Wa and Gottfried strains replicated poorly in PIEs while the replication of attenuated G9P[13] and OSU strains in PIEs was relatively efficient. However, the replication of all 4 attenuate strains was less affected by the PIE HBGA phenotypes. HBGA synthesis inhibitor 2-F-Peracetyl-Fucose (2F) treatment demonstrated that HBGAs are essential for G1P[8] Wa replication; however, they may only serve as a cofactor for PRVs G9P[13] and OSU G5P[7]. Interestingly, contrasting outcomes were observed following sialidase treatment which significantly enhanced G9P[13] replication, but inhibited the growth of G5P[7]. These observations suggest that some additional receptors recognized by G9P[13] become unmasked after removal of terminal SA. Overall, our results confirm that differential HBGAs-RV and SA-RV interactions determine replication efficacy of virulent group A RVs in PIEs. Consequently, targeting individual glycans for development of therapeutics may not yield uniform results for various RV strains.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Gastroenterite/virologia , Infecções por Rotavirus/virologia , Rotavirus/patogenicidade , Ácidos Siálicos/metabolismo , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Células Epiteliais/virologia , Humanos , Intestino Delgado/virologia , Rotavirus/genética , Rotavirus/fisiologia , Suínos , Virulência , Replicação Viral
13.
Virus Res ; 291: 198193, 2021 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-33053412

RESUMO

Rotavirus (RV) replication occurs in cytoplasmic compartments, known as viroplasms, that are composed of viral and cellular proteins. Viroplasm formation requires RV nonstructural proteins NSP2 and NSP5 and cellular lipid droplets (LDs); however, the mechanisms required for viroplasm assembly remain largely unknown. We previously identified two conformationally-distinct forms of NSP2 (dNSP2, vNSP2) found in RV-infected cells that interact differentially with hypo- and hyperphosphorylated NSP5, respectively, and indicate a coordinated phosphorylation-dependent mechanism regulating viroplasm assembly. We also reported that phosphorylation of dNSP2 on serine 313 by the cellular kinase CK1α triggers the localization of vNSP2 to sites of viroplasm assembly and its association with hyperphosphorylated NSP5. To directly evaluate the role of CK1α-mediated NSP2 phosphorylation on viroplasm formation, we used a recently published plasmid-based reverse genetics method to generate a recombinant rotavirus (rRV) with a phosphomimetic NSP2 mutation (rRV NSP2 S313D). The rRV NSP2 S313D virus is significantly delayed in viroplasm formation, virus replication, and interferes with wild type RV replication during co-infection. The rRV NSP2 S313A virus was not rescued. Taking advantage of the delay in viroplasm formation, the NSP2 S313D phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (1) viroplasm assembly correlates with NSP5 hyperphosphorylation, and (2) that vNSP2 S313D co-localizes with RV-induced LDs without NSP5, suggesting that vNSP2 phospho-S313 is sufficient for interacting with LDs and may be the virus factor required for RV-induced LD formation. Further studies with the rRV NSP2 S313D virus are expected to reveal new aspects of viroplasm and LD initiation and assembly.


Assuntos
Citoplasma/virologia , Plasmídeos/genética , Genética Reversa/métodos , Rotavirus/genética , Rotavirus/fisiologia , Humanos , Fosforilação , Infecções por Rotavirus/virologia , Replicação Viral
14.
Biochem Biophys Res Commun ; 534: 740-746, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33250174

RESUMO

Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Luciferases/genética , Peptídeos/genética , Rotavirus/genética , Animais , Antivirais/farmacologia , Cricetinae , Humanos , Camundongos , Microrganismos Geneticamente Modificados , Testes de Neutralização , Ribavirina/farmacologia , Rotavirus/fisiologia , Infecções por Rotavirus/tratamento farmacológico , Infecções por Rotavirus/virologia , Proteínas não Estruturais Virais/genética , Replicação Viral/genética
15.
Nat Prod Res ; 35(6): 1014-1018, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31496276

RESUMO

The antiviral effect against RVA in cell culture was evaluated by using an aqueous extract of Patallus mollis sea cucumber, applying the titration methodology. This technique is used to measures the ability of the extract dilutions to inhibit the cytopathic effect (CPE) of the virus, expressed as percentage of inhibition (IP). The mean extract cytotoxic concentration (CC50) used in the antiviral assay was 27,042.10 µg/mL and the PI of the antiviral activity extract was greater than 99.9% for each concentration. To determine the viral action mode, the cells were previously treated with the extracts in different stages during the viral infection cycle. The result analysis suggests that the extract inhibits 99% of the virus during the absorption and viral inactivation phase. These results show the P. mollis extract has a remarkable antiviral effect against the RVA in cell culture. So that, it is crucial to investigate its action mechanisms.


Assuntos
Antivirais/farmacologia , Rotavirus/efeitos dos fármacos , Pepinos-do-Mar/anatomia & histologia , Animais , Efeito Citopatogênico Viral/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Rotavirus/fisiologia , Metabolismo Secundário/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
16.
Sci Rep ; 10(1): 22002, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33319798

RESUMO

Rotavirus (RV) is considered a major cause of acute viral gastroenteritis in young animals. RV is classified into nine species, five of which have been identified in pigs. Most studies worldwide have highlighted diarrhoea outbreaks caused by RVA, which is considered the most important RV species. In the present study, we described the detection and characterization of porcine RVB as a primary causative agent of diarrhoea outbreaks in pig herds in Brazil. The study showed a high frequency (64/90; 71.1%) of RVB diagnosis in newborn piglets associated with marked histopathological lesions in the small intestines. Phylogenetic analysis of the VP7 gene of wild-type RVB strains revealed a high diversity of G genotypes circulating in one geographic region of Brazil. Our findings suggest that RVB may be considered an important primary enteric pathogen in piglets and should be included in the routine differential diagnosis of enteric diseases in piglets.


Assuntos
Diarreia/epidemiologia , Diarreia/veterinária , Surtos de Doenças/veterinária , Infecções por Rotavirus/veterinária , Infecções por Rotavirus/virologia , Rotavirus/fisiologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Animais , Animais Recém-Nascidos , Sequência de Bases , Diarreia/patologia , Diarreia/virologia , Filogenia , Rotavirus/genética , Rotavirus/isolamento & purificação , Rotavirus/ultraestrutura , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/patologia , Suínos , Doenças dos Suínos/patologia , Proteínas Virais/metabolismo
17.
PLoS One ; 15(11): e0240714, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33170860

RESUMO

BACKGROUND: Despite contributing to a significant reduction in rotavirus associated diarrhoea in highly burdened low- and middle-income countries, live attenuated, oral rotavirus vaccines have lower immunogenicity and efficacy in these settings in comparison to more developed countries. Breastmilk has been implicated among factors contributing to this lowered oral vaccine efficacy. We conducted in-vitro experiments to investigate the inhibitory effects of maternal antibody and other non-antibody components in breastmilk on rotavirus vaccine strain (Rotarix) multiplication in MA104 cell culture system and assessed associations with in-vivo vaccine seroconversion in vaccinated infants. METHODS: Breastmilk samples were collected from mothers before routine rotavirus vaccination of their infant at 6 weeks of age. For each sample, whole breastmilk, purified IgA, purified IgG and IgG and IgA depleted breastmilk samples were prepared as exposure preparations. A 96 well microtitre plate was set up for each sample including a control in which only MA104 cells were grown as well as a virus control with MA104 cells and virus only. The outcome of interest was 50% inhibition dilution of each of the exposure preparations calculated as the titer at which 50% of virus dilution was achieved. Samples from 30 women were tested and correlated to vaccine seroconversion status of the infant. HIV status was also correlated to antiviral breastmilk proteins. RESULTS: The mean 50% inhibitory dilution titer when whole breastmilk was added to virus infected MA104 cells was 14.3 (95% CI: 7.1, 22.7). Incubation with purified IgG resulted in a mean 50% inhibitory dilution of 5 (95%CI -1.6, 11.6). Incubating with purified IgA resulted in a mean 50% inhibitory dilution of 6.5 (95% CI -0.7, 13.7) and IgG and IgA depleted breastmilk did not yield any inhibition with a titer of 1.06 (95%CI 0.9, 1.2). Higher milk IgA levels contributed to a failure of infants to seroconvert. HIV was also not associated with any antiviral breastmilk proteins. DISCUSSION AND CONCLUSION: Whole breastmilk and breastmilk purified IgG and IgA fractions showed inhibitory activity against the rotavirus vaccine Rotarix™ whilst IgA and IgG depleted breastmilk with non-antibody breastmilk fraction failed to show any inhibition activity in-vitro. These findings suggest that IgA and IgG may have functional inhibitory properties and indicates a possible mechanism of how mothers in rotavirus endemic areas with high titres of IgA and IgG may inhibit viral multiplication in the infant gut and would potentially contribute to the failure of their infants to serocovert. There was not association of HIV with either lactoferrin, lactadherin or tenascin-C concentrations.


Assuntos
Imunoglobulina A/farmacologia , Imunoglobulina G/farmacologia , Leite Humano/imunologia , Vacinas contra Rotavirus/imunologia , Rotavirus/fisiologia , Administração Oral , Linhagem Celular , Feminino , Humanos , Técnicas In Vitro , Lactente , Rotavirus/efeitos dos fármacos , Rotavirus/imunologia , Soroconversão , Vacinas Atenuadas/imunologia , Replicação Viral
18.
Cell ; 183(7): 1867-1883.e26, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33248023

RESUMO

Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic T cell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and T cells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.


Assuntos
Atresia Biliar/imunologia , Atresia Biliar/terapia , Fígado/imunologia , Animais , Antígenos CD20/metabolismo , Linfócitos B/imunologia , Atresia Biliar/sangue , Atresia Biliar/tratamento farmacológico , Biópsia , Receptor 1 de Quimiocina CX3C/metabolismo , Morte Celular , Linhagem Celular , Proliferação de Células , Transdiferenciação Celular , Criança , Pré-Escolar , Estudos de Coortes , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/metabolismo , Lactente , Inflamação/patologia , Células Matadoras Naturais/imunologia , Macrófagos do Fígado/patologia , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/complicações , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Depleção Linfocítica , Linfopoese , Masculino , Camundongos Endogâmicos BALB C , Fagocitose , RNA/metabolismo , Rituximab/administração & dosagem , Rituximab/farmacologia , Rituximab/uso terapêutico , Rotavirus/fisiologia , Análise de Célula Única , Células Th1/imunologia , Células Th17/imunologia
19.
Science ; 370(6519)2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33214249

RESUMO

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5'-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.


Assuntos
Difosfato de Adenosina/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Infecções por Rotavirus/metabolismo , Rotavirus/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Feminino , Células HEK293 , Humanos , Jejuno/metabolismo , Jejuno/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Comunicação Parácrina , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo
20.
Sci Rep ; 10(1): 20296, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219309

RESUMO

Bats are natural reservoirs for potential zoonotic viruses. In this study, next-generation sequencing was performed to obtain entire genome sequences of picornavirus from a picornavirus-positive bat feces sample (16BF77) and to explore novel viruses in a pooled bat sample (16BP) from samples collected in South Korea, 2016. Fourteen mammalian viral sequences were identified from 16BF77 and 29 from 16BP, and verified by RT-PCR. The most abundant virus in 16BF77 was picornavirus. Highly variable picornavirus sequences encoding 3Dpol were classified into genera Kobuvirus, Shanbavirus, and an unassigned group within the family Picornaviridae. Amino acid differences between these partial 3Dpol sequences were ≥ 65.7%. Results showed that one bat was co-infected by picornaviruses of more than two genera. Retrovirus, coronavirus, and rotavirus A sequences also were found in the BP sample. The retrovirus and coronavirus genomes were identified in nine and eight bats, respectively. Korean bat retroviruses and coronavirus demonstrated strong genetic relationships with a Chinese bat retrovirus (RfRV) and coronavirus (HKU5-1), respectively. A co-infection was identified in one bat with a retrovirus and a coronavirus. Our results indicate that Korean bats were multiply infected by several mammal viruses.


Assuntos
Quirópteros/virologia , Fezes/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Boca/virologia , Vírus de RNA/genética , Animais , Encéfalo/virologia , Coronavirus/classificação , Coronavirus/genética , Coronavirus/fisiologia , Geografia , Interações Hospedeiro-Patógeno , Intestinos/virologia , Fígado/virologia , Pulmão/virologia , Filogenia , Picornaviridae/classificação , Picornaviridae/genética , Picornaviridae/fisiologia , Vírus de RNA/classificação , Vírus de RNA/fisiologia , República da Coreia , Retroviridae/classificação , Retroviridae/genética , Retroviridae/fisiologia , Rotavirus/classificação , Rotavirus/genética , Rotavirus/fisiologia
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