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1.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934842

RESUMO

The eIF4F complex is a translation initiation factor that closely regulates translation in response to a multitude of environmental conditions including viral infection. How translation initiation factors regulate rotavirus infection remains poorly understood. In this study, the knockdown of the components of the eIF4F complex using shRNA and CRISPR/Cas9 were performed, respectively. We have demonstrated that loss-of-function of the three components of eIF4F, including eIF4A, eIF4E and eIF4G, remarkably promotes the levels of rotavirus genomic RNA and viral protein VP4. Consistently, knockdown of the negative regulator of eIF4F and programmed cell death protein 4 (PDCD4) inhibits the expression of viral mRNA and the VP4 protein. Mechanically, we confirmed that the silence of the eIF4F complex suppressed the protein level of IRF1 and IRF7 that exert potent antiviral effects against rotavirus infection. Thus, these results demonstrate that the eIF4F complex is an essential host factor restricting rotavirus replication, revealing new targets for the development of new antiviral strategies against rotavirus infection.


Assuntos
Fator de Iniciação 4F em Eucariotos/metabolismo , Regulação da Expressão Gênica , Fator Regulador 1 de Interferon/genética , Fator Regulador 7 de Interferon/genética , Infecções por Rotavirus/genética , Antivirais/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Células CACO-2 , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Proteínas de Ligação a RNA/metabolismo
2.
PLoS One ; 14(1): e0211002, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30673764

RESUMO

Rotavirus A (RVA) infection is a major cause of diarrhea-related illness in young children. RVA is also one of the most common enteric viruses detected on pig farms and contributes to substantial morbidity and mortality in piglets. Long-term multi-site surveillance of RVA on Thai swine farms to determine the diversity of RVA strains in circulation is currently lacking. In this study, we characterized the 11 segments of the RVA genome from 24 Thai porcine RVA strains circulating between 2011 and 2016. We identified G9 (15/24) and P[13] (12/24) as the dominant genotypes. The dominant G and P combinations were G9P[13] (n = 6), G9P[23] (n = 6), G3P[13] (n = 5), G9P[19] (n = 3), G4P[6] (n = 2), G4P[19] (n = 1), and G5P[13] (n = 1). Genome constellation of the Thai strains showed the predominance of Wa-like genotype (Gx-P[x]-I1/I5-R1-C1-M1-A8-N1-T1/T7-E1/E9-H1) with evidence of reassortment between the porcine and human RVA strains (e.g., G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[19]-I5-R1-C1-M1-A8-N1-T7-E9-H1). To assess the potential effectiveness of rotavirus vaccination, the Thai RVA strains were compared to the RVA strains represented in the swine rotavirus vaccine, which showed residue variations in the antigenic epitope on VP7 and shared amino acid identity below 90% for G4 and G5 strain. Several previous studies suggested these variations might effect on virus neutralization specificity and vaccine efficacy. Our study illustrates the importance of RVA surveillance beyond the G/P genotyping on commercial swine farms, which is crucial for controlling viral transmission.


Assuntos
Antígenos Virais/genética , Epitopos/genética , Infecções por Rotavirus/genética , Rotavirus/genética , Animais , Genoma Viral , Humanos , Infecções por Rotavirus/virologia , Suínos , Doenças dos Suínos
3.
Proc Natl Acad Sci U S A ; 115(51): E12015-E12023, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30509975

RESUMO

The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (i) >90% decrease in RV replication, (ii) disrupted vNSP2 and NSP5 interaction, (iii) dispersion of vNSP2 throughout the cytoplasm, and (iv) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.


Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Rotavirus/genética , Rotavirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Animais , Caseína Quinase Ialfa/genética , Caseína Quinase Ialfa/metabolismo , Linhagem Celular , Cristalografia por Raios X , Citoplasma/metabolismo , Citoplasma/virologia , Inativação Gênica , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Modelos Moleculares , Fosforilação , Fosfotransferases/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/genética , Infecções por Rotavirus/genética , Infecções por Rotavirus/metabolismo , Proteínas não Estruturais Virais/genética
4.
Elife ; 72018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30460894

RESUMO

Rotaviruses (RVs), a leading cause of severe diarrhea in young children and many mammalian species, have evolved multiple strategies to counteract the host innate immunity, specifically interferon (IFN) signaling through RV non-structural protein 1 (NSP1). However, whether RV structural components also subvert antiviral response remains under-studied. Here, we found that MAVS, critical for the host RNA sensing pathway upstream of IFN induction, is degraded by the RV RNA methyl- and guanylyl-transferase (VP3) in a host-range-restricted manner. Mechanistically, VP3 localizes to the mitochondria and mediates the phosphorylation of a previously unidentified SPLTSS motif within the MAVS proline-rich region, leading to its proteasomal degradation and blockade of IFN-λ production in RV-infected intestinal epithelial cells. Importantly, VP3 inhibition of MAVS activity contributes to enhanced RV replication and to viral pathogenesis in vivo. Collectively, our findings establish RV VP3 as a viral antagonist of MAVS function in mammals and uncover a novel pathogen-mediated inhibitory mechanism of MAVS signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Capsídeo/genética , Interações Hospedeiro-Patógeno , Interferons/genética , Infecções por Rotavirus/genética , Rotavirus/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células COS , Proteínas do Capsídeo/imunologia , Caspase 1/genética , Caspase 1/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Células HEK293 , Células HT29 , Humanos , Íleo/imunologia , Íleo/virologia , Interferons/imunologia , Camundongos , Células NIH 3T3 , Proteínas NLR/genética , Proteínas NLR/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Rotavirus/crescimento & desenvolvimento , Rotavirus/imunologia , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/virologia , Transdução de Sinais
5.
Sci Rep ; 8(1): 12961, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154494

RESUMO

Human strains of rotavirus A (RVAs) recognize fucosylated glycans belonging to histo-blood group antigens (HBGAs) through their spike protein VP8*. Lack of these ligands due to genetic polymorphisms is associated with resistance to gastroenteritis caused by P[8] genotype RVAs. With the aim to delineate the contribution of HBGAs in the process, we analyzed the glycan specificity of VP8* proteins from various P genotypes. Binding to saliva of VP8* from P[8] and P[4] genotypes required expression of both FUT2 and FUT3 enzymes, whilst binding of VP8* from the P[14] genotype required FUT2 and A enzymes. We further defined a glycan motif, GlcNAcß3Galß4GlcNAc, recognized by P[6] clinical strains. Conversion into Lewis antigens by the FUT3 enzyme impaired recognition, explaining their lower binding to saliva of Lewis positive phenotype. In addition, the presence of neutralizing antibodies was associated with the presence of the FUT2 wild type allele in sera from young healthy adults. Nonetheless, in vitro infection of transformed cell lines was independent of HBGAs expression, indicating that HBGAs are not human RV receptors. The match between results from saliva-based binding assays and the epidemiological data indicates that the polymorphism of human HBGAs controls susceptibility to RVAs, although the exact mechanism remains unclear.


Assuntos
Antígenos de Grupos Sanguíneos , Gastroenterite , Proteínas de Ligação a RNA , Infecções por Rotavirus , Rotavirus , Proteínas não Estruturais Virais , Adolescente , Adulto , Animais , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Células CACO-2 , Feminino , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Gastroenterite/genética , Gastroenterite/metabolismo , Gastroenterite/virologia , Humanos , Masculino , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Rotavirus/genética , Rotavirus/metabolismo , Infecções por Rotavirus/genética , Infecções por Rotavirus/metabolismo , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
6.
Virus Res ; 253: 28-37, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29859235

RESUMO

Active virus-host interactions determine the outcome of pathogen invasions. It has been shown that in isolated dendritic cells (DCs), rotavirus can induce the expression of tumor necrosis factor α (TNF-α), a vital cytokine mediating host immune responses. However, the role of TNF-α in rotavirus infection is unknown. In this study, we demonstrated that TNF-α has potent anti-rotavirus effects, independent of type I interferon production. Blocking of TNF-α by infliximab, a clinically available TNFα antibody, totally abrogated this effect. Mechanistic studies revealed that the anti-rotavirus effect of TNF-α was achieved by NFκB-regulated genes via the activation of classical nuclear factor κB (NF-κB) signaling. Our study reveals the pivotal role and the mechanism-of-actions of TNF-α in the host defense against rotavirus. Thus, this knowledge may contribute to the better understanding of the complexity of rotavirus-host interactions.


Assuntos
NF-kappa B/imunologia , Infecções por Rotavirus/imunologia , Rotavirus/fisiologia , Fator de Necrose Tumoral alfa/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , NF-kappa B/genética , Rotavirus/genética , Infecções por Rotavirus/genética , Infecções por Rotavirus/virologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética
7.
J Virol ; 92(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29769336

RESUMO

Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), which are nucleated by two essential viral nonstructural proteins, NSP2 and NSP5. However, the precise composition of the VM, the intracellular localization of host proteins during virus infection, and their association with VMs or role in rotavirus growth remained largely unexplored. Mass spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs), AU-rich element-binding proteins (ARE-BPs), and cytoplasmic proteins from uninfected MA104 cell extracts in the pulldown (PD) complexes of the purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of PD complexes from RNase-treated and untreated cell extracts, analyses of coimmunoprecipitation complexes using RNase-treated infected cell lysates, and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA independent. Time course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed a surprising sequestration of the majority of the relocalized host proteins in viroplasms. Analyses of ectopic overexpression and small interfering RNA (siRNA)-mediated downregulation of expression revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces the cytoplasmic relocalization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than is currently understood.IMPORTANCE Rotavirus replicates exclusively in the cytoplasm. Knowledge on the relocalization of nuclear proteins to the cytoplasm or the role(s) of host proteins in rotavirus infection is very limited. In this study, it is demonstrated that rotavirus infection induces the cytoplasmic relocalization of a large number of nuclear RNA-binding proteins (hnRNPs and AU-rich element-binding proteins). Except for a few, most nuclear hnRNPs and ARE-BPs, nuclear transport proteins, and some cytoplasmic proteins directly interact with the viroplasmic proteins NSP2 and NSP5 in an RNA-independent manner and become sequestered in the viroplasms of infected cells. The host proteins differentially affected viral gene expression and virus growth. This study demonstrates that rotavirus induces the relocalization and sequestration of a large number of host proteins in viroplasms, affecting host processes in both compartments and generating conditions conducive for virus growth in the cytoplasm of infected cells.


Assuntos
Citoplasma , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas , Interações Hospedeiro-Parasita , Infecções por Rotavirus , Rotavirus/fisiologia , Animais , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/virologia , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Infecções por Rotavirus/genética , Infecções por Rotavirus/metabolismo
8.
Sci Rep ; 8(1): 67, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29311575

RESUMO

Detection of viral infection by host cells leads to secretion of type I interferon, which induces antiviral gene expression. The class I major histocompatibility complex (MHCI) is required for viral antigen presentation and subsequent infected cell killing by cytotoxic T lymphocytes. STAT1 activation by interferon can induce NLRC5 expression, promoting MHCI expression. Rotavirus, an important pathogen, blocks interferon signalling through inhibition of STAT1 nuclear translocation. We assessed MHCI expression in HT-29 intestinal epithelial cells following rotavirus infection. MHCI levels were upregulated in a partially type I interferon-dependent manner in bystander cells lacking rotavirus antigen, but not in infected cells. MHCI and NLRC5 mRNA expression also was elevated in bystander, but not infected, cells, suggesting a transcriptional block in infected cells. STAT1 was activated in bystander and infected cells, but showed nuclear localisation in bystander cells only. Overall, the lack of MHCI upregulation in rotavirus-infected cells may be at least partially due to rotavirus blockade of interferon-induced STAT1 nuclear translocation. The reduced MHCI protein levels in infected cells support the existence of an additional, non-transcriptional mechanism that reduces MHCI expression. It is possible that rotavirus also may suppress MHCI expression in vivo, which might limit T cell-mediated killing of rotavirus-infected enterocytes.


Assuntos
Antígenos Virais/imunologia , Expressão Gênica , Genes MHC Classe I , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Infecções por Rotavirus/genética , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Animais , Antígenos Virais/genética , Biomarcadores , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Interferon Tipo I/metabolismo , Mucosa Intestinal/virologia , Camundongos , Infecções por Rotavirus/virologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Replicação Viral
9.
Indian J Pediatr ; 85(5): 364-368, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29185230

RESUMO

OBJECTIVES: To determine the prevalence and genotype distribution of rotaviruses in children presenting to authors' hospital in Middle Black Sea region of Turkey. The results may supply important information about vaccine studies in Turkey. METHODS: Rotavirus antigen was detected by latex agglutination test and rotavirus RNA was detected by RT-PCR test. On the other hand, rotavirus positive samples were genotyped by semi-nested multiplex polymerase chain reaction. RESULTS: The highest rate of rotavirus positivity (46.9%) was observed among children in the 13 to 24 mo age group. All the positive-strains were in G1-4, G8-9, P [4], P [8], and P [9] genotypes. The most common G and P combination in present study was G9P[8] (n = 24, 28.9%). CONCLUSIONS: The present results indicated that the most prevalent genotypes were G1, G9, P8, G9P[8] and G1P[8] in authors' region. Rotavirus vaccines used in this region must include mainly these genotypes.


Assuntos
Genótipo , Epidemiologia Molecular , Infecções por Rotavirus/genética , Antígenos Virais , Pré-Escolar , Fezes , Gastroenterite , Humanos , Lactente , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rotavirus/isolamento & purificação , Infecções por Rotavirus/epidemiologia , Turquia
10.
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3212-3225, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28890396

RESUMO

MicroRNAs (miRNAs) are short RNAs of approximately 22 nucleotides that post-transcriptionally regulate gene expression by controlling mRNA stability or translation. They play critical roles in intricate networks of host-pathogen interactions and innate immunity. Rotaviruses (RVs) are the leading cause of severe diarrhea among infants and young children worldwide. This study was undertaken to demonstrate the importance of cellular miRNAs during RV (human Wa RV or Rhesus RV) strains infection. Twenty-nine differentially regulated miRNAs were identified during RV infection, and miR-525-3p was downregulated and validated by quantitative real-time polymerase chain reaction (qRT-PCR). MiR-525-3p mimic inhibited RV replication in dose-dependent manner. Correspondingly, the miR-525-3p inhibitors enhanced RV replication. We confirmed that miR-525-3p was complementary to the 3' untranslated region (UTR) of nonstructural protein 1(NSP1) of RV (Wa or Rhesus) strains. Interestingly, miR-525-3p induced type I interferon (IFN) expression and proinflammatory cytokines during RV infection through IFN regulatory factor (IRF) 3/IRF7 and NF-κB activation, which can induce an antiviral state to further suppress RV infection. In addition, RV suppressed miR-525-3p expression to evade host innate immunity through the action of the RV protein NSP1. These results suggest that miR-525-3p has the potential to be used as an antiviral therapeutic against RV infection.


Assuntos
MicroRNAs/imunologia , Infecções por Rotavirus/genética , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Proteínas não Estruturais Virais/imunologia , Replicação Viral/imunologia , Regiões 3' não Traduzidas , Células CACO-2 , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Células HEK293 , Células HT29 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Transporte Proteico/imunologia , Rotavirus/genética , Transdução de Sinais , Proteínas não Estruturais Virais/genética , Ensaio de Placa Viral
11.
Benef Microbes ; 8(4): 645-656, 2017 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-28670908

RESUMO

Rotavirus-related diarrhoea is considered one of the most important diseases in field animal production. In addition to the classic vaccine strategy, a number of studies have utilised probiotics, such as Lactobacillus rhamnosus GG (LGG), to prevent rotavirus-induced diarrhoea. Although it has been partially revealed that Toll-like receptors (TLRs) are involved in the LGG-mediated protection against rotavirus infection, the details of the underlying immunologic mechanisms remain largely unknown. In this study, three-to-four-week-old female BALB/c mice were divided into three groups and orally administered phosphate buffered saline (PBS), PBS plus rotavirus or LGG plus rotavirus, respectively. The differentiation and maturation of dendritic cells (DCs) were then determined by FACS, the expression levels of TLR-3 and nuclear factor kappa beta (NF-κB) were evaluated using real time PCR, and the production of inflammatory cytokines in mesenteric lymph nodes (MLNs) were determined by ELISA. The results demonstrated that rotavirus infection significantly increased the percentage of CD11c+CD11b+CD8a- DCs and decreased the percentage of CD11c+CD11b-CD8a+ DCs in MLNs. By contrast, the presence of LGG significantly decreased the percentage of CD11c+CD11b+CD8a- DCs and increased the percentage of CD11c+CD11b-CD8a+ DCs, which indicates that the differentiation of DCs is involved in the protective effects of LGG. Rotavirus infection also resulted in the increased expression of surface markers such as CD40, CD80 and MHC-II in DCs, and the administration of LGG significantly increased the expression level further. The mRNA levels of TLR-3 and NF-κB in the intestine and MLNs were also significantly increased in the presence of rotavirus, which was further increased in the presence of LGG. The production of inflammatory cytokines was also determined, and the results showed that rotavirus caused the increased production of interleukin (IL)-12 and tumour necrosis factor alpha; this effect was further enhanced by LGG. Meanwhile, although rotavirus infection led to the increased production of IL-6 and IL-10, the presence of LGG significantly decreased the mRNA levels of these cytokines. By contrast, rotavirus infection resulted in the decreased production of interferon gamma (IFN-γ), and the administration of LGG significantly increased the levels of IFN-γ. Taken together, the protective effects of LGG were partially due to the modulation of the differentiation and maturation of DCs, the increased production of TLR-3 and NF-κB, and the modulation of inflammatory cytokines.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Lactobacillus rhamnosus/fisiologia , Probióticos/administração & dosagem , Infecções por Rotavirus/tratamento farmacológico , Rotavirus/fisiologia , Animais , Células Dendríticas/efeitos dos fármacos , Feminino , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Rotavirus/genética , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/fisiopatologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
12.
Sci Rep ; 7(1): 6412, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28743921

RESUMO

The genetic susceptibility to and vaccine effectiveness against rotavirus gastroenteritis were different in distinct ethnic groups. The case-control study was aimed to evaluate the effectiveness of rotavirus vaccines and associations between the histo-blood group antigens and susceptibility to rotavirus infections in a Taiwanese population. Cases were children <18 years old who were hospitalized because of laboratory-confirmed rotavirus infection. Controls were healthy children matched to cases by age and gender. The secretor status and Lewis antigen and ABO types were determined by molecular methods. A total of 68 cases and 133 controls were included. Rotavirus immunization was recorded in 8 (12%) cases and 77 (58%) controls, indicating a vaccine effectiveness of 90.3% (95% confidence interval [CI], 78.1% - 95.7%). The secretor and Lewis-positive genotypes were independently associated with increased risk of rotavirus infections (matched odds ratio [mOR] 28.5, 95% CI 2.94-277, P = 0.003 and mOR 16.8, 95% CI 1.08-2601, P = 0.04, respectively). The distribution of ABO blood types did not differ significantly between cases and controls (P = 0.47). In conclusion, Taiwanese children with the secretor genotype and Lewis-positive genotype were at increased risk of moderate-to-severe rotavirus infections. The illness can be effectively prevented by immunization in this population.


Assuntos
Gastroenterite/virologia , Infecções por Rotavirus/genética , Vacinas contra Rotavirus/uso terapêutico , Sistema ABO de Grupos Sanguíneos/genética , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Fucosiltransferases/genética , Gastroenterite/genética , Gastroenterite/prevenção & controle , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Polimorfismo de Nucleotídeo Único , Infecções por Rotavirus/prevenção & controle , Taiwan
13.
Vet Q ; 37(1): 252-261, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28643555

RESUMO

BACKGROUND: Rotavirus C (RVC), a known etiological agent of diarrheal outbreaks, mainly inflicts swine population globally with sporadic incidence in human, cattle, ferret, mink and dog. OBJECTIVE: To demonstrate the presence of RVC in Indian swine population and characterization of its selected structural (VP6) and non-structural (NSP4 and NSP5) genes. METHODS: A total of 108 diarrheic samples from different regions of India were used. Isolated RNA was loaded onto polyacrylamide gel to screen for the presence of RVs through the identification of specific electrophoretic genomic migration pattern. To characterize the RVC strains, VP6 gene and NSP4 and NSP5 genes were amplified, sequenced and analyzed. RESULTS: Based on VP6 gene specific diagnostic RT-PCR, the presence of RVC was confirmed in 12.0% (13/108) piglet fecal specimens. The nucleotide sequence analysis of VP6 gene, encoding inner capsid protein, from selected porcine RVC (PoRVC) strains revealed more than 93% homologies to human RVC strains (HuRVC) of Eurasian origin. These strains were distant from hitherto reported PoRVCs and clustered with HuRVCs, owning I2 genotype. However, the two non-structural genes, i.e. NSP4 and NSP5, of these strains were found to be of swine type, signifying a re-assortment event that has occurred in the Indian swine population. CONCLUSION: The findings indicate the presence of human-like RVC in Indian pigs and division of RVC clade with I2 genotype into further sub-clades. To the best of our knowledge, this appears to be the first report of RVC in Indian swine population. Incidence of human-like RVC VP6 gene in swine supports its subsequent zoonotic prospective.


Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Diarreia/veterinária , Infecções por Rotavirus/veterinária , Rotavirus/genética , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Animais , Antígenos Virais/isolamento & purificação , Proteínas do Capsídeo/isolamento & purificação , Bases de Dados de Ácidos Nucleicos , Diarreia/virologia , Fezes/virologia , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Humanos , Índia , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rotavirus/isolamento & purificação , Infecções por Rotavirus/genética , Infecções por Rotavirus/virologia , Análise de Sequência , Suínos , Toxinas Biológicas/genética , Toxinas Biológicas/isolamento & purificação , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação
14.
Proc Natl Acad Sci U S A ; 114(18): E3642-E3651, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28416666

RESUMO

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.


Assuntos
Dinaminas/metabolismo , Endocitose , Neuropeptídeos/metabolismo , Infecções por Rotavirus/metabolismo , Rotavirus/metabolismo , Internalização do Vírus , Animais , Cricetinae , Dinaminas/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Rotavirus/genética , Infecções por Rotavirus/genética
15.
Virus Genes ; 53(3): 367-376, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28289928

RESUMO

Rotavirus (RV) is the most common cause of severe gastroenteritis and fatal dehydration in human infants and neonates of different species. However, the pathogenesis of rotavirus-induced diarrhea is poorly understood. Secretory diarrhea caused by rotavirus may lead to a combination of excessive secretion of fluid and electrolytes into the intestinal lumen. Fluid absorption in the small intestine is driven by Na+-coupled transport mechanisms at the luminal membrane, including Na+/H+ exchanger (NHE). Here, we performed qRT-PCR to detect the transcription of NHEs. Western blotting was employed for protein detection. Furthermore, immunocytochemistry was used to validate the NHE's protein expression. Finally, intracellular Ca2+ concentration was detected by confocal laser scanning microscopy. The results demonstrated that the NHE6 mRNA and protein expressed in the human colon adenocarcinoma cell line (Caco-2). Furthermore, RV-Wa induced decreased expression of the NHE1 and NHE6 in Caco-2 cell in a time-dependent manner. In addition, intracellular Ca2+ concentration in RV-Wa-infected Caco-2 cells was higher than that in the mock-infected cells. Furthermore, RV-Wa also can downregulate the expression of calmodulin (CaM) and calmodulin kinase II (CaMKII) in Caco-2 cells. These findings provides important insights into the mechanisms of rotavirus-induced diarrhea. Further studies on the underlying pathophysiological mechanisms that downregulate NHEs in RV-induced diarrhea are required.


Assuntos
Regulação para Baixo , Regulação da Expressão Gênica/genética , Rotavirus/patogenicidade , Trocador 1 de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/genética , Animais , Células CACO-2 , Cálcio/análise , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular , Citosol/química , Diarreia/virologia , Humanos , Macaca mulatta , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Infecções por Rotavirus/genética , Infecções por Rotavirus/virologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Transcrição Genética
16.
Sci Rep ; 7: 45559, 2017 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-28358023

RESUMO

Host genetic factors, such as histo-blood group antigens (HBGAs), are associated with susceptibility to norovirus (NoV) and rotavirus (RV) infections. Recent advances point to the gut microbiome as a key player necessary for a viral pathogen to cause infection. In vitro NoV attachment to host cells and resulting infections have been linked to interactions with certain bacterial types in the gut microbiota. We investigated the relationship between host genotype, gut microbiota, and viral infections. Saliva and fecal samples from 35 adult volunteers were analysed for secretor status genotype, the gut microbiota composition by 16S rRNA gene sequencing, and salivary IgA titers to NoV and RV. Higher levels of IgA against NoV and RV were related to secretor-positive status. No significant differences were found between the FUT2 genotype groups, although the multivariate analysis showed a significant impact of host genotype on specific viral susceptibilities in the microbiome composition. A specific link was found between the abundance of certain bacterial groups, such as Faecalibacterium and Ruminococcus spp., and lower IgA titers against NoV and RV. As a conclusion, we can state that there is a link between host genetics, gut microbiota, and susceptibility to viral infections in humans.


Assuntos
Infecções por Caliciviridae/genética , Infecções por Caliciviridae/microbiologia , Fucosiltransferases/genética , Microbioma Gastrointestinal , Infecções por Rotavirus/genética , Infecções por Rotavirus/microbiologia , Adulto , Fezes , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Saliva
17.
Proc Natl Acad Sci U S A ; 114(4): E570-E579, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28069942

RESUMO

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/ß) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine.


Assuntos
Interferons/genética , Intestino Delgado/imunologia , Infecções por Rotavirus/genética , Animais , Linhagem Celular , Humanos , Imunidade Inata , Interferons/imunologia , Rotavirus/fisiologia , Infecções por Rotavirus/imunologia , Análise de Sequência de RNA , Replicação Viral
18.
Rev Fac Cien Med Univ Nac Cordoba ; 74(4): 345-349, 2017 12 21.
Artigo em Espanhol | MEDLINE | ID: mdl-29902141

RESUMO

Vaccination against rotavirus in Argentina is obligatory from January 2015. From 418 stools in children with acute diarrhea were collected from 2000 to 2010 in city of La Rioja, Argentina, Rotavirus was detected. The 90 rotavirus positive stools samples (21.53%) were amplified by RT-PCR and genotyped by PCR-Mix for G1, G2, G3, G4, G9 and G8. The results show that during 2000-2003, the most frequent genotype was G1 and but since 2008, the G2, G3 and G9 genotypes in single and mixed infections were detected. In La Rioja, the vaccines could prevent dehydration in older children the year but children under one year could be vulnerable to the emergence of strains with genic aberrations due to the implementation of vaccination in our region.


Assuntos
Diarreia/virologia , Infecções por Rotavirus/virologia , Vacinas contra Rotavirus/normas , Rotavirus/isolamento & purificação , Doença Aguda , Argentina , Criança , Pré-Escolar , Demografia/estatística & dados numéricos , Diarreia/genética , Fezes/virologia , Genótipo , Hospitalização/estatística & dados numéricos , Humanos , Lactente , Infecções por Rotavirus/genética
19.
Virus Genes ; 53(2): 179-189, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28000081

RESUMO

Rotavirus (RV) is the predominant cause of infantile gastroenteritis with multiple pathogenic factors, among which enterotoxin NSP4 is the most significant factor. NSP4 has been shown to induce elevation of the intracellular calcium concentration, alteration of the cytoskeleton organization, and cytopathic effect among other processes. However, increasing evidence suggests that RVs can escape from the gastrointestinal tract and invade other organs and tissues to cause extra-intestinal diseases. In this study, we investigated whether NSP4 has a pathogenic effect on extra-intestinal cells and examined possible molecular mechanisms in vitro. Our results showed that NSP486-175 has important functions in increasing intracellular Ca2+ concentration, altering actin cytoskeleton organization and inducing cellular damage in H9c2(2-1) cells. Blockade of the integrin α2 receptor using a specific antibody attenuated the increase of intracellular Ca2+ concentration and alleviated the observed cytopathic effects, suggesting that integrin α2 may be a receptor for NSP486-175. Collectively, these results indicate that extracellular NSP486-175 can induce elevation of the intracellular Ca2+ concentration, cause cytotoxic changes, and disrupt the actin cytoskeleton in H9c2(2-1) cells, which may constitute a possible mechanism for RV extra-intestinal pathogenesis.


Assuntos
Citoesqueleto de Actina/genética , Glicoproteínas/metabolismo , Infecções por Rotavirus/genética , Rotavirus/genética , Toxinas Biológicas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Citoesqueleto de Actina/patologia , Animais , Células CACO-2 , Cálcio/metabolismo , Trato Gastrointestinal/patologia , Trato Gastrointestinal/virologia , Glicoproteínas/genética , Humanos , Intestinos/patologia , Intestinos/virologia , Rotavirus/patogenicidade , Infecções por Rotavirus/patologia , Infecções por Rotavirus/virologia , Toxinas Biológicas/genética , Proteínas não Estruturais Virais/genética
20.
Sci Rep ; 6: 34591, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27708367

RESUMO

Rotaviruses are known to recognize human histo-blood group antigens (HBGAs) as a host ligand that is believed to play an important role in rotavirus host susceptibility and host range. In this study, paired fecal and saliva samples collected from children with viral gastroenteritis, as well as paired serum and saliva samples collected from the general population in south China were studied to evaluate potential association between rotavirus infections and human HBGA phenotypes. Rotavirus was detected in 75 (28%) of 266 fecal samples and P[8] rotaviruses were found to be the predominant genotype. The HBGA phenotypes of the rotavirus-infected children were determined through their saliva samples. Secretor statuses were found to correlate with the risk of rotavirus infection and all P[8]/P[4] rotavirus infected children were secretors. Accordingly, recombinant VP8* proteins of the P[8]/P[4] rotaviruses bound saliva samples from secretor individuals. Furthermore, correlation between serum P[8]/P[4]-specific IgG and host Lewis and secretor phenotypes has been found among 206 studied serum samples. Our study supported the association between rotavirus infection and the host HBGA phenotypes, which would help further understanding of rotavirus host range and epidemiology.


Assuntos
Fezes/virologia , Genótipo , Infecções por Rotavirus/genética , Infecções por Rotavirus/metabolismo , Rotavirus/genética , Rotavirus/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Infecções por Rotavirus/patologia
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