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1.
Nihon Saikingaku Zasshi ; 74(3): 167-175, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31787706

RESUMO

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum and related species cause botulism, a neuroparalytic disease associated with a high mortality. BoNTs are always produced as large protein complexes (progenitor toxin complexes, PTCs) through association with non-toxic components (NAPs) including hemagglutinin (HA) and non-toxic non-hemagglutinin (NTNHA). Food-borne botulism is caused by the ingestion of PTCs. PTCs in the gastrointestinal tract cross the intestinal epithelial barrier, enter the blood stream, and reach the nerve endings, where BoNTs cleave the SNAREs required for vesicle fusion. Consequently, BoNTs inhibit neurotransmitter release and cause paralysis. To cause food-borne botulism, BoNTs must traverse the intestinal epithelial barrier. However, the mechanism used to cross this barrier remains unclear. Using an in vitro epithelial barrier system, we previously showed that the interaction of HA with E-cadherin results in disruption of tight junctions. Furthermore, we previously reported that microfold (M) cells in the follicle-associated epithelium (FAE) of mouse Peyer's patches (PPs) are major sites where type A1 BoNT breaches the intestinal epithelial barrier. Here, I would like to demonstrate an ingenious invasion mechanism of the BoNT complex.


Assuntos
Toxinas Botulínicas/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal , Mucosa Intestinal/citologia , Complexos Multiproteicos/metabolismo , Neurotoxinas/metabolismo , Animais , Caderinas , Células Cultivadas , Cães , Hemaglutininas , Humanos , Camundongos , Terminações Nervosas/metabolismo , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Nódulos Linfáticos Agregados/metabolismo , Proteínas SNARE/metabolismo
2.
Vet Microbiol ; 239: 108462, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767100

RESUMO

In contrast to human influenza viruses that replicate in the respiratory tract and are airborne transmitted, avian viruses also replicate in gut epithelial cells and are transmitted via the fecal-oral route. On this route, the virus is exposed to destructive fluids of the digestive tract, which are acidic and contain the proteases pepsin (gizzard) or chymotrypsin and trypsin (intestine). Only the latter enzyme activates virus by cleaving hemagglutinin (HA) into HA1 and HA2 subunits. We mimicked the passage of viruses through the gastrointestinal tract by treating them with digestive fluids from chicken and determined titers and integrity of HA by western-blot. Gizzard fluid completely inactivated virions and degrades HA even at a high dilution, but only if the pH was kept acidic. If the fluid is diluted with neutral buffer (mimicking virus uptake with seawater) particles were more resistant. Virions containing an uncleaved HA were even activated suggesting that gastric juice contains a trypsin-like protease. Undiluted intestinal fluid inactivated particles and destroyed HA, but diluted fluid activated virions. A virus isolated from the duck´s intestine is more tolerant against intestinal fluid compared to fowl plague virus suggesting that the former is better adapted to grow in the intestine. We also demonstrate that influenza viruses replicate to high titers in a novel chicken epithelial gut cell line. While viruses with a monobasic HA cleavage site require addition of trypsin, these cells effectively process HA with a polybasic cleavage site, which could be blocked with an inhibitor of the cellular furin protease.


Assuntos
Trato Gastrointestinal/virologia , Hemaglutininas/metabolismo , Influenza Aviária/virologia , Animais , Galinhas , Células Epiteliais/citologia , Células Epiteliais/virologia , Suco Gástrico/química , Suco Gástrico/enzimologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Secreções Intestinais/química , Secreções Intestinais/enzimologia , Inativação de Vírus , Replicação Viral/fisiologia
3.
Vet Ital ; 55(3): 195-201, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31599544

RESUMO

Each year millions of people are infected by influenza viruses, and this causes a substantial economic and health burden on our society. Influenza epidemics and pandemics are attributable to the ongoing evolution of influenza viruses through antigenic drift and shift, respectively. One of the reasons for the continuous circulation of influenza viruses in the human population is the incomplete protection conferred by currently available seasonal influenza vaccines against possible arising drifted or shifted influenza strains. Recently, tremendous efforts have been focused on the development of a more effective broadly reactive or universal influenza vaccine. The main objective of underdevelopment vaccines is to protect the human population not only from currently circulating virus strains but also from possible future variants without the need for their continuous update. Different approaches have been developed to reach this goal and elicit an effective and cross-protective immune response. Among these, consensus-based prophylactic approaches to effectively prevent influenza infections are the major focus of this review.


Assuntos
Hemaglutininas/uso terapêutico , Influenza Humana/prevenção & controle , Infecções por Orthomyxoviridae/veterinária , Orthomyxoviridae/efeitos dos fármacos , Animais , Humanos , Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/prevenção & controle
4.
Adv Exp Med Biol ; 1222: 69-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31637606

RESUMO

This study seeks to define the level of antihemagglutinin antibodies, using the hemagglutination inhibition assay (HAI), in the serum of patients, stratified into seven age groups, in Poland during the influenza epidemic season of 2017/18. A quadrivalent influenza vaccine has been introduced in Poland as of this epidemic season, making it possible for the first time to conduct the analysis for four antigens: A/Michigan/45/2015 (H1N1) pdm09, A/Hong Kong/4801/2014 (H3N2), B/Brisbane/60/2008 - Victoria lineage, and B/Phuket/3073/2013 - Yamagata lineage. We found that the level of individual antihemagglutinin antibodies was different among the seven age groups studied; with the highest in patients of 5-9 years and 10-14 years of age. Interestingly, the protection factor, defined as the percentage of people with the level of antihemagglutinin antibodies of at least 1:40 after vaccination or due to a previous infection, was the highest for the antigen A/Hong Kong/4801/2014 (H3N2) in the same age groups (74% and 75%, respectively). Taking into account the dismal 3.6% of the vaccinated population in Poland, these findings point toward the sustained presence of an immune system response in patients after a prior influenza virus infection.


Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Feminino , Hemaglutininas , Humanos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza B , Influenza Humana/sangue , Masculino , Pessoa de Meia-Idade , Polônia/epidemiologia , Estações do Ano , Vacinação/estatística & dados numéricos , Adulto Jovem
5.
Sensors (Basel) ; 19(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627298

RESUMO

Rather than the internal genome nucleic acids, the biomolecules on the surface of the influenza virus itself should be detected for a more exact and rapid point-of-care yes/no decision for influenza virus-induced infectious diseases. This work demonstrates the ultrasensitive electrical detection of the HA1 domain of hemagglutinin (HA), a representative viral surface protein of the influenza virus, using the top-down complementary metal oxide semiconductor (CMOS) processed silicon nanowire (SiNW) field-effect transistor (FET) configuration. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) was employed as a probe that specifically binds both to the aldehyde self-aligned monolayer on the SiNWs and to HA1 simultaneously. CMP-NANA was serially combined with two kinds of linkers, namely 3-aminopropyltriethoxysilane and glutaraldehyde. The surface functionalization used was verified using the purification of glutathione S-transferase-tagged HA1, contact angle measurement, enzyme-linked immunosorbent assay test, and isoelectric focusing analysis. The proposed functionalized SiNW FET showed high sensitivities of the threshold voltage shift (ΔVT) ~51 mV/pH and the ΔVT = 112 mV (63 mV/decade) with an ultralow detectable range of 1 fM of target protein HA1.


Assuntos
Técnicas Biossensoriais , Hemaglutininas/isolamento & purificação , Infecções por Orthomyxoviridae/diagnóstico , Orthomyxoviridae/isolamento & purificação , Animais , Humanos , Nanofios/química , Orthomyxoviridae/patogenicidade , Sistemas Automatizados de Assistência Junto ao Leito , Silício
6.
Zhonghua Yu Fang Yi Xue Za Zhi ; 53(9): 929-933, 2019 Sep 06.
Artigo em Chinês | MEDLINE | ID: mdl-31474076

RESUMO

Objective: Analyze the genetic characteristic of Hemagglutinin(H) gene of measles viruses isolated in Henan Province in 2017. Methods: Swab samples collected from 7 lab confirmed measles cases, and we got the measles virus by Vero/Slam inoculation. Fragment of H genes were amplified by reverse transcription polymerase chain reaction(RT-PCR), then the PCR products were sequenced and analyzed. Results: The age of the 7 measles confirmed cases were between 1 and 50 years old, and all of them were males. All the 7 measles viruses were identified as H1a genotype, and the average distance of the nucleotides and the amino acids was 0.005, respectively. Compared with the Shanghai-191/China-vaccine, there were some changes in isolated virus, such as 240(th), 397(th) and 381(st) sites in the amino acid sequence. Conclusion: The measles genotype which isolated in Henan Province in 2017 was H1a. There were some difference from Shanghai-191/China-vaccine in the nucleotides sequence of H gene, which suggested that it's necessary to strengthen the monitor the variation of measles virus.


Assuntos
Hemaglutininas , Vírus do Sarampo , Sarampo/virologia , Adolescente , Adulto , Criança , Pré-Escolar , China , Genótipo , Hemaglutininas/genética , Humanos , Lactente , Masculino , Vacina contra Sarampo/genética , Vírus do Sarampo/genética , Pessoa de Meia-Idade , Filogenia , Adulto Jovem
7.
Chem Pharm Bull (Tokyo) ; 67(11): 1201-1207, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434835

RESUMO

Oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor in our earlier studies. In the present work, 20 compounds were prepared by structural modifications of OA, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells were evaluated. Based on the biological result, structure-activity relationship (SAR) was discussed. Compound 10 with six-carbon chain and a terminal hydroxyl group showed the strongest anti-influenza activity with an IC50 of 2.98 µM, which is an order of magnitude more potent than OA. Hemagglutination inhibition and Surface plasmon resonance (SPR) assay indicated that compound 10 might interfere with influenza invasion by interacting with HA protein.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Cães , Relação Dose-Resposta a Droga , Hemaglutininas/efeitos dos fármacos , Hemaglutininas/metabolismo , Vírus da Influenza A/metabolismo , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Estrutura Molecular , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
8.
Nanoscale ; 11(29): 13878-13884, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31304500

RESUMO

Stimulation of dendritic cells (DCs) by antigens (Ags) promotes an Ag-specific immune response that kills Ag-expressing pathogens. These biologically inspired nanocarriers have received much attention as tools to deliver cancer Ags to DCs. A polymer-templated protein nanoball having hemagglutinin (H1-NB) that mimics the influenza virus can be used as a cancer Ag delivery vehicle, as DCs show effective phagocytic activities against H1-NB without any adjuvant. In the present study, H1-NB containing ovalbumin (OVA), a model Ag (H1-OVA-NB), was prepared as an anti-cancer agent and evaluated for its effect on anticancer immunity. H1-OVA-NB treatments in C57BL/6 mice enhanced OVA-specific immune activation and efficiently inhibited B16-OVA tumor growth compared to control groups. Our results indicate that H1-NB is an effective carrier for Ag delivery to DCs and promotes immunotherapy to fight cancer.


Assuntos
Antígenos/imunologia , Células Dendríticas/imunologia , Hemaglutininas/química , Imunoterapia , Nanopartículas/química , Polímeros/química , Animais , Antígenos/química , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Portadores de Fármacos/química , Hemaglutininas/genética , Hemaglutininas/metabolismo , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/metabolismo , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo
9.
Antonie Van Leeuwenhoek ; 112(11): 1655-1662, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31230158

RESUMO

Actinobacillus seminis, a commensal of ovine and caprine reproductive organs, is able to induce epididymitis in the small ruminants that it infects. In this work, we characterised two protein bands of approximately 150 kDa and 65 kDa. These proteins cross-reacted with a polyclonal serum against Gallibacterium anatis hemagglutinin and with a polyclonal serum from sheep with epididymitis, indicating that the proteins are expressed in vivo; the two proteins also interacted with biotin-labeled sheep fibrinogen and fibronectin, suggesting that they may function as adhesins. The participation of these proteins as adhesins was confirmed by a cultured human bladder cell-A. seminis adhesion assay and adherence inhibition by preincubation of A. seminis with polyclonal antiserum to the 150 kDa protein. Both proteins presented sequence identity with an A. seminis GroEL protein by mass spectrometry analysis and agglutinated glutaraldehyde-fixed sheep red blood cells. Immunogold labeling was observed by transmission electron microscopy on bacterial cells that were negatively stained, and a peroxidase reaction was detected in A. seminis biofilms, when an anti-A. seminis 150 kDa protein serum was used, indicating the presence of this protein on the surface of A. seminis and in biofilms. The A. seminis GroEL-homologue is a multifunctional protein that likely acts as a hemagglutinin.


Assuntos
Actinobacillus seminis/fisiologia , Eritrócitos/imunologia , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/metabolismo , Aglutinação , Testes de Aglutinação , Animais , Anticorpos Antibacterianos , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes , Adesão Celular , Eritrócitos/metabolismo , Proteínas de Choque Térmico/isolamento & purificação , Hemaglutinação , Hemaglutininas/metabolismo , Ovinos
10.
BMC Res Notes ; 12(1): 358, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234908

RESUMO

OBJECTIVE: Urinary tract infection (UTI) is one of the most frequent disease encounters in pregnant mothers, and the most drug resistant, biofilm and hemagglutinin producer Uropathogenic Escherichia coli (UPEC) is the major etiologic agent. Therefore, the aim of this study was to assess the association between the antimicrobial resistance, and biofilm and hemagglutinin production of Uropathogenic Escherichia coli. RESULTS: UTI among the study participants was 27.3%; and UPEC was found the major etiologic agent followed by coagulase negative staphylococcus. Risk factors, previous history of catheterization and previous history of UTI were found significantly associated with UTI, recurrent UTI, drug resistance and biofilm formation. Of the tested antibiotics, nitrofurantoin was the most effective drug for UPEC. Nearly 100% of the biofilm producers were resistant to norfloxacin, cotrimoxazole, and gentamicin.


Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana , Hemaglutininas/metabolismo , Escherichia coli Uropatogênica/fisiologia , Adolescente , Adulto , Feminino , Humanos , Modelos Logísticos , Análise Multivariada , Adulto Jovem
11.
J Gen Virol ; 100(6): 958-967, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31140969

RESUMO

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), is a highly contagious disease in chickens and is a great threat to the poultry industry. Fusion of the viral and target cell membranes is a prerequisite for NDV's entry into host cells. This process is directly mediated by the fusion (F) protein. Although several domains of F are known to regulate membrane fusion activity, the roles of the DI-DII linker (residues 376-381) of the NDV F protein in membrane fusion still remain unclear. To investigate the roles of this linker in NDV F-induced cell-cell fusion, mutations were engineered into this linker by site-directed mutagenesis. These mutants were analysed with respect to cell surface expression and membrane fusion activity. Each of the mutated F proteins in this linker was expressed at the cell surface at a similar level to wild-type (WT) F. However, most of them resulted in significant alterations in fusion activity. In particular, the mutants G377S, A378D, L379A and T380P were able to independently mediate cell fusion in the absence of HN protein in BHK-21 cells. Taken together, the results indicated that the DI-DII linker region has an important effect on the fusion activity of NDV F and mutants in this region could alter the requirement for HN for the promotion of membrane fusion.


Assuntos
Hemaglutininas/genética , Proteínas de Fusão de Membrana/genética , Mutação/genética , Neuraminidase/genética , Vírus da Doença de Newcastle/genética , Animais , Fusão Celular/métodos , Linhagem Celular , Membrana Celular/genética , Cricetinae , Doença de Newcastle/virologia , Células Vero
12.
Western Pac Surveill Response J ; 10(1): 32-38, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31110840

RESUMO

Introduction: There are two methods of reverse transcription polymerase chain reaction (RT-PCR) that have been the common methods to detect influenza infections: conventional and real-time RT-PCR. From December 2017 to March 2018, several missed diagnoses of influenza A(H1)pdm09 using real-time RT-PCR were reported in northern Viet Nam. This study investigated how these missed detections occurred to determine their effect on the surveillance of influenza. Methods: The haemagglutinin (HA) segments of A(H1N1)pdm09 from both real-time RT-PCR positive and negative samples were isolated and sequenced. The primer and probe sets in the HA gene were checked for mismatches, and phylogenetic analyses were performed to determine the molecular epidemiology of these viruses. Results: There were 86 positive influenza A samples; 32 were A(H1)pdm09 positive by conventional RT-PCR but were negative by real-time RT-PCR. Sequencing was conducted on 23 influenza (H1N1)pdm09 isolates that were recovered from positive samples. Eight of these were negative for A(H1)pdm09 by real-time RT-PCR. There were two different mismatches in the probe target sites of the HA gene sequences of all isolates (n = 23) with additional mismatches only at position 7 (template binding site) identified for all eight negative real-time RT-PCR isolates. The prime target sites had no mismatches. Phylogenetic analysis of the HA gene showed that both the positive and negative real-time RT-PCR isolates were grouped in clade 6B.1; however, the real-time RT-PCR negative viruses were located in a subgroup that referred to substitution I295V. Conclusion: Constant monitoring of genetic changes in the circulating influenza A(H1N1)pdm09 viruses is important for maintaining the sensitivity of molecular detection assays.


Assuntos
Diagnóstico Tardio/tendências , Influenza Humana/diagnóstico , Análise de Sequência de DNA/normas , Testes de Hemaglutinação/métodos , Hemaglutininas/análise , Hemaglutininas/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/epidemiologia , Influenza Humana/mortalidade , Mutação/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/tendências , Vietnã
13.
Vet Res ; 50(1): 37, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118100

RESUMO

The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPRmt. Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion-fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.


Assuntos
Retículo Endoplasmático/virologia , Hemaglutininas/metabolismo , Mitocôndrias/metabolismo , Neuraminidase/metabolismo , Doença de Newcastle/metabolismo , Vírus da Doença de Newcastle/metabolismo , Resposta a Proteínas não Dobradas , Células A549/metabolismo , Células A549/virologia , Animais , Western Blotting , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Mitocôndrias/virologia
14.
Cell ; 177(5): 1086-1088, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100263

RESUMO

A universal vaccine against influenza remains a critical target, and efforts have recently focused on the stem of the hemagglutinin glycoprotein. In this issue of Cell and a related Cell Host & Microbe article, three studies identify broad protective epitopes in the hemagglutinin head domain that are exposed by trimer "breathing."


Assuntos
Vacinas contra Influenza , Influenza Humana , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas , Humanos , Imunoglobulina G
15.
Nat Commun ; 10(1): 1660, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971703

RESUMO

Influenza A viruses evolve rapidly to escape host immunity, causing reinfection. The form and duration of protection after each influenza virus infection are poorly understood. We quantify the dynamics of protective immunity by fitting individual-level mechanistic models to longitudinal serology from children and adults. We find that most protection in children but not adults correlates with antibody titers to the hemagglutinin surface protein. Protection against circulating strains wanes to half of peak levels 3.5-7 years after infection in both age groups, and wanes faster against influenza A(H3N2) than A(H1N1)pdm09. Protection against H3N2 lasts longer in adults than in children. Our results suggest that influenza antibody responses shift focus with age from the mutable hemagglutinin head to other epitopes, consistent with the theory of original antigenic sin, and might affect protection. Imprinting, or primary infection with a subtype, has modest to no effect on the risk of non-medically attended infections in adults.


Assuntos
Anticorpos Antivirais/sangue , Proteção Cruzada/imunologia , Memória Imunológica/imunologia , Influenza Humana/imunologia , Modelos Biológicos , Adolescente , Adulto , Fatores Etários , Idoso , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Criança , Pré-Escolar , Feminino , Seguimentos , Hemaglutininas/imunologia , Hong Kong/epidemiologia , Humanos , Incidência , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Tempo , Adulto Jovem
16.
Nat Commun ; 10(1): 1799, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996301

RESUMO

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Hemaglutininas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Influenza Humana/imunologia , Oligossacarídeos/metabolismo , Animais , Proteínas de Bactérias/genética , Bioensaio/métodos , Células CHO , Cricetulus , Cães , Glicosiltransferases/genética , Voluntários Saudáveis , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Microscopia Intravital/métodos , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oligossacarídeos/imunologia , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodos
17.
Arch Virol ; 164(5): 1309-1321, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877453

RESUMO

Full-length infectious cDNA clones for recombinant HY12 bovine enteroviruses designated as rHY12-3A-2-HA, rHY12-3A-3-HA, and rHY12-3A-9-HA were constructed by the insertion of an epitope from influenza virus hemagglutinin (HA) at the N-terminus of the HY12-encoded 3A protein at amino acid positions 2, 3, and 9. The recombinant HY12 viruses expressing the HA epitope were rescued and characterized using immunoperoxidase monolayer assay, western blotting, and electron microscopy. The three rescued recombinant marker viruses showed similar characteristics, such as TCID50 titer, plaque size, and growth properties, to those of parental rHY12 virus. Comparative analysis of the nucleotide sequences demonstrated the three recombinant marker viruses remained stable for 15 passages with no genetic changes. The recombinant viruses remained viable in various permissive cell lines, including BHK-21, Vero, and PK15 cells, suggesting that the insertion of the HA epitope tag had no effect on virus infectivity. Mice infected with the recombinant marker viruses and the parental virus produced anti-HY12-virus antibodies, while the recombinant marker viruses also produced anti-HA-epitope-tag antibodies. Taken together, these results demonstrate that HY12 viruses containing genetic markers may be useful tools for future investigations of the mechanisms of viral pathogenesis and virus replication, as well as for vaccine development.


Assuntos
Anticorpos Antivirais/imunologia , Enterovirus Bovino/genética , Enterovirus Bovino/imunologia , Epitopos/imunologia , Hemaglutininas/imunologia , Proteínas Virais/imunologia , Animais , Linhagem Celular , Cricetinae , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas/genética , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos ICR , Suínos , Células Vero , Proteínas Virais/genética
18.
Bull Exp Biol Med ; 166(5): 631-636, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30903496

RESUMO

We compared three cold-adapted live attenuated influenza vaccine strains prepared by reverse genetics methods on the basis of master donor virus A/Leningrad/134/17/57 and influenza H7N9 strains A/Anhui/1/2013 and A/Shanghai/1/2013. Two strains based on A/Anhui/1/2013 differed by amino acid positions 123 and 149 in HA1 (123N/149N; 123D/149D). All strains efficiently replicated in developing chicken embryos; A/Shanghai/1/2013-based strain and A/Anhui/1/2013-123N/149N variant were characterized by reduced replication in MDCK cells. Strains based on A/Anhui/1/2013 virus agglutinated erythrocytes with α2,3- and α2,6-linked sialic acid residues, whereas strain A/Shanghai/1/2013 only α2,3. In experiments with BALB/c mice, Anhui-123D/149D strain was most immunogenic and induced high crossreactive humoral immune response, therefore it can be recommended as the model virus for the construction of recombinant vector vaccines based on live attenuated influenza vaccine.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/imunologia , Substituição de Aminoácidos , Animais , Hemaglutininas/química , Hemaglutininas/imunologia , Humanos , Imunidade Humoral , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Atenuadas/química , Vacinas Atenuadas/imunologia
19.
Mol Biol Evol ; 36(6): 1172-1186, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30851115

RESUMO

Seasonal influenza viruses undergo frequent mutations on their surface hemagglutinin (HA) proteins to escape the host immune response. In these mutations, a few key amino acid sites are associated with significant antigenic cluster transitions. To recognize the cluster-transition determining sites of seasonal influenza A/H3N2 and A/H1N1 viruses systematically and quickly, we developed a computational model named RECDS (recognition of cluster-transition determining sites) to evaluate the contribution of a specific amino acid site on the HA protein in the whole history of antigenic evolution. In RECDS, we ranked all of the HA sites by calculating the contribution scores derived from the forest of gradient boosting classifiers trained by various sequence- and structure-based features. With the RECDS model, we found out that the sites determining influenza antigenicity were mostly around the receptor-binding domain both for the influenza A/H3N2 and A/H1N1 viruses. Specifically, half of the cluster-transition determining sites of the influenza A/H1N1 virus were located in the vestigial esterase domain and basic path area on the HA, which indicated that the differential driving force of the antigenic evolution of the A/H1N1 virus refers to the A/H3N2 virus. Beyond that, the footprints of substitutions responsible for antigenic evolution were inferred according to the phylogenetic trees for the cluster-transition determining sites. The monitoring of genetic variation occurring at these cluster-transition determining sites in circulating influenza viruses on a large scale will potentially reduce current assay workloads in influenza surveillance and the selection of new influenza vaccine strains.


Assuntos
Antígenos Virais/genética , Evolução Molecular , Hemaglutininas/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Algoritmos , Técnicas Genéticas , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Software
20.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30927348

RESUMO

Bacteria use a variety of mechanisms to translocate proteins from the cytoplasm, where they are synthesized, to the cell surface or extracellular environment or directly into other cells, where they perform their ultimate functions. Type V secretion systems (T5SS) use ß-barrel transporter domains to export passenger domains across the outer membranes of Gram-negative bacteria. Distinct among T5SS are type Vb or two-partner secretion (TPS) systems in which the transporter and passenger are separate proteins, necessitating a mechanism for passenger-translocator recognition in the periplasm and providing the potential for reuse of the translocator. This review describes current knowledge of the TPS translocation mechanism, using Bordetella filamentous hemagglutinin (FHA) and its transporter FhaC as a model. We present the hypothesis that the TPS pathway may be a general mechanism for contact-dependent delivery of toxins to target cells.


Assuntos
Bordetella/metabolismo , Hemaglutininas/metabolismo , Via Secretória/fisiologia , Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Bordetella/patogenicidade , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Bactérias Gram-Negativas , Proteínas de Membrana Transportadoras , Modelos Moleculares , Sistemas de Secreção Tipo V/metabolismo , Virulência , Fatores de Virulência de Bordetella/metabolismo , Coqueluche/microbiologia
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