Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.353
Filtrar
1.
Molecules ; 26(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562349

RESUMO

As a member of the Orthomyxoviridae family of viruses, influenza viruses (IVs) are known causative agents of respiratory infection in vertebrates. They remain a major global threat responsible for the most virulent diseases and global pandemics in humans. The virulence of IVs and the consequential high morbidity and mortality of IV infections are primarily attributed to the high mutation rates in the IVs' genome coupled with the numerous genomic segments, which give rise to antiviral resistant and vaccine evading strains. Current therapeutic options include vaccines and small molecule inhibitors, which therapeutically target various catalytic processes in IVs. However, the periodic emergence of new IV strains necessitates the continuous development of novel anti-influenza therapeutic options. The crux of this review highlights the recent studies on the biology of influenza viruses, focusing on the structure, function, and mechanism of action of the M2 channel and neuraminidase as therapeutic targets. We further provide an update on the development of new M2 channel and neuraminidase inhibitors as an alternative to existing anti-influenza therapy. We conclude by highlighting therapeutic strategies that could be explored further towards the design of novel anti-influenza inhibitors with the ability to inhibit resistant strains.


Assuntos
Influenza Humana/tratamento farmacológico , Orthomyxoviridae/efeitos dos fármacos , Infecções Respiratórias/tratamento farmacológico , Proteínas da Matriz Viral/genética , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Humanos , Influenza Humana/virologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Orthomyxoviridae/genética , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Proteínas da Matriz Viral/antagonistas & inibidores
2.
Eur J Med Chem ; 212: 113097, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385836

RESUMO

Encouraged by our earlier discovery of N1-selective inhibitors, the 150-cavity of influenza virus neuraminidases (NAs) could be further exploited to yield more potent oseltamivir derivatives. Herein, we report the design, synthesis and biological evaluation of a series of novel oseltamivir derivatives via the structural modifications at C5-NH2 of oseltamivir targeting 150-cavity. Among them, compound 5c bearing 4-(3-methoxybenzyloxy)benzyl group exhibited the most potent activity, which was lower or modestly improved activities than oseltamivir carboxylate (OSC) against N1 (H1N1), N1 (H5N1) and N1 (H5N1-H274Y). Specifically, there was 30-fold loss of activity against the wild-type strain H1N1. However, 5c displayed 4.85-fold more potent activity than OSC against H5N1-H274Y NA. Also, 5c demonstrated low cytotoxicity in vitro and no acute toxicity in mice. Molecular docking studies provided insights into the high potency of 5c against N1 and N1-H274Y mutant NAs. Besides, the in silico prediction of physicochemical properties and CYP enzymatic inhibitory ability of representative compounds were conducted to evaluate their drug-like properties.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/efeitos dos fármacos , Oseltamivir/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neuraminidase/genética , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Oseltamivir/análogos & derivados , Oseltamivir/química , Relação Estrutura-Atividade
3.
PLoS Biol ; 18(8): e3000788, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841232

RESUMO

Women with bacterial vaginosis (BV), an imbalance of the vaginal microbiome, are more likely to be colonized by potential pathogens such as Fusobacterium nucleatum, a bacterium linked with intrauterine infection and preterm birth. However, the conditions and mechanisms supporting pathogen colonization during vaginal dysbiosis remain obscure. We demonstrate that sialidase activity, a diagnostic feature of BV, promoted F. nucleatum foraging and growth on mammalian sialoglycans, a nutrient resource that was otherwise inaccessible because of the lack of endogenous F. nucleatum sialidase. In mice with sialidase-producing vaginal microbiotas, mutant F. nucleatum unable to consume sialic acids was impaired in vaginal colonization. These experiments in mice also led to the discovery that F. nucleatum may also "give back" to the community by reinforcing sialidase activity, a biochemical feature of human dysbiosis. Using human vaginal bacterial communities, we show that F. nucleatum supported robust outgrowth of Gardnerella vaginalis, a major sialidase producer and one of the most abundant organisms in BV. These results illustrate that mutually beneficial relationships between vaginal bacteria support pathogen colonization and may help maintain features of dysbiosis. These findings challenge the simplistic dogma that the mere absence of "healthy" lactobacilli is the sole mechanism that creates a permissive environment for pathogens during vaginal dysbiosis. Given the ubiquity of F. nucleatum in the human mouth, these studies also suggest a possible mechanism underlying links between vaginal dysbiosis and oral sex.


Assuntos
Proteínas de Bactérias/genética , Disbiose/microbiologia , Fusobacterium/metabolismo , Gardnerella vaginalis/metabolismo , Neuraminidase/genética , Polissacarídeos/metabolismo , Vaginose Bacteriana/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , Disbiose/patologia , Feminino , Fusobacterium/genética , Fusobacterium/isolamento & purificação , Fusobacterium/patogenicidade , Gardnerella vaginalis/genética , Gardnerella vaginalis/isolamento & purificação , Gardnerella vaginalis/patogenicidade , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/genética , Neuraminidase/metabolismo , RNA Ribossômico 16S/genética , Ácidos Siálicos/metabolismo , Simbiose/genética , Vagina/microbiologia , Vaginose Bacteriana/patologia
4.
PLoS Pathog ; 16(8): e1008816, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853241

RESUMO

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Ácidos Siálicos/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/isolamento & purificação , Células Madin Darby de Rim Canino , Neuraminidase/química , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Ligação Proteica
5.
BMC Infect Dis ; 20(1): 550, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727378

RESUMO

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H5N8/genética , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Neuraminidase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Aves/virologia , Primers do DNA/genética , Feminino , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
6.
BMC Infect Dis ; 20(1): 478, 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32631240

RESUMO

BACKGROUND: Extended use of oseltamivir in an immunocompromised host could reportedly induce neuraminidase gene mutation possibly leading to oseltamivir-resistant influenza A/H3N2 virus. To our knowledge, no report is available on the clinical course of a severely immunocompromised patient with a dual E119D/R292K neuraminidase mutated-influenza A/H3N2 during the administration of peramivir. CASE PRESENTATION: A 49-year-old male patient was admitted for second allogeneic hematopoietic cell transplantation for active acute leukemia. The patient received 5 mg prednisolone and 75 mg cyclosporine and had severe lymphopenia (70/µL). At the time of hospitalization, the patient was diagnosed with upper tract influenza A virus infection, and oseltamivir treatment was initiated immediately. However, the patient was intolerant to oseltamivir. The following day, treatment was changed to peramivir. Despite a total period of neuraminidase-inhibitor administration of 16 days, the symptoms and viral shedding continued. Changing to baloxavir marboxil resolved the symptoms, and the influenza diagnostic test became negative. Subsequently, sequence analysis of the nasopharyngeal specimen revealed the dual E119D/R292K neuraminidase mutant influenza A/H3N2. CONCLUSIONS: In a highly immunocompromised host, clinicians should take care when peramivir is used for extended periods to treat influenza virus A/H3N2 infection as this could potentially leading to a dual E119D/R292K substitution in neuraminidase protein. Baloxavir marboxil may be one of the agents that can be used to treat this type of mutated influenza virus infection.


Assuntos
Antivirais/uso terapêutico , Ciclopentanos/uso terapêutico , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Guanidinas/uso terapêutico , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Ácidos Carbocíclicos , Ciclopentanos/efeitos adversos , Ciclopentanos/farmacologia , Dibenzotiepinas , Farmacorresistência Viral/genética , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacologia , Guanidinas/efeitos adversos , Guanidinas/farmacologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Hospedeiro Imunocomprometido , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Morfolinas , Mutação , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Oseltamivir/uso terapêutico , Piridonas , Transplante Homólogo/métodos , Resultado do Tratamento , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética
7.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641475

RESUMO

Some avian influenza (AI) viruses have a deletion of up to 20 to 30 amino acids in their neuraminidase (NA) stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses have only a 2- or 3-amino-acid deletion, and such deletions were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8-amino-acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses that had various NA stalk lengths and that were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low-molecular-weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with the NA stalk length but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry into host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with a hemagglutinin (HA) with a higher α2,6-linked sialylglycan affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicate that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, is required for optimal G1 virus entry into human airway cells.IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage being the most prevalent worldwide. In this study, we isolated G1 variants carrying an 8-amino-acid deletion in their NA stalk, which is, to our knowledge, the longest deletion found in H9N2 viruses in the field. The NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species specific and dependent on the corresponding HA binding affinity. Our results suggest that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.


Assuntos
Regulação Viral da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/virologia , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Sequência de Aminoácidos , Animais , Galinhas , Genótipo , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hemaglutininas , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H9N2/metabolismo , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/genética , Influenza Aviária/metabolismo , Influenza Aviária/patologia , Camundongos , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Fenótipo , Filogenia , Receptores Virais , Deleção de Sequência , Relação Estrutura-Atividade , Virulência , Internalização do Vírus , Replicação Viral
8.
Nat Commun ; 11(1): 3418, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647286

RESUMO

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.


Assuntos
Anticorpos Antivirais/imunologia , Resistência a Múltiplos Medicamentos/imunologia , Farmacorresistência Viral/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Mutação/genética , Neuraminidase/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Afinidade de Anticorpos/imunologia , Antígenos Virais/metabolismo , Líquidos Corporais/virologia , Análise Mutacional de DNA , Cães , Epitopos/química , Epitopos/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H3N2/enzimologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Imagem Óptica , Ligação Proteica , Análise Espectral Raman
9.
PLoS One ; 15(6): e0234869, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579578

RESUMO

The continuous variation of the seasonal influenza viruses, particularly A(H1N1)pdm09, persistently threatens human life and health around the world. In local areas of southwest china, the large time-scale genomic research on A(H1N1)pdm09 is still insufficient. Here, we sequenced 45 whole-genome sequences of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018, by next-generation sequencing technology to characterize molecular mechanisms of their origin and evolution. Our phylogenetic analyses suggest that the A(H1N1)pdm09 strains circulating in Lincang belong to clade 6B and the subclade 6B.1A predominates in 2018. Further, the strains in 2018 possess elevated evolutionary rate as compared to strains in other years. Several newly emerged mutations for HA (hemagglutinin) in 2018 are revealed (i.e., S183P and R221K). Intriguingly, the substitution R221K falls into the RBS (receptor binding site) of HA protein, which could affect antigenic properties of influenza A(H1N1)pdm09 viruses, and another substitution S183P near to RBS with a high covering frequency (11/14 strains) in 2018 is exactly located at the epitope B. Notably, the NA (neuraminidase) protein harbors a new mutation I23T, potentially involved in N-glycosylation. Based on the background with a higher evolutionary rate in 2018 strains, we deeply evaluate the potential vaccine efficacy against Lincang strains and discover a substantive decline of the vaccine efficacy in 2018. Our analyses reaffirm that the real-time molecular surveillance and timely updated vaccine strains for prevention and control of influenza A(H1N1)pdm09 are crucial in the future.


Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H1N1/genética , Sequenciamento Completo do Genoma , Sequência de Aminoácidos , China , Análise Mutacional de DNA , Demografia , Epitopos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/imunologia , Mutação/genética , Neuraminidase/genética , Filogenia , Resultado do Tratamento
10.
PLoS Pathog ; 16(6): e1008592, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555740

RESUMO

The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility.


Assuntos
Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza B/metabolismo , Infecções por Orthomyxoviridae , Oseltamivir/farmacologia , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Furões , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza B/genética , Macaca fascicularis , Macrolídeos , Masculino , Mutação de Sentido Incorreto , Neuraminidase/genética , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia
11.
PLoS Pathog ; 16(6): e1008411, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598381

RESUMO

In the 2014-2015 influenza season a novel neuraminidase (NA) genotype was detected in global human influenza A surveillance. This novel genotype encoded an N-linked glycosylation site at position 245-247 in the NA protein from clade 3c.2a H3N2 viruses. In the years following the 2014-2015 season, this novel NA glycosylation genotype quickly dominated the human H3N2 population of viruses. To assess the effect this novel N-linked glycan has on virus fitness and antibody binding, recombinant viruses with (NA Gly+) or without (NA Gly-) the 245 NA glycan were created. Viruses with the 245 NA Gly+ genotype grew to a significantly lower infectious virus titer on primary, differentiated human nasal epithelial cells (hNEC) compared to viruses with the 245 NA Gly- genotype, but growth was similar on immortalized cells. The 245 NA Gly+ blocked human and rabbit monoclonal antibodies that target the enzymatic site from binding to their epitope. Additionally, viruses with the 245 NA Gly+ genotype had significantly lower enzymatic activity compared to viruses with the 245 NA Gly- genotype. Human monoclonal antibodies that target residues near the 245 NA glycan were less effective at inhibiting NA enzymatic activity and virus replication of viruses encoding an NA Gly+ protein compared to ones encoding NA Gly- protein. Additionally, a recombinant H6N2 virus with the 245 NA Gly+ protein was more resistant to enzymatic inhibition from convalescent serum from H3N2-infected humans compared to viruses with the 245 NA Gly- genotype. Finally, the 245 NA Gly+ protected from NA antibody mediated virus neutralization. These results suggest that while the 245 NA Gly+ decreases virus replication in hNECs and decreases enzymatic activity, the 245 NA glycan blocks the binding of monoclonal and human serum NA specific antibodies that would otherwise inhibit enzymatic activity and virus replication.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana , Neuraminidase , Proteínas Virais , Replicação Viral , Animais , Cães , Células HEK293 , Humanos , Influenza Humana/genética , Influenza Humana/imunologia , Células Madin Darby de Rim Canino , Neuraminidase/genética , Neuraminidase/imunologia , Coelhos , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
12.
J Med Microbiol ; 69(7): 986-998, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32459617

RESUMO

Introduction. Influenza viruses evolve rapidly and change their antigenic characteristics, necessitating biannual updates of flu vaccines.Aim. The aim of this study was to characterize influenza viruses circulating in Bulgaria during the 2018/2019 season and to identify amino acid substitutions in them that might impact vaccine effectiveness.Methodology. Typing/subtyping of influenza viruses were performed using real-time Reverse Transcription-PCR (RT-PCR) and results of phylogenetic and amino acid sequence analyses of influenza strains are presented.Results. A(H1N1)pdm09 (66 %) predominated over A(H3N2) (34 %) viruses, with undetected circulation of B viruses in the 2018/2019 season. All A(H1N1)pdm09 viruses studied fell into the recently designated 6B.1A subclade with over 50 % falling in four subgroups: 6B.1A2, 6B.1A5, 6B.1A6 and 6B.1A7. Analysed A(H3N2) viruses belonged to subclades 3C.2a1b and 3C.2a2. Amino acid sequence analysis of 36 A(H1N1)pdm09 isolates revealed the presence of six-ten substitutions in haemagglutinin (HA), compared to the A/Michigan/45/2015 vaccine virus, three of which occurred in antigenic sites Sa and Cb, together with four-nine changes at positions in neuraminidase (NA), and a number of substitutions in internal proteins. HA1 D222N substitution, associated with increased virulence, was identified in two A(H1N1)pdm09 viruses. Despite the presence of several amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically similar to the vaccine virus. The 28 A(H3N2) viruses characterized carried substitutions in HA, including some in antigenic sites A, B, C and E, in NA and internal protein sequences.Conclusion. The results of this study showed the genetic diversity of circulating influenza viruses and the need for continuous antigenic and molecular surveillance.


Assuntos
Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Influenza Humana/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Antígenos Virais/genética , Bulgária/epidemiologia , Monitoramento Epidemiológico , Evolução Molecular , Variação Genética/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas , História do Século XXI , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/imunologia , Influenza Humana/história , Influenza Humana/virologia , Neuraminidase/genética , Filogenia , RNA Viral/genética , Estações do Ano , Análise de Sequência de DNA/métodos
13.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188732

RESUMO

The discovery in 1976 of waterfowl as the primary reservoir of influenza A viruses (IAVs) has since spurred decades of waterfowl surveillance efforts by researchers dedicated to understanding the ecology of IAV and its subsequent threat to human and animal health. Here, we employed a multidecade, continental-scale approach of surveillance data to understand trends of seasonal IAV subtype diversity. Between 1976 and 2015, IAVs were detected in 8,427 (10.8%) of 77,969 samples from migratory waterfowl throughout the Central and Mississippi Migratory Flyways in the United States and Canada. A total of 96 hemagglutinin (HA)/neuraminidase (NA) subtype combinations were isolated, which included most HA (H1 to H14) and all 9 NA subtypes. We observed an annual trend of high influenza prevalence, involving a few dominant subtypes, on northern breeding grounds during summer with progressively lowered influenza prevalence, comprised of a highly diverse profile of subtypes, as waterfowl migrate toward southern wintering grounds. Isolates recovered during winter had the highest proportion of mixed and rare HA/NA combinations, indicating increased opportunity for reassortment of IAVs. In addition, 70% of H5 and 49% of H7 IAV isolates were recovered from samples collected during fall and spring, respectively; these are subtypes that can have significant implications for public health and agriculture sectors. Annual cyclical dominance of subtypes on northern breeding grounds is revealed through the longitudinal nature of this study. Our novel findings exhibit the unrealized potential for discovery using existing IAV surveillance data.IMPORTANCE Wild aquatic birds are the primary natural reservoir of influenza A viruses (IAVs) and are therefore responsible for the dispersal and maintenance of IAVs representing a broad range of antigenic and genetic diversity. The aims of IAV surveillance in waterfowl not only relate to understanding the risk of spillover risk to humans, but also to improving our understanding of basic questions related to IAV evolution and ecology. By evaluating several decades of surveillance data from wild aquatic birds sampled along North American migratory flyways, we discovered an annual trend of increasing subtype diversity during southbound migration, peaking on southern wintering grounds. Winter sampling revealed the highest proportion of mixed and rare infections that suggest higher opportunity for spillover. These findings allow improvements to surveillance efforts to robustly capture IAV diversity that will be used for vaccine development and cultivate a more thorough understanding of IAV evolution and persistence mechanisms.


Assuntos
Aves/virologia , Variação Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Influenza Aviária , Neuraminidase/genética , Filogenia , Proteínas Virais/genética , Migração Animal , Animais , Canadá/epidemiologia , Influenza Aviária/epidemiologia , Influenza Aviária/genética , Prevalência , Estados Unidos/epidemiologia
14.
Gene ; 742: 144538, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32184168

RESUMO

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.


Assuntos
Ciclídeos/metabolismo , Proteínas de Peixes/metabolismo , Neuraminidase/metabolismo , Animais , Ciclídeos/genética , Ciclídeos/crescimento & desenvolvimento , Clonagem Molecular , Evolução Molecular , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Neuraminidase/química , Neuraminidase/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Especificidade por Substrato/genética
15.
Arch Virol ; 165(4): 891-911, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32060794

RESUMO

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Animais , Biologia Computacional , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/imunologia , Coelhos , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
16.
Sci Rep ; 10(1): 2161, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034220

RESUMO

While molecular-targeted drugs have demonstrated strong therapeutic efficacy against diverse diseases such as cancer and infection, the appearance of drug resistance associated with genetic variations in individual patients or pathogens has severely limited their clinical efficacy. Therefore, precision medicine approaches based on the personal genomic background provide promising strategies to enhance the effectiveness of molecular-targeted therapies. However, identifying drug resistance mutations in individuals by combining DNA sequencing and in vitro analyses is generally time consuming and costly. In contrast, in silico computation of protein-drug binding free energies allows for the rapid prediction of drug sensitivity changes associated with specific genetic mutations. Although conventional alchemical free energy computation methods have been used to quantify mutation-induced drug sensitivity changes in some protein targets, these methods are often adversely affected by free energy convergence. In this paper, we demonstrate significant improvements in prediction performance and free energy convergence by employing an alchemical mutation protocol, MutationFEP, which directly estimates binding free energy differences associated with protein mutations in three types of a protein and drug system. The superior performance of MutationFEP appears to be attributable to its more-moderate perturbation scheme. Therefore, this study provides a deeper level of insight into computer-assisted precision medicine.


Assuntos
Resistência a Medicamentos , Simulação de Acoplamento Molecular/métodos , Mutação , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/química , Aldeído Redutase/genética , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/química , Quinase do Linfoma Anaplásico/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular/normas , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Neuraminidase/genética , Sensibilidade e Especificidade
17.
Lett Appl Microbiol ; 70(4): 252-258, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31990997

RESUMO

Neuraminidase (NA) is an integral membrane protein of influenza A virus (IAV) and primarily aids in the release of progeny virions, following the intracellular viral replication cycle. In an attempt to discover new functions of NA, we conducted a classical yeast two-hybrid screen and found acute myeloid leukaemia marker 1 (AML1) as a novel interacting partner of IAV-NA. The interaction was further validated by co-immunoprecipitation in IAV-infected cells and in an in vitro coupled transcription/translation system. Interestingly, we found an increase in the expression of AML1 upon IAV infection in a dose-dependent manner. As expected, we also observed an increase in the IFN-ß levels, the first line of defence against viral infections. Subsequently, when AML1 was downregulated using siRNA, the IFN-ß levels were found to be remarkably reduced. Our study also shows that AML1 is induced upon IAV infection and results in the induction of IFN-ß. Thus, AML1 is proposed to be an important player in IFN induction and has a role in an antiviral response against IAV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Influenza epidemics and pandemics are constant threats to human health. Development of antiviral therapeutics has focused on important and major IAV proteins as targets. However, the rate at which this virus mutates makes the task challenging. Thus, next-generation approaches aim at host cellular proteins that aid the virus in its replication. This study reports a new host-virus interaction, of acute myeloid leukaemia marker 1 (AML1) with influenza A neuraminidase (IAV-NA). We have found that this interaction has a direct effect on the upregulation of host IFN-ß response. Further studies may lead to a greater understanding of this new innate defence pathway in infected cells.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Humana/metabolismo , Interferon beta/metabolismo , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Interações Hospedeiro-Patógeno , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/genética , Influenza Humana/virologia , Interferon beta/genética , Neuraminidase/genética , Ligação Proteica , Regulação para Cima , Proteínas Virais/genética
18.
PLoS One ; 15(1): e0227597, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31951605

RESUMO

Human infections with avian influenza viruses including H5, H7 and H9 hemagglutinin subtypes occur at a low rate. Among human infections with H7 viruses, regional outbreaks with H7N2, H7N3, H7N7 and H7N9 have been documented. Early in 2018, a human infection with a novel H7N4 avian influenza virus was reported in Jiangsu, China. This study is aimed at understanding the probable origin and molecular features of this emerging H7N4 virus. Genomic segments encoding hemagglutinin (HA) and neuraminidase (NA) of H7Nx and HxN4 viruses were compared with this H7N4 strain by alignment and phylogenetic tree analysis. Phylogenetic analysis indicated that the human H7N4 virus probably originated from multiple reassortments of avian H7N7 and H8N4 viruses for its HA and NA, respectively, and likely a regional uncharacterized virus for its internal segments. Our data excluded that circulating avian H9N2 viruses were the origin of the H7N4 internal segments, unlike the human H5N1 and H7N9 viruses that both had H9N2 backbones. This index case provided a unique opportunity to examine viral mutations by directly comparing the human isolate with its closest viral relatives isolated from avian species from the patient's farm, which may suggest critical mutations required for viral adaptation in humans. Whole-genome scanning was performed and the sequences of the human and related avian H7N4 isolates were compared. Mutations in PB2 (E627K), PB2 (K683T), PB1-F2 (N47S), HA (N283D), HA(K321E), NA(A137V), NA(K296R) and M2 (C19Y) were identified in the human isolate while no mutations were found in PB1, NP, NS1, and NS2 of the human H7N4 compared to the avian H7N4 viruses. Our data in this report provide further evidence for the genesis of this novel H7N4 virus with a multi-reassortment model and show molecular changes that might be responsible for the transmission of this virus from chickens or ducks to and subsequent replication in humans.


Assuntos
Vírus da Influenza A/genética , Mutação , Filogenia , Vírus Reordenados/genética , Adaptação Biológica/genética , Animais , China , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Neuraminidase/genética , Proteínas Virais/genética
19.
Biochem Biophys Res Commun ; 523(2): 487-492, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31889533

RESUMO

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5-7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.


Assuntos
Glicosídeos/metabolismo , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Sphingobacterium/enzimologia , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CHO , Cricetulus , Concentração de Íons de Hidrogênio , Hidrólise , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/farmacologia , Ácidos Neuramínicos , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Neuraminidase/genética , Sphingobacterium/genética , Especificidade por Substrato , Temperatura
20.
Fish Physiol Biochem ; 46(2): 563-574, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31792756

RESUMO

Fish store triglycerides (TGs) in the liver, muscle, and adipose tissue and TGs constitute an energy source upon metabolic demand. The liver generally plays important roles in lipid metabolism. Recent studies have suggested the possibility of hepatic lipid metabolic regulation by ganglioside in mammals; however, ganglioside-mediated regulation of lipid metabolism is unclear in fish. This study aimed to clarify the role of ganglioside in fish TG metabolism, with particular reference to Neu3a, a ganglioside-specific sialidase expressed in the fish liver. Under fasting conditions, there was a decrease in hepatic TG contents, and neu3a mRNA level was significantly up-regulated in the medaka liver. To determine the role of Neu3a in hepatic lipid metabolism, Neu3a stable transfectants were generated using fish liver Hepa-T1 cells. After treating Neu3a cells with oleic acid, reduction of TG was detected in comparison with the mock cells. Furthermore, lipase activity was greater in Neu3a cells than in mock cells. To examine which ganglioside regulates these events, alterations of ganglioside composition in Neu3a cells were analyzed. Neu3a cells exhibited increased level of lactosylceramide (LacCer), a Neu3 enzymatic product originating from GM3. In addition, exposure of LacCer toward Hepa-T1 cells resulted in an increase of neutral lipase activity. The present results suggest that Neu3a up-regulation in medaka under fasting condition accelerates hepatic TG degradation for energy production via GM3 desialylation.


Assuntos
Neuraminidase/metabolismo , Oryzias/fisiologia , Triglicerídeos/metabolismo , Animais , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Neuraminidase/genética , RNA Mensageiro/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...