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1.
Int J Infect Dis ; 90: 84-96, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31669593

RESUMO

BACKGROUND: This study compared the genomes of influenza viruses that caused mild infections among outpatients and severe infections among hospitalized patients in Singapore, and characterized their molecular evolution and receptor-binding specificity. METHODS: The complete genomes of influenza A/H1N1, A/H3N2 and B viruses that caused mild infections among outpatients and severe infections among inpatients in Singapore during 2012-2015 were sequenced and characterized. Using various bioinformatics approaches, we elucidated their evolutionary, mutational and structural patterns against the background of global and vaccine strains. RESULTS: The phylogenetic trees of the 8 gene segments revealed that the outpatient and inpatient strains overlapped with representative global and vaccine strains. We observed a cluster of inpatients with A/H3N2 strains that were closely related to vaccine strain A/Texas/50/2012(H3N2). Several protein sites could accurately discriminate between outpatient versus inpatient strains, with site 221 in neuraminidase (NA) achieving the highest accuracy for A/H3N2. Interestingly, amino acid residues of inpatient but not outpatient isolates at those sites generally matched the corresponding residues in vaccine strains, except at site 145 of hemagglutinin (HA). This would be especially relevant for future surveillance of A/H3N2 strains in relation to their antigenicity and virulence. Furthermore, we observed a trend in which the HA proteins of influenza A/H3N2 and A/H1N1 exhibited enhanced ability to bind both avian and human host cell receptors. In contrast, the binding ability to each receptor was relatively stable for the HA of influenza B. CONCLUSIONS: Overall, our findings extend our understanding of the molecular and structural evolution of influenza virus strains in Singapore within the global context of these dynamic viruses.


Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenzavirus B/genética , Adolescente , Adulto , Idoso , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hospitalização , Humanos , Influenza Humana/virologia , Pessoa de Meia-Idade , Mutação , Neuraminidase/genética , Pacientes Ambulatoriais , Filogenia , Receptores Virais/química , Singapura , Proteínas Virais/genética , Adulto Jovem
2.
SAR QSAR Environ Res ; 30(12): 899-917, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31645133

RESUMO

Influenza A virus (IAV) has caused epidemic infections worldwide, with many strains resistant to inhibitors of a surface protein, neuraminidase (NA), due to point mutations on its structure. A novel NA inhibitor named peramivir was recently approved, but no exhaustive computational research regarding its binding affinity with wild-type and mutant NA has been conducted. In this study, a thorough investigation of IAV-NA PDB entries of 9 subtypes is described, providing a list of residues constituting the protein-ligand binding sites. The results of induced-fit docking approach point out key residues of wild-type NA participating in hydrogen bonds and/or ionic interactions with peramivir, among which Arg 368 is responsible for a peramivir-NA ionic interaction. Mutations on this residue greatly reduced the binding affinity of peramivir with NA, with 3 mutations R378Q, R378K and R378L (NA6) capable of deteriorating the docking performance of peramivir by over 50%. 200 compounds from 6-scaffolds were docked into these 3 mutant versions, revealing 18 compounds giving the most promising results. Among them, CMC-2012-7-1527-56 (benzoic acid scaffold, IC50 = 32 nM in inhibitory assays with IAV) is deemed the most potential inhibitor of mutant NA resisting both peramivir and zanamivir, and should be further investigated.


Assuntos
Antivirais/química , Ciclopentanos/química , Inibidores Enzimáticos/química , Guanidinas/química , Neuraminidase/química , Proteínas Virais/química , Sítios de Ligação , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Mutação , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Relação Quantitativa Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética
3.
Microbiology ; 165(11): 1181-1197, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31517596

RESUMO

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: Tannerella forsythia, Treponema denticola and Porphyromonas gingivalis. These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant P. gingivalis sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids - a phenomenon overcome by the NanS sialate-esterase from T. forsythia. Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and P. gingivalis was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of P. gingivalis on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by P. gingivalis and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host-pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Neuraminidase/metabolismo , Porphyromonas gingivalis/enzimologia , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interações Microbianas , Mucinas/metabolismo , Mutação , Neuraminidase/genética , Polissacarídeos/metabolismo , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/patogenicidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/metabolismo , Tannerella forsythia/enzimologia , Fatores de Virulência/genética , Zanamivir/farmacologia
4.
Cell Biochem Biophys ; 77(4): 319-333, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31559538

RESUMO

Influenza virus is known for its intermittent outbreaks affecting billions of people worldwide. Several neuraminidase inhibitors have been used in practice to overcome this situation. However, advent of new resistant mutants has limited its clinical utilization. In the recent years drug repurposing technique has attained the limelight as it is cost effective and reduces the time consumed for drug discovery. Here, we present multi-dimensional repurposing strategy that integrates the results of ligand-, energy-, receptor cavity, and shape-based pharmacophore algorithm to effectively identify novel drug candidate for influenza. The pharmacophore hypotheses were generated by utilizing the PHASE module of Schrödinger. The generated hypotheses such as AADP, AADDD, and DDRRNH, respectively, for ligand-, e-pharmacophore and receptor cavity based approach alongside shape of oseltamivir were successfully utilized to screen the DrugBank database. Subsequently, these models were evaluated for their differentiating ability using Enrichment calculation. Receiver operating curve and enrichment factors from the analysis indicate that the models possess better capability to screen actives from decoy set of molecules. Eventually, the hits retrieved from different hypotheses were subjected to molecular docking using Glide module of Schrödinger Suite. The results of different algorithms were then combined to eliminate false positive hits and to demonstrate reliable prediction performance than existing approaches. Of note, Pearson's correlation coefficients were calculated to examine the extent of correlation between the glide score and IC50 values. Further, the interaction profile, pharmacokinetic, and pharmacodynamics properties were analyzed for the hit compounds. The results from our analysis showed that alprostadil (DB00770) exhibits better binding affinity toward NA protein than the existing drug molecules. The biological activity of the hit was also predicted using PASS algorithm that renders the antiviral activity of the compound. Further, the results were validated using mutation analysis and molecular dynamic simulation studies. Indeed, this integrative filtering is able to exceed accuracy of other state-of-the-art methods for the drug discovery.


Assuntos
Descoberta de Drogas , Reposicionamento de Medicamentos , Algoritmos , Alprostadil/química , Alprostadil/metabolismo , Alprostadil/uso terapêutico , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Oseltamivir/química , Oseltamivir/metabolismo , Ligação Proteica
5.
Emerg Microbes Infect ; 8(1): 1428-1437, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31560252

RESUMO

Switching of receptor binding preference has been widely considered as one of the necessary mutations for avian influenza viruses, enabling efficient transmissions between human hosts. By stably overexpressing B4GalNT2 gene in MDCK cells, surface α2,3-siallylactose receptors were modified without affecting α2,6-receptor expression. The cell line MDCK-B4GalNT2 was used as a tool to screen for α2,3-receptor requirements in a panel of influenza viruses with previously characterized glycan array data. Infection of viruses with α2,3-receptor binding capability was inhibited in MDCK-B4GalNT2 cells, with the exception of A/WSN/33 (WSN). Infection with the 2009 pandemic H1N1 strains, A/California/04/2009 (Cal04) and A/Hong Kong/415742/2009 (HK09), despite showing α2,6-receptor binding, was also found to be inhibited. Further investigation showed that viral inhibition was due to a reduction in viral entry rate and viral attachment. Recombinant WSN virus with the neuraminidase (NA) gene swapped to A/Puerto Rico/8/1934 (PR8) and Cal04 resulted in a significant viral inhibition in MDCK-B4GalNT2 cells. With oseltamivir, the NA active site was found to be important for the replication results of WSN, but not Cal04.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , N-Acetilgalactosaminiltransferases/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Células Madin Darby de Rim Canino , N-Acetilgalactosaminiltransferases/metabolismo , Neuraminidase/genética , Oseltamivir/farmacologia , Replicação Viral/efeitos dos fármacos
6.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509989

RESUMO

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.


Assuntos
Galectina 1/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária , Linfócitos T/metabolismo , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Feminino , Expressão Gênica , Glicosilação , Humanos , Lectinas/metabolismo , Lúpus Eritematoso Sistêmico/genética , Masculino , Pessoa de Meia-Idade , Neuraminidase/genética , Neuraminidase/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Propriedades de Superfície , Adulto Jovem
7.
Indian J Med Microbiol ; 37(1): 42-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424009

RESUMO

Introduction: Influenza A(H1N1)pdm09 virus, since its identification in April 2009, has continued to cause significant outbreaks of respiratory tract infections including pandemics in humans. In the course of its evolution, the virus has acquired many mutations with an ability to cause increased disease severity. A regular molecular surveillance of the virus is essential to mark the evolutionary changes that may cause a shift to the viral behavior. Materials and Methods: Samples of Throat/Nasal swabs were collected from a total of 3715 influenza-like illness cases and screened by Real-time Reverse Transcription-Polymerase Chain Reaction for influenza viruses. Nucleotide sequence analysis was done to identify changes in antigenicity of the virus strains. Results: The present study describes the molecular characteristics of influenza A(H1N1)pdm09 viruses detected in Assam of Northeast India during 2009-2015. Influenza A viruses were detected in 11.4% (425/3715), of which influenza A(H1N1)pdm09 viruses were detected in 41.4% (176/425). The nucleotide sequencing of influenza A(H1N1)pdm09 viruses revealed a total of 17 and 22 amino acid substitutions in haemagglutinin (HA) and neuraminidase (NA) genes of the virus, respectively, compared to contemporary vaccine strain A/California/07/2009. The important mutations detected in HA genes of A/Assam(H1N1)pdm09 strains included E391K, K180Q and S202T. Mutation 'N248D' which has an ability to develop oseltamivir resistance was also detected in NA gene of A/Assam(H1N1)pdm09 strains. Conclusions: Regular molecular surveillance of influenza A(H1N1)pdm09 is important to monitor the viral behavior in terms of increase virulence, drug resistance pattern and emergence of novel strains.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/epidemiologia , Neuraminidase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antivirais/farmacologia , Sequência de Bases , Criança , Pré-Escolar , Feminino , Variação Genética/genética , Humanos , Índia/epidemiologia , Lactente , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Oseltamivir/farmacologia , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Adulto Jovem
8.
Arch Virol ; 164(11): 2881-2885, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31456087

RESUMO

Aquatic birds are known to be a reservoir for the most common influenza A viruses (IAVs). In the annual surveillance program, we collected the feces of migratory birds for the detection of IAVs in South Korea in November 2016. A novel reassorted H3N3 avian influenza virus (AIV) containing genes from viruses of wild and domestic birds was identified and named A/aquatic bird/South Korea/sw006/2016(H3N3). The polymerase basic 2 (PB2) and non-structural (NS) genes of this isolate are most closely related to those of wild-bird-origin AIV, while the polymerase basic 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), and matrix (M) genes are most closely related to those of domestic-bird-origin AIV. A/aquatic bird/South Korea/sw006/2016 contains PA, NP, M, and NS genes were most closely related to those of AIV subtype H4 and PB2, PB1, and HA genes that are most closely related to those of AIV subtype H3N8, while the NA gene was most closely related to those of subtype H10, which was recently detected in humans in China. These results suggest that novel reassortment of AIV strains occurred due to interaction between wild and domestic birds. Hence, we emphasize the need for continued surveillance of avian influenza virus in bird populations.


Assuntos
Genoma Viral/genética , Vírus da Influenza A Subtipo H3N8/genética , Influenza Aviária/virologia , Vírus Reordenados/genética , Animais , Aves/virologia , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Neuraminidase/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Sequenciamento Completo do Genoma
9.
Artigo em Inglês | MEDLINE | ID: mdl-31297339

RESUMO

The most frequent form of hemolytic-uremic syndrome (HUS) is associated with infections caused by Shiga-like toxin-producing Enterohaemorrhagic Escherichia coli (STEC). In rarer cases HUS can be triggered by Streptococcus pneumoniae. While production of Shiga-like toxins explains STEC-HUS, the mechanisms of pneumococcal HUS are less well-known. S. pneumoniae produces neuraminidases with activity against cell surface sialic acids that are critical for factor H-mediated complement regulation on cells and platelets. The aim of this study was to find out whether S. pneumoniae neuraminidase NanA could trigger complement activation and hemolysis in whole blood. We studied clinical S. pneumoniae isolates and two laboratory strains, a wild-type strain expressing NanA, and a NanA deletion mutant for their ability to remove sialic acids from various human cells and platelets. Red blood cell lysis and activation of complement was measured ex vivo by incubating whole blood with bacterial culture supernatants. We show here that NanA expressing S. pneumoniae strains and isolates are able to remove sialic acids from cells, and platelets. Removal of sialic acids by NanA increased complement activity in whole blood, while absence of NanA blocked complement triggering and hemolytic activity indicating that removal of sialic acids by NanA could potentially trigger pHUS.


Assuntos
Neuraminidase/sangue , Neuraminidase/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/genética , Plaquetas/metabolismo , Proteínas do Sistema Complemento/efeitos dos fármacos , Eritrócitos , Células HEK293 , Hemólise , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Inflamação , Neuraminidase/genética , Neuraminidase/farmacologia , Infecções Pneumocócicas/microbiologia , Deleção de Sequência , Ácidos Siálicos
10.
Vet Microbiol ; 235: 21-24, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282375

RESUMO

Occurrence of avian influenza (AI) with Neuraminidase (NA) mutations which confer reduced neuraminidase inhibitor (NAI) susceptibility has remained a cause of concern. The susceptibility to NAIs of 67 highly pathogenic avian influenza H5N1 viruses isolated during 2006-2012 in India was tested in phenotypic fluorescence-based NA inhibition assay, sequence analysis and in ovo. One isolate showed a novel NA I117T amino acid substitution (N2 numbering) and eight isolates showed previously known NAI-resistance marker mutations (I117V, E119D, N294S, total 9/67). The overall incidence of resistant variants was 13.4%. The novel I117T substitution reduced oseltamivir susceptibility by 18.6-fold and zanamivir susceptibility by 11.8-fold, compared to the wild type AI H5N1virus, thus showed cross-resistance to both oseltamivir and zanamivir in NA inhibition assays. However, the other two isolates with I117V substitution were sensitive to both the NAIs. In addition, the comparison of growth of the I117T and I117V variants in presence of NAI's in the in ovo assays exhibited difference in growth levels. The present study reports the natural occurrence of a novel I117T mutation in AI H5N1 virus conferring cross-resistance to oseltamivir and zanamivir highlighting the urgent need of antiviral surveillance of AI viruses.


Assuntos
Antivirais/farmacologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Neuraminidase/genética , Oseltamivir/farmacologia , Proteínas Virais/genética , Zanamivir/farmacologia , Substituição de Aminoácidos , Animais , Galinhas , Farmacorresistência Viral , Índia , Virus da Influenza A Subtipo H5N1/genética , Concentração Inibidora 50 , Mutação de Sentido Incorreto , Zigoto
11.
Virology ; 534: 80-86, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31220651

RESUMO

The immunopathological mechanisms as well as the role played by influenza A virus infection of human leukocytes and induction of apoptosis have not been fully elucidated. We confirm here that the percentage of cells that are infected is less than the percent of apoptotic cells. Depletion of monocytes/macrophages and depletion of cells expressing influenza neuraminidase from the cultures after exposure to virus decreased lymphocyte apoptosis. Treatment of virus-exposed leukocyte cultures with anti-neuraminidase antibodies but not with anti-hemagglutinin antibodies, reduced lymphocyte production of active caspase-3 and induction of apoptosis. Different strains of virus induced different levels of apoptosis. Variations in induction of apoptosis correlated with production and expression of viral neuraminidase by infected leukocytes. The data suggest that cell surface expression of neuraminidase plays an important role in the induction of apoptosis in human lymphocytes. The benefit, or cost, to the host of lymphocyte apoptosis warrants continued investigation.


Assuntos
Apoptose , Membrana Celular/virologia , Vírus da Influenza A/enzimologia , Influenza Humana/virologia , Linfócitos/citologia , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Influenza Humana/enzimologia , Influenza Humana/genética , Influenza Humana/fisiopatologia , Linfócitos/virologia , Neuraminidase/genética , Proteínas Virais/genética
12.
PLoS Pathog ; 15(6): e1007860, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181126

RESUMO

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.


Assuntos
Vírus da Influenza A Subtipo H2N2 , Vírus da Influenza A Subtipo H3N2 , Neuraminidase , Receptores Virais , Proteínas Virais , Vírion , Animais , Sítios de Ligação , Cães , Humanos , Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Células Madin Darby de Rim Canino , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo
13.
Pak J Pharm Sci ; 32(2 (Supplementary)): 825-829, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31103978

RESUMO

Trans-sialidase of Trypanosoma cruzi (TcTS) is a key enzyme in the infection process from parasite to host; therefore, it has been considered an important target for developing new anti-Chagas drugs. Different compounds with trypanocidal activity and/or inhibition of TcTS have been reported; however, some benzoic acid derivatives have shown high enzymatic inhibition but low trypanocidal activity and viceversa. These results show that each compound may possess a different mechanism of action. Based on the above, the compound 4-amino-3-nitrobenzoic acid (16), a potent TcTS inhibitor (77% inhibition in enzymatic assays) was selected to evaluate its effects on the expression level of the TS gene in T. cruzi epimastigotes and determine its involvement in the mechanism of action. Results showed an increase in the expression level of the TcTS gene, which confirmed that compound 16, has a direct effect on TcTS.


Assuntos
Glicoproteínas/genética , Neuraminidase/genética , Nitrobenzoatos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Nitrobenzoatos/química , Reação em Cadeia da Polimerase em Tempo Real , Tripanossomicidas/química
14.
Transbound Emerg Dis ; 66(5): 1884-1893, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31059176

RESUMO

Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub-lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.


Assuntos
Surtos de Doenças/veterinária , Doenças dos Cavalos/epidemiologia , Vírus da Influenza A Subtipo H3N8/genética , Infecções por Orthomyxoviridae/veterinária , Vacinação/veterinária , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Doenças dos Cavalos/virologia , Cavalos , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Malásia/epidemiologia , Nasofaringe/virologia , Neuraminidase/genética , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Filogenia , Alinhamento de Sequência/veterinária , Proteínas Virais/genética
15.
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
16.
Virus Genes ; 55(4): 440-447, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31025287

RESUMO

The untranslated regions within viral segments are the essential promoter elements required for the initiation of viral replication and transcription. The end of the UTR sequence and part of the ORF sequence constitute the packaging signal for progeny viruses. To explore the influence of single-point and multi-site joint mutations in the UTR of the NA gene on the viral expression, we select clones with upregulated expression of the reporter gene and analyze their sequence characteristics. Bioinformatics methods were used to analyze polymorphisms in the untranslated region (UTR) of the neuraminidase gene of the H9N2 influenza A virus. Using the RNA polymerase I reporting system with enhanced green fluorescence protein (EGFP) gene as the reporter gene, libraries containing random mutations at sites within the N2 UTR were constructed using random mutagenesis. The mutants were selected from the randomized mutagenesis libraries for the N2-UTR. The N2-UTR-RNA polymerase I fluorescence reporter system was identified by sequencing and transfected into infected MDCK cells. The expression of the reporter EGFP was observed using fluorescence microscopy, and the relative fluorescence intensity was measured using a multifunctional microplate reader to analyze the expression of the reporter gene (EGFP) qualitatively and quantitatively. Herein, an RNA polymerase reporter system was constructed to rescue the mutated viruses and measure their tissue culture infective dose (TCID50). The results showed that the U13 → C13 mutation in the 3'end of the NA gene promoted the expression of viral RNA and protein, and mutation of other sites within the UTR could differentially regulate viral genomic transcription and translation. These data showed that the U13 → C13 mutation within the variable region of the 3'UTR of the NA gene in the H9N2 influenza virus promotes viral genomic expression and infection.


Assuntos
Vírus da Influenza A Subtipo H9N2/genética , Neuraminidase/genética , Proteínas Virais/genética , Replicação Viral/genética , Regiões 3' não Traduzidas , Animais , Cães , Regulação Viral da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Células Madin Darby de Rim Canino , Mutagênese , RNA Viral
17.
Vet Microbiol ; 232: 151-155, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030840

RESUMO

Influenza virus-like sequences of H17N10 and H18N11 were identified in bats, despite there has been no live virus isolated. The genetic analysis indicated that they have distinct but relatively close evolutionary relationships to known influenza A viruses. However, the infectivity and adaptation of bat influenza viruses in avian species remain unclear. In this study, two modified bat influenza viruses cH9cN2/H17 and cH9cN2/H18 containing HA and NA coding regions replaced with those of H9N2 influenza A virus were generated in the background of the H17N10 or H18N11 viruses. These two modified viruses replicated less efficiently than wild type H9N2 virus in cultured chicken cells. The mini-genome assay showed that viral ribonucleoproteins (vRNPs) of H9N2 has significantly higher polymerase activity than that of bat influenza viruses in avian cells. In chicken study, compared with H9N2 virus, which replicated and transmitted efficiently in chickens, the cH9cN2/H17 and cH9cN2/H18 viruses only replicated in chicken tracheas with lower titers. Pathological examination showed that the H9N2 caused severer lesions in lung and trachea than the modified bat influenza viruses. Notably, the cH9cN2/H18 transmitted among chickens, but not cH9cN2/H17, and chicken IFN-ß antagonism results showed that H18N11 NS1 protein inhibited chicken IFN-ß response more efficiently than H17N10 NS1 protein in avian cells. Taken together, our data indicated that the internal genes of bat influenza viruses adapted poorly to chickens, while the internal genes of H18N11 seemed to adapt to chickens better than H17N10.


Assuntos
Adaptação Fisiológica/genética , Quimera/genética , Quirópteros/virologia , Vírus da Influenza A Subtipo H9N2/genética , Orthomyxoviridae/genética , Animais , Sequência de Bases , Galinhas/virologia , Hemaglutininas Virais/genética , Influenza Aviária/virologia , Neuraminidase/genética , Doenças das Aves Domésticas/virologia , Ribonucleoproteínas
18.
BMC Infect Dis ; 19(1): 314, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971204

RESUMO

BACKGROUND: Influenza surveillance is necessary for detection of emerging variants of epidemiologic and clinical significance. This study describes the epidemiology of influenza types A and B, and molecular characteristics of surface glycoproteins (hemagglutinin [HA] and neuraminidase [NA]) of influenza A subtypes: pH1N1 and H3N2 circulated in Arabian Gulf, Levant and North Africa regions during 2009-2017. METHODS: Analysis of phylogenetics and evolution of HA and NA genes was done using full HA and NA sequences (n = 1229) downloaded from Influenza Research Database (IRD). RESULTS: In total, 130,354 influenza positive cases were reported to WHO during study period. Of these, 50.8% were pH1N1 positive, 15.9% were H3N2 positives and 17.2% were influenza B positive. With few exceptions, all three regions were showing the typical seasonal influenza peak similar to that reported in Northern hemisphere (December-March). However, influenza activity started earlier (October) in both Gulf and North Africa while commenced later during November in Levant countries. The molecular analysis of the HA genes (influenza A subtypes) revealed similar mutations to those reported worldwide. Generally, amino acid substitutions were most frequently found in head domain in H1N1 pandemic viruses, while localized mainly in the stem region in H3N2 viruses. Expectedly, seasons with high pH1N1 influenza activity was associated with a relatively higher number of substitutions in the head domain of the HA in pH1N1 subtype. Furthermore, nucleotide variations were lower at the antigenic sites of pH1N1 viruses compared to H3N2 viruses, which experienced higher variability at the antigenic sites, reflecting the increased immunological pressure because of longer circulation and continuous vaccine changes. Analysis of NA gene of pH1N1 viruses revealed sporadic detections of oseltamivir-resistance mutation, H275Y, in 4% of reported sequences, however, none of NAI resistance mutations were found in the NA of H3N2 viruses. CONCLUSIONS: Molecular characterization of H1N1 and H3N2 viruses over 9 years revealed significant differences with regard to position and function of characterized substitutions. While pH1N1 virus substitutions were mainly found in HA head domain, H3N2 virus substitutions were mostly found in HA stem domain. Additionally, more fixed substitutions were encountered in H3N2 virus compared to larger number of non-fixed substitutions in pH1N1.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/epidemiologia , Mutação , África do Norte/epidemiologia , Substituição de Aminoácidos , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Oriente Médio/epidemiologia , Neuraminidase/genética , Filogenia , Estações do Ano
19.
J Gen Virol ; 100(5): 752-759, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30994443

RESUMO

Influenza A virus mutates rapidly, allowing it to escape natural and vaccine-induced immunity. Neuraminidase (NA) is a surface protein capable of cleaving the glycosidic linkages of neuraminic acids to release newly formed virions from infected cells. Genetic variants within a viral population can influence the emergence of pandemic viruses as well as drug susceptibility and vaccine effectiveness. In the present study, 55 clinical specimens from patients infected with the 2009 pandemic influenza A/H1N1 virus, abbreviated as A(H1N1)pdm09, during the 2015-2016 outbreak season in Taiwan were collected. Whole genomes were obtained through next-generation sequencing. Based on the published sequences from A(H1N1)pdm09 strains worldwide, a mixed population of two distinct variants at NA position 151 was revealed. We initially reasoned that such a mixed population may have emerged during cell culture. However, additional investigations confirmed that these mixed variants were detectable in the specimens of patients. To further investigate the role of the two NA-151 variants in a dynamic population, a reverse genetics system was employed to generate recombinant A(H1N1)pdm09 viruses. It was observed that the mixture of the two distinct variants was characterized by a higher replication rate compared to the recombinant viruses harbouring a single variant. Moreover, an NA inhibition assay revealed that a high frequency of the minor NA-151 variant in A(H1N1)pdm09 was associated with a reduced susceptibility to NA inhibitors. We conclude that two distinct NA-151 variants can be identified in patient specimens and that such variants may increase viral replication and NA activity.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Neuraminidase/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Cães , Variação Genética/genética , Células HEK293 , Humanos , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/virologia , Dinâmica Populacional , Replicação Viral/genética
20.
mBio ; 10(2)2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967460

RESUMO

The effectiveness of influenza vaccines against circulating A(H1N1)pdm09 viruses was modest for several seasons despite the absence of antigenic drift of hemagglutinin (HA), the primary vaccine component. Since antibodies against HA and neuraminidase (NA) contribute independently to protection against disease, antigenic changes in NA may allow A(H1N1)pdm09 viruses to escape from vaccine-induced immunity. In this study, analysis of the specificities of human NA-specific monoclonal antibodies identified antigenic sites that have changed over time. The impact of these differences on in vitro inhibition of enzyme activity was not evident for polyclonal antisera until viruses emerged in 2013 without a predicted glycosylation site at amino acid 386 in NA. Phylogenetic and antigenic cartography demonstrated significant antigenic changes that in most cases aligned with genetic differences. Typical of NA drift, the antigenic difference is observed in one direction, with antibodies against conserved antigenic domains in A/California/7/2009 (CA/09) continuing to inhibit NA of recent A(H1N1)pdm09 viruses reasonably well. However, ferret CA/09-specific antiserum that inhibited the NA of A/Michigan/45/2015 (MI/15) very well in vitro, protected mice against lethal MI/15 infection poorly. These data show that antiserum against the homologous antigen is most effective and suggest the antigenic properties of NA should not be overlooked when selecting viruses for vaccine production.IMPORTANCE The effectiveness of seasonal influenza vaccines against circulating A(H1N1)pdm09 viruses has been modest in recent years, despite the absence of antigenic drift of HA, the primary vaccine component. Human monoclonal antibodies identified antigenic sites in NA that changed early after the new pandemic virus emerged. The reactivity of ferret antisera demonstrated antigenic drift of A(H1N1)pdm09 NA from 2013 onward. Passive transfer of serum raised against A/California/7/2009 was less effective than ferret serum against the homologous virus in protecting mice against a virus with the NA of more recent virus, A/Michigan/45/2015. Given the long-standing observation that NA-inhibiting antibodies are associated with resistance against disease in humans, these data demonstrate the importance of evaluating NA drift and suggest that vaccine effectiveness might be improved by selecting viruses for vaccine production that have NAs antigenically similar to those of circulating influenza viruses.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Deriva Genética , Vírus da Influenza A Subtipo H1N1/imunologia , Neuraminidase/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Modelos Animais de Doenças , Furões , Evasão da Resposta Imune , Imunização Passiva , Vírus da Influenza A Subtipo H1N1/genética , Camundongos , Neuraminidase/genética , Infecções por Orthomyxoviridae/prevenção & controle , Análise de Sobrevida , Resultado do Tratamento , Proteínas Virais/genética
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