RESUMO
A single-nucleotide polymorphism (SNP) rs12252-C of interferon-induced transmembrane protein 3 (IFITM3), resulting in a truncated IFITM3 protein lacking 21 N-terminus amino acids, is associated with severe influenza infection in the Chinese population. However, the effect of IFITM3 rs12252-C on influenza vaccination and the underlying mechanism is poorly understood. Here, we constructed a mouse model with a deletion of 21 amino acids at the N-terminus (NΔ21) of IFITM3 and then compared the antibody response between Quadrivalent influenza vaccine (QIV) immunized wild-type (WT) mice and NΔ21 mice. Significantly higher levels of haemagglutination inhibition (HI) titre, neutralizing antibodies (NAb), and immunoglobulin G (IgG) to H1N1, H3N2, B/Victory, and B/Yamagata viruses were observed in NΔ21 mice compared to WT mice. Correspondingly, the numbers of splenic germinal centre (GC) B cells, plasma cells, memory B cells, QIV-specific IgG+ antibody-secreting cells (ASC), and T follicular helper cells (TFH) in NΔ21 mice were higher compared with WT mice. Moreover, the 21-amino-acid deletion caused IFITM3 translocation from the endocytosis compartment to the periphery of cells, which also prevented the degradation of a co-stimulatory molecule of B cell receptor (BCR) CD81 on the cell surface. More importantly, a more interaction was observed between NΔ21 protein and CD81 compared to the interaction between IFITM3 and CD81. Overall, our study revealed a potential mechanism of NΔ21 protein enhancing humoral immune response by relocation to prevent the degradation of CD81, providing insight into SNP affecting influenza vaccination.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Influenza Humana , Animais , Camundongos , Humanos , Imunidade Humoral , Vírus da Influenza A Subtipo H3N2/genética , Imunoglobulina G , Aminoácidos , Anticorpos AntiviraisRESUMO
Egg-adaptive mutations in influenza hemagglutinin (HA) often emerge during the production of egg-based seasonal influenza vaccines, which contribute to the largest share in the global influenza vaccine market. While some egg-adaptive mutations have minimal impact on the HA antigenicity (e.g. G186V), others can alter it (e.g. L194P). Here, we show that the preference of egg-adaptive mutation in human H3N2 HA is strain-dependent. In particular, Thr160 and Asn190, which are found in many recent H3N2 strains, restrict the emergence of L194P but not G186V. Our results further suggest that natural amino acid variants at other HA residues also play a role in determining the preference of egg-adaptive mutation. Consistently, recent human H3N2 strains from different clades acquire different mutations during egg passaging. Overall, these results demonstrate that natural mutations in human H3N2 HA can influence the preference of egg-adaptation mutation, which has important implications in seed strain selection for egg-based influenza vaccine.
Assuntos
Vacinas contra Influenza , Influenza Humana , Aminoácidos/genética , Animais , Galinhas , Ovos , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Vacinas contra Influenza/genética , MutaçãoRESUMO
Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAs.
Assuntos
COVID-19 , Interferon Tipo I , MicroRNAs , Antivirais/farmacologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Interferon Tipo I/genética , RNA de Cadeia Dupla , SARS-CoV-2Assuntos
Antivirais/uso terapêutico , Dibenzotiepinas/uso terapêutico , Farmacorresistência Viral , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Morfolinas/uso terapêutico , Piridonas/uso terapêutico , Triazinas/uso terapêutico , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/virologia , Japão , Masculino , Testes de Sensibilidade Microbiana , Mutação , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genéticaRESUMO
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 , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza B , Infecções por Orthomyxoviridae , Oseltamivir , Animais , Feminino , Masculino , Substituição de Aminoácidos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Furões , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza B/genética , Vírus da Influenza B/metabolismo , Macaca fascicularis , Macrolídeos , 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 , Oseltamivir/farmacologiaRESUMO
Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in the polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold) but also on in vitro replicative fitness. Although I38L conferred undiminished growth, other substitutions led to mild attenuation. In a ferret model, control viruses outcompeted those carrying I38M or I38T substitutions, although their advantage was limited. These findings offer insights into the attributes of baloxavir-resistant viruses needed for informed risk assessment.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Infecções por Orthomyxoviridae/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Replicação Viral/genética , Substituição de Aminoácidos , Animais , Dibenzotiepinas , Modelos Animais de Doenças , Cães , Furões , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino , Masculino , Testes de Sensibilidade Microbiana , Morfolinas , Infecções por Orthomyxoviridae/virologia , Piridonas , RNA Polimerase Dependente de RNA/genética , Estações do Ano , Resultado do Tratamento , Proteínas Virais/genéticaRESUMO
BACKGROUND: In May 2017, 17 dogs in a German Shepherd breeding kennel in northern China developed respiratory clinical signs. The owner treated the dogs with an intravenous injection of Shuang-Huang-lian, a traditional Chinese medicine, and azithromycin. The respiratory signs improved 3 days post-treatment, however, cysts were observed in the necks of eight dogs, and three of them died in the following 2 days. CASE PRESENTATION: Quantitative real-time PCR was used to detect canine influenza virus (CIV). All of the dogs in this kennel were positive and the remaining 14 dogs had seroconverted. Two of the dogs were taken to the China Agricultural University Veterinary Teaching Hospital for further examination. Two strains of influenza virus (A/canine/Beijing/0512-133/2017 and A/canine/Beijing/0512-137/2017) isolated from the nasal swabs of these dogs were sequenced and identified as avian-origin H3N2 CIV. For the two dogs admitted to the hospital, hematology showed mild inflammation and radiograph results indicated pneumonia. Cyst fluid was plated for bacterial culture and bacterial 16 s rRNA gene PCR was performed, followed by Sanger sequencing. The results indicated an Enterococcus faecalis infection. Antimicrobial susceptibility tests were performed and dogs were treated with enrofloxacin. All 14 remaining dogs recovered within 16 days. CONCLUSIONS: Coinfection of H3N2 CIV and Enterococcus faecalis was detected in dogs, which has not been reported previously. Our results highlight that CIV infection might promote the secondary infection of opportunistic bacteria and cause more severe and complicated clinical outcomes.
Assuntos
Coinfecção/veterinária , Doenças do Cão/virologia , Infecções por Bactérias Gram-Positivas/veterinária , Vírus da Influenza A Subtipo H3N2 , Infecções por Orthomyxoviridae/veterinária , Animais , China/epidemiologia , Coinfecção/epidemiologia , Coinfecção/microbiologia , Coinfecção/virologia , Surtos de Doenças/veterinária , Doenças do Cão/epidemiologia , Doenças do Cão/microbiologia , Cães/virologia , Enterococcus faecalis , Feminino , Infecções por Bactérias Gram-Positivas/complicações , Infecções por Bactérias Gram-Positivas/microbiologia , Vírus da Influenza A Subtipo H3N2/genética , Masculino , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza virus infection in Japan in February 2018. Two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA) were detected in baloxavir-treated children in December 2018. This mutation is known to confer reduced susceptibility to baloxavir, and the two mutant viruses exhibited 76- and 120-fold reduced susceptibility to baloxavir.
Assuntos
Antivirais/uso terapêutico , Endonucleases/antagonistas & inibidores , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Substituição de Aminoácidos/genética , Antivirais/administração & dosagem , Dibenzotiepinas , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Endonucleases/genética , Humanos , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/diagnóstico , Japão , Testes de Sensibilidade Microbiana , Morfolinas , Piridonas , Resultado do TratamentoRESUMO
BACKGROUND: Artemisia scoparia Waldst and Kit is a famous traditional Chinese medicine widely distributed in Xinjiang, China. Flavonoids extracted from it exhibits inhibitory activities against several influenza virus strains. Despite this fact, the antiviral properties of CST, one of such flavonoids, against the influenza virus has not been reported. Thus, the aim of this study is to investigate the anti-influenza virus efficacy and antiviral mechanism of CST. METHODS: The inhibitory activity of CST against influenza viruses was assessed by using viral titers and performing Western blot, qRT-PCR, and immunofluorescence assays in Madin-Darby canine kidney (MDCK) cells and a human monocytic cell line (THP-1). The mechanism of CST against influenza virus was analyzed by hemagglutination inhibition (HI) assay, neuraminidase (NA) inhibition assay, and Western blot. RESULTS: CST reduced viral titers and influenza A virus (IAV) RNA and protein synthesis in a dose-dependent manner. Mechanistically, CST had no inhibitory effect on the attachment and release processes of the viral life cycle, as indicated by the HI and NA assays. Conversely, the CST-mediated inhibition of IAV is possibly linked to the inactivation of the NF-κB/p65 signal pathway. CST also suppressed the activation of JNK MAPK and P38 MAPK in vitro. In line with NF-κB/p65 inhibition, the expression levels of proinflammatory cytokines (TNF-α, IL-1ß, IL-8, and IL-10) and the inflammation-related protein COX-2 were downregulated by CST. CONCLUSIONS: CST inhibited IAV replication by downregulating the NF-κB signal transduction pathway. CST may be a potential agent or supplement against IAV infection.
Assuntos
Antivirais/farmacologia , Artemisia/química , Flavonas/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Fator de Transcrição RelA/genética , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Cães , Relação Dose-Resposta a Droga , Flavonas/isolamento & purificação , Regulação da Expressão Gênica , Testes de Inibição da Hemaglutinação , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Células Madin Darby de Rim Canino , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Neuraminidase/metabolismo , Extratos Vegetais/química , Transdução de Sinais , Células THP-1 , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Carga Viral/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Influenza virus evolves rapidly due to the accumulated genetic variations on the viral sequence. Unlike in North America and Europe, influenza season in the tropical Southeast Asia spans both the rainy and cool seasons. Thus, influenza epidemiology and viral evolution sometimes differ from other regions, which affect the ever-changing efficacy of the vaccine. To monitor the current circulating influenza viruses in this region, we determined the predominant influenza virus strains circulating in Thailand between January 2016 and June 2017 by screening 7,228 samples from patients with influenza-like illness. During this time, influenza A(H3N2) virus was the predominant influenza virus detected. We then phylogenetically compared the hemagglutinin (HA) gene from a subset of these A(H3N2) strains (n = 62) to the reference sequences and evaluated amino acid changes in the dominant antigenic epitopes on the HA protein structure. The divergence of the circulating A(H3N2) from the A/Hong Kong/4801/2014 vaccine strain formed five genetic groups (designated I to V) within the 3C.2a clade. Our results suggest a marked drift of the current circulating A(H3N2) strains in Thailand, which collectively contributed to the declining predicted vaccine effectiveness (VE) from 74% in 2016 down to 48% in 2017.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Aminoácidos/química , Epitopos/imunologia , Variação Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Filogenia , RNA Viral/genética , Estações do Ano , Tailândia/epidemiologiaRESUMO
BACKGROUND: Infants exhibit elevated influenza virus loads and prolonged viral shedding, which may increase the risk for resistance development, especially in cases of suboptimal exposure to antiviral therapy. METHODS: We performed a prospective surveillance of hospitalized infants undergoing oseltamivir therapy during the 2008-2009 and 2011-2012 influenza seasons at two paediatric hospitals in Germany. A total of 37 infants less than 1 year of age with laboratory confirmed influenza A(H3N2) infection received oseltamivir as per physician's order for 5 days (2008-2009 season: 2 mg/kg twice daily; 2011-2012 season: 2.0 mg/kg; 2.5 mg/kg and 3.0 mg/kg twice daily for infants <1 month; 2-3 months and 4-12 months, respectively). Virus load, the susceptibility to neuraminidase inhibitors (NAIs), and the presence of molecular markers of resistance to NAIs was assessed for influenza viruses recovered from respiratory samples collected at baseline and during follow-up visits. RESULTS: Overall, 73% of the infants continued to shed viral RNA detectable by reverse transcription (RT)-PCR after dose number 10 of oseltamivir; 12 infants shed viruses, 2 of them (both 9 months of age) shed resistant viruses. Resistance was characterized by ≥1,000-fold increase of 50% inhibitory concentration (IC50) for oseltamivir, up to 50-fold for zanamivir and elevated Km values when compared to susceptible A(H3N2) strains. Sanger sequencing revealed the selection of the NA-R292K substitution in both instances (after dose number 10 on day 6). CONCLUSIONS: Our data suggest that it may be relevant to monitor antiviral resistance systematically in all infants, considering that the European Medicines Agency has recently extended the licensure for oseltamivir to include full-term infants.
Assuntos
Farmacorresistência Viral , Hospitalização , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Oseltamivir/farmacologia , Oseltamivir/uso terapêutico , Antivirais/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Lactente , Recém-Nascido , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/diagnóstico , Influenza Humana/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Neuraminidase/antagonistas & inibidores , Vigilância em Saúde Pública , RNA Viral , Carga ViralRESUMO
There was an increase in severe and fatal influenza cases in Greece during the 2011-2015 post-pandemic period. To investigate causality, we determined neuraminidase (NA) inhibitor susceptibility and resistance-conferring NA and hemagglutinin (HA) mutations in circulating influenza type A viruses during the pandemic (2009-2010) and post-pandemic periods in Greece. One hundred thirty-four influenza A(H1N1)pdm09 and 95 influenza A(H3N2) viruses submitted to the National Influenza Reference Laboratory of Southern Greece were tested for susceptibility to oseltamivir and zanamivir. Antiviral resistance was assessed by neuraminidase sequence analysis, as well as the fluorescence-based 50 % inhibitory concentration (IC50) method. Five influenza A(H1N1)pdm09 viruses (2.2 %) showed significantly reduced inhibition by oseltamivir (average IC50 300.60nM vs. 1.19nM) by Gaussian kernel density plot analysis. These viruses were isolated from immunocompromised patients and harbored the H275Y oseltamivir resistance-conferring NA substitution. All A(H1N1)pdm09 viruses were zanamivir-susceptible, and all A(H3N2) viruses were susceptible to both drugs. Oseltamivir-resistant viruses did not form a distinct cluster by phylogenetic analysis. Permissive mutations were detected in immunogenic and non immunogenic NA regions of both oseltamivir- resistant and susceptible viruses in the post-pandemic seasons. Several amino acid substitutions in the HA1 domain of the HA gene of post-pandemic viruses were identified. This study indicated low resistance to NAIs among tested influenza viruses. Antiviral resistance emerged only in immunocompromised patients under long-term oseltamivir treatment. Sequential sample testing in this vulnerable group of patients is recommended to characterise resistance or reinfection and viral evolution.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Influenza Humana/virologia , Idoso , Feminino , Genótipo , Grécia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Hospedeiro Imunocomprometido , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Concentração Inibidora 50 , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Neuraminidase/genética , Oseltamivir/farmacologia , Proteínas Virais/genética , Zanamivir/farmacologiaRESUMO
The human influenza A (H3N2) virus dominated the 2014-2015 winter season in many countries and caused massive morbidity and mortality because of its antigenic variation. So far, very little is known about the antigenic patterns of the recent H3N2 virus. By systematically mapping the antigenic relationships of H3N2 strains isolated since 2010, we discovered that two groups with obvious antigenic divergence, named SW13 (A/Switzerland/9715293/2013-like strains) and HK14 (A/Hong Kong/5738/2014-like strains), co-circulated during the 2014-2015 winter season. HK14 group co-circulated with SW13 in Europe and the United States during this season, while there were few strains of HK14 in mainland China, where SW13 has dominated since 2012. Furthermore, we found that substitutions near the receptor-binding site on hemagglutinin played an important role in the antigenic variation of both the groups. These findings provide a comprehensive understanding of the recent antigenic evolution of H3N2 virus and will aid in the selection of vaccine strains.
Assuntos
Variação Antigênica , Antígenos Virais/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/virologia , Sequência de Aminoácidos , Aminoácidos/química , Antígenos Virais/genética , Sítios de Ligação , China , Simulação por Computador , Epidemias , Epitopos/química , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza , Influenza Humana/epidemiologia , Dados de Sequência Molecular , Filogenia , Estações do Ano , Homologia de Sequência de AminoácidosRESUMO
UNLABELLED: In order to improve the bioavailability levels of polyprenols (derived from ginkgo leaves (GBP)) in the human body, a GBP nanoemulsion was prepared, and its antiviral activity was evaluated against influenza A H3N2 and hepatitis B virus in vitro. METHODS: A GBP nanoemulsion was prepared by inversed-phase emulsification (IPE). Next, we investigated the antiviral activity of the GBP nanoemulsion on influenza A H3N2 and hepatitis B virus in vitro by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenlytetrezolium bromide) method. ELISA and the fluorescent quantitative PCR method were used to measure the content of HBsAg, HBeAg and DNA virus in human samples. RESULTS: The GBP nanoemulsion exhibited uniformity at an average particle size 97 nm with a hydrophilic-lipophilic balance (HLB) of 9.5. GBP is non-toxic to normal cells, hepatitis B virus DNA, hepatitis B virus antigen and HepG2215. Furthermore, GBP could reach a 70% virucidal activity and a 74.9% protection rate (*** p < 0.001) on MDCK cells infected with H3N2 virus at a high concentration of 100 µg/mL. GBP had a good inhibition rate on HBsAg (52.11%, ** p < 0.01) at 50 µg/mL and Day 9 of incubation, and a 67.32% inhibition effect on HBeAg at a high concentration of 100 µg/mL and Day 9. GBP had good inhibition on HBV DNA with CT 18.6 and lower copies (** p < 0.01) at a middle concentration of 12.5 to 25 µg/mL. CONCLUSIONS: The GBP nanoemulsion was very stable and non-toxic and had very strong antiviral activity against influenza A H3N2 and hepatitis B virus in vitro. The inhibitory effects and reactive mechanisms were similar to the drug, 3TC; by lengthening the incubation time and increasing the drug concentration, GBP has promising potential as an antiviral drug.
Assuntos
Antivirais/química , Antivirais/farmacologia , Ginkgo biloba/química , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Terpenos/química , Terpenos/farmacologia , Animais , Linhagem Celular Tumoral , DNA Viral/efeitos dos fármacos , DNA Viral/genética , Cães , Antígenos de Superfície da Hepatite B/efeitos dos fármacos , Antígenos de Superfície da Hepatite B/genética , Antígenos E da Hepatite B/efeitos dos fármacos , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Técnicas In Vitro , Vírus da Influenza A Subtipo H3N2/genética , Células Madin Darby de Rim Canino , Tamanho da Partícula , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/químicaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Chaenomeles sinensis Koehne (Chinese quince) are distributed throughout China and Japan. It has traditionally been known to have a therapeutic effect against respiratory symptoms caused by infectious diseases. AIM OF THE STUDY: The polyphenol-rich extract, CSD3, from Chaenomeles sinensis has previously been shown to neutralize influenza virus infectivity. The aim of this study was to clarify which step(s) in the replication cycle in vitro were inhibited. MATERIALS AND METHODS: We examined cell-binding, hemagglutination and hemolytic activities and infectivity of A/Udorn/72(H3N2) virus after pre-treatment with CSD3. We also investigated the time course of synthesis for viral mRNA, cRNA, and vRNA in Madin-Darby canine kidney epithelial cells (MDCK) cells infected with CSD3-treated virus. Finally, we studied the effect of CSD3-treatment on the ultrastructure of the influenza virion. RESULTS: Pre-treatment with CSD3 mildly reduced cell-binding, hemagglutination and hemolytic activities. These activities were reduced by 70% to be equivalent to 30% of the control at 1µg/ml. CSD3 severely reduced infectivity to 1% of the control at 1µg/ml. Primary transcription in MDCK cells infected with CSD3 (1µg/ml)-treated virus was decreased to about 1% of that in cells infected with mock-treated virus. Synthesis of viral cRNA, vRNA and secondary mRNA was also severely decreased. Electron microscopy revealed that the integrity of the virus envelope was damaged by CSD3 and was permeable to uranyl acetate. CONCLUSIONS: The main target step(s) of CSD3 in the replication cycle is after cell-binding but before or at primary transcription. Involvement of the increased permeability of virus envelope as the inhibition mechanism was proposed. CSD3 could be useful in preventing influenza virus infection, and be employed as a lozenge or mouthwash for daily use.
Assuntos
Antivirais/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Polifenóis/farmacologia , Rosaceae/química , Transcrição Gênica/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Técnicas de Cultura de Células , Galinhas , Cães , Medicamentos de Ervas Chinesas/isolamento & purificação , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/virologia , Hemaglutinação por Vírus/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/ultraestrutura , Células Madin Darby de Rim Canino , Microscopia Eletrônica de Transmissão , Polifenóis/isolamento & purificação , RNA Viral/biossíntese , RNA Viral/genética , Vírion/ultraestrutura , Replicação Viral/efeitos dos fármacosRESUMO
The anti-influenza A/HK (H3N2) virus activity of ß-santalol was evaluated in MDCK cells and investigated the effect of ß-santalol on synthesis of viral mRNAs. ß-Santalol was investigated for its antiviral activity against influenza A/HK (H3N2) virus using a cytopathic effect (CPE) reduction method. ß-Santalol exhibited anti-influenza A/HK (H3N2) virus activity of 86% with no cytotoxicity at the concentration of 100 µg/ml reducing the formation of a visible CPE. Oseltamivir also showed moderate antiviral activity of about 83% against influenza A/HK (H3N2) virus at the concentration of 100 µg/ml. Furthermore, the mechanism of anti-influenza virus action in the inhibition of viral mRNA synthesis was analyzed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and the data indicated an inhibitory effect in late viral RNA synthesis compared with oseltamivir in the presence of 100 µg/ml of ß-santalol. ß-Santalol should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Sesquiterpenos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Cães , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Testes de Sensibilidade Microbiana , Oseltamivir/farmacologia , Óleos de Plantas/química , Óleos de Plantas/farmacologia , Sesquiterpenos Policíclicos , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Santalum/química , Sesquiterpenos/químicaRESUMO
BACKGROUND: Oseltamivir, a specific influenza neuraminidase inhibitor, is an effective treatment for seasonal influenza. Emergence of drug-resistant influenza viruses after treatment has been reported, particularly in children in Japan, where the dosing schedule is different from that used throughout the rest of the world. We investigated the emergence of drug-resistant infection in children treated with a tiered weight-based dosing regimen. METHODS: We analyzed sequential clinical nasopharyngeal samples, obtained before and after tiered weight-based oseltamivir therapy, from children with acute influenza during 2005-2007. We isolated viruses, tested for drug resistance with use of a fluorescence-based neuraminidase inhibition assay, performed neuraminidase gene sequencing, and determined quantitative viral loads. RESULTS: Sixty-four children (34 with influenza A subtype H3N2, 11 with influenza A subtype H1N1, and 19 with influenza B virus) aged 1-12 years (median age, 3 years, 1 month) were enrolled. By days 4-7 after initiation of treatment, of 64 samples tested, 47 (73.4%) and 26 (40.6%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. By days 8-12 after initiation of treatment, of 53 samples tested, 18 (33.9%) and 1 (1.8%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. We found no statistically significant differences in the reduction of viral shedding or time to clearance of virus between viral subtypes. Antiviral-resistant viruses were recovered from 3 (27.3%) of 11 children with influenza A subtype H1N1, 1 (2.9%) of 34 children with influenza A subtype H3N2, and 0 (0%) of 19 children with influenza B virus, all of whom were treated with oseltamivir (P = .004). There was no evidence of prolonged illness in children infected with drug-resistant virus. CONCLUSIONS: Drug resistance emerges at a higher rate in influenza A subtype H1N1 virus than in influenza A subtype H3N2 or influenza B virus after tiered weight-based oseltamivir therapy. Virological surveillance for patterns of drug resistance is essential for determination of antiviral treatment strategies and for composition of pandemic preparedness stockpiles.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Oseltamivir/uso terapêutico , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Influenza Humana/virologia , Masculino , Testes de Sensibilidade Microbiana , Neuraminidase/genética , Análise de Sequência de DNA , Carga Viral , Proteínas Virais/genéticaRESUMO
The internal influenza virus proteins M1 and RNP free from surface protein impurities were isolated from subviral particles (virions free from HA and NA ectomenes). The spikeless particles had no propensity to aggregate in the solution at pH 5.0 as compared with native viruses. The subviral particles of B/Hong Kong/330/01 influenza virus, which belonged to B/Victoria/2/87-lineage, were obtained by proteolytic treatment with the enzyme bromelain under the same conditions as in cases of influenza B viruses of B/Jamagata/16/88 lineage. A chromatographic analysis of the tryptic hydrolyzates obtained for matrix (M1) proteins of A(H1N1) and A(H3N2) influenza viruses revealed differences that were greatest between the protein M1 molecules isolated from influenza viruses of different subtypes of hemagglutinine. These findings suggest there are variations in the structure of this conservative internal viral protein M1 during evolution.