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1.
Cells ; 13(2)2024 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-38247817

RESUMO

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.


Assuntos
Proteínas M de Coronavírus , Animais , Cães , Humanos , Células Madin Darby de Rim Canino/metabolismo , Células Madin Darby de Rim Canino/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio , Miosinas , Proteínas rab de Ligação ao GTP/genética , Saccharomyces cerevisiae , Suínos , Proteínas da Matriz Viral , SARS-CoV-2/metabolismo , Vírus da Hepatite Murina/metabolismo , Células A549/metabolismo , Células A549/virologia , Vírus da Diarreia Epidêmica Suína/metabolismo
2.
Int J Mol Sci ; 21(17)2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854295

RESUMO

Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin-Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC's MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.


Assuntos
Adenovirus Caninos/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Células Madin Darby de Rim Canino/citologia , Cultura de Vírus/métodos , Animais , Bancos de Espécimes Biológicos , Adesão Celular , Meios de Cultura Livres de Soro , Cães , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células Madin Darby de Rim Canino/classificação , Células Madin Darby de Rim Canino/virologia , Replicação Viral , Sequenciamento do Exoma
3.
Viruses ; 12(7)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32708703

RESUMO

Canine adenoviruses (CAdVs) are divided into pathotypes CAdV1 and CAdV2, which cause infectious hepatitis and laryngotracheitis in canid animals, respectively. They can be the backbones of viral vectors that could be applied in recombinant vaccines or for gene transfer in dogs and in serologically naïve humans. Although conventional plasmid-based reverse genetics systems can be used to construct CAdV vectors, their large genome size creates technical difficulties in gene cloning and manipulation. In this study, we established an improved reverse genetics system for CAdVs using bacterial artificial chromosomes (BACs), in which genetic modifications can be efficiently and simply made through BAC recombineering. To validate the utility of this system, we used it to generate CAdV2 with the early region 1 gene deleted. This mutant was robustly generated and attenuated in cell culture. The results suggest that our established BAC-based reverse genetics system for CAdVs would be a useful and powerful tool for basic and advanced practical studies with these viruses.


Assuntos
Adenovirus Caninos/genética , Cromossomos Artificiais Bacterianos/genética , Genética Reversa/métodos , Infecções por Adenoviridae/veterinária , Infecções por Adenoviridae/virologia , Animais , Clonagem Molecular , Cães , Genoma Viral/genética , Hepatite Infecciosa Canina/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino/virologia
4.
Arch Pharm Res ; 43(5): 489-502, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32248350

RESUMO

Curcumin derivatives have been shown to inhibit replication of human influenza A viruses (IAVs). However, it is not clear whether curcumin and its derivatives can inhibit neuraminidase (NA) of influenza virus. In this study, a meaningful 3D quantitative structure-activity relationship model (comparative molecular field analysis R2 = 0.997, q2 = 0.527, s = 0.064, F = 282.663) was built to understand the chemical-biological interactions between their activities and neuraminidase. Molecular docking was used to predict binding models between curcumin derivatives and neuraminidase. Real-time polymerase chain reactions showed that the five active curcumin derivatives might have direct effects on viral particle infectivity in H1N1-infected lung epithelial (MDCK) cells. Neuraminidase activation assay showed that five active curcumin derivatives decreased H1N1-induced neuraminidase activation in MDCK cells. Indirect immunofluorescence assay indicated that two active curcumin derivatives (tetramethylcurcumin and curcumin) down-regulated the nucleoprotein expression. Curcumin inhibited IAV in vivo. The therapeutic mechanism of curcumin in the treatment of influenza viral pneumonia is related to improving the immune function of infected mice and regulating secretion of tumor necrosis-α, interleukin-6, and interferon-γ. These results indicate that curcumin derivatives inhibit IAV by blocking neuraminidase in the cellular model and curcumin also has anti-IAV activity in the animal model.


Assuntos
Antivirais/farmacologia , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Curcumina/química , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Vírus da Influenza A Subtipo H1N1/enzimologia , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Neuraminidase/metabolismo
5.
Biochem Biophys Res Commun ; 523(1): 183-189, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31843192

RESUMO

Canarium album (Lour.) Raeusch (C. album) as a normally medicinal and edible plant has been used widely in Asian countries and is considered a source of phytochemicals that are beneficial to human health. Here, we showed at the first time isocorilagin, a polyphenolic compound isolated from C. album, displayed antiviral activity against diverse strains of influenza A virus (IAV), including A/Puerto Rico/8/34 (H1N1), A/Aichi/2/68 (H3N2) and NA-H274Y (H1N1) with IC50 value of 9.19 ±â€¯1.99, 23.72 ±â€¯2.51 and 4.64 ±â€¯3.01 µM, respectively. Further mechanistic studies revealed that it clearly inhibited neuraminidase activity of IAV and directly influenced the virus release. The molecular docking studies presented isocorilagin could bind to the highly conserved residues in the active sites of NA, implying that isocorilagin may be effective against various influenza strains and not susceptible to produce drug resistance. Taken together, the results strongly suggest that isocorilagin has potential to be an effective, safe and affordable neuraminidase inhibitor against a diverse panel of IAV strains. More importantly, our work expands the biological activities of C. album extracts and provide a new option for the development of anti-influenza drug.


Assuntos
Antivirais/farmacologia , Burseraceae/química , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Taninos/farmacologia , Animais , Antivirais/química , Antivirais/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Neuraminidase/metabolismo , Relação Estrutura-Atividade , Taninos/química , Taninos/isolamento & purificação
6.
Planta Med ; 85(3): 195-202, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30130818

RESUMO

In an in vitro screening for anti-influenza agents from European polypores, the fruit body extract of Gloeophyllum odoratum dose-dependently inhibited the cytopathic effect of the H3N2 influenza virus A/Hong Kong/68 (HK/68) in Madin Darby canine kidney cells with a 50% inhibitory concentration (IC50) of 15 µg/mL, a noncytotoxic concentration. After a chromatographic work-up, eight lanostane triterpenes (1: -8: ) were isolated and their structures were elucidated based on high-resolution electrospray ionization mass spectrometry analyses, and one- and two-dimensional nuclear magnetic resonance experiments. Constituents 1: (gloeophyllin K) and 2: (gloeophyllin L) are reported here for the first time, and compounds 5: , 7: , and 8: have not been described for the investigated fungal material so far. The highest activity was determined for trametenolic acid B (3: ) against HK/68 and the 2009 pandemic H1N1 strain A/Jena/8178/09 with IC50 values of 14 and 11 µM, respectively. In a plaque reduction assay, this compound was able to bind to cell-free viruses and to neutralize their infectivity.


Assuntos
Antivirais/farmacologia , Basidiomycota/química , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Antivirais/isolamento & purificação , Cães , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Células Madin Darby de Rim Canino/virologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Triterpenos/isolamento & purificação , Ensaio de Placa Viral
7.
Med Chem ; 14(6): 595-603, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29332597

RESUMO

BACKGROUND: T-705 (Favipiravir) is a broad spectrum antiviral agent approved for stockpiling in Japan and currently in Phase 3 testing in the United States. Against influenza, it acts as a prodrug, converted intracellularly to selectively inhibit viral RNA-dependent RNA polymerase or similar enzymes. This is regarded as a novel antiviral mechanism of action, reducing crossresistance to other existing anti-influenza drugs. OBJECTIVE: To develop new analogs, a class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized in this work. RESULTS: Anti-influenza activity and Anti-HIV activity of these compounds were evaluated. Compound 1a displayed activity against A H1N1 with an IC50 of 40.4 µmol/L. Compound 1b showed weak activity against HIV with a viral suppression rate of 70-80% at 30 µmol/L. CONCLUSION: A class of 1,3-oxathiolane nucleoside derivatives of T-705 was designed and synthesized, and one of them was identified as a novel scaffold against viral infection.


Assuntos
Fármacos Anti-HIV/farmacologia , Nucleosídeos/farmacologia , Tiofenos/farmacologia , Amidas/farmacologia , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/toxicidade , Cães , Células HEK293 , HIV/efeitos dos fármacos , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Isomerismo , Células Madin Darby de Rim Canino/virologia , Nucleosídeos/síntese química , Nucleosídeos/química , Nucleosídeos/toxicidade , Pirazinas/farmacologia , Ribavirina/farmacologia , Tiofenos/síntese química , Tiofenos/química , Tiofenos/toxicidade
8.
ACS Nano ; 12(2): 1188-1202, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29357226

RESUMO

Silver nanoparticles (AgNPs) are microbicidal agents which could be potentially used as an alternative to antivirals to treat human infectious diseases, especially influenza virus infections where antivirals have generally proven unsuccessful. However, concerns about the use of AgNPs on humans arise from their potential toxicity, although mechanisms are not well-understood. We show here, in the context of an influenza virus infection of lung epithelial cells, that AgNPs down-regulated influenza induced CCL-5 and -IFN-ß release (two cytokines important in antiviral immunity) through RIG-I inhibition, while enhancing IL-8 production, a cytokine important for mobilizing host antibacterial responses. AgNPs activity was independent of coating and was not observed with gold nanoparticles. Down-stream analysis indicated that AgNPs disorganized the mitochondrial network and prevented the antiviral IRF-7 transcription factor influx into the nucleus. Importantly, we showed that the modulation of RIG-I-IRF-7 pathway was concomitant with inhibition of either classical or alternative autophagy (ATG-5- and Rab-9 dependent, respectively), depending on the epithelial cell type used. Altogether, this demonstration of a AgNPs-mediated functional dichotomy (down-regulation of IFN-dependent antiviral responses and up-regulation of IL-8-dependent antibacterial responses) may have practical implications for their use in the clinic.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/química , Mitocôndrias/efeitos dos fármacos , Orthomyxoviridae/efeitos dos fármacos , Prata/farmacologia , Tretinoína/farmacologia , Animais , Antivirais/química , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cães , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Testes de Sensibilidade Microbiana , Mitocôndrias/metabolismo , Prata/química , Tretinoína/química
9.
Org Biomol Chem ; 15(5): 1130-1139, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28054092

RESUMO

Reactions of O-t-butyldimethylsilyl-protected thymidine, 2'-deoxyuridine, and 3'-azidothymidine (AZT) with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) leads to activation of the C4 amide carbonyl by formation of putative O4-(benzotriazol-1-yl) derivatives. Subsequent substitution with alkyl and aryl amines, thiols, and alcohols leads to facile functionalization at this position. Reactions with amines and thiols were conducted either as a two-step, one-pot transformation, or as a one-step conversion. Reactions with alcohols were conducted as two-step, one-pot transformations. In the course of these investigations, the formation of 1-(4-pyrimidinyl)-1H-benzotriazole-3-oxide derivatives from the pyrimidine nucleosides was identified. However, these too underwent conversion to the desired products. Products obtained from AZT were converted to the 3'-amino derivatives by catalytic reduction. All products were assayed for their abilities to inhibit cancer cell proliferation and for antiviral activities. Many were seen to be active against HIV-1 and HIV-2, and one was active against herpes simplex virus-1 (HSV-1).


Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Compostos Organofosforados/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Amidas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Camundongos , Testes de Sensibilidade Microbiana , Conformação Molecular , Compostos Organofosforados/química , Nucleosídeos de Pirimidina/química , Relação Estrutura-Atividade
10.
Sci Rep ; 6: 23529, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-27004747

RESUMO

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.


Assuntos
Adenovirus Caninos/fisiologia , Glucose/farmacocinética , Glutamina/farmacocinética , Células Madin Darby de Rim Canino/virologia , Análise do Fluxo Metabólico/métodos , Animais , Isótopos de Carbono/farmacocinética , Proliferação de Células , Transformação Celular Viral , Cães , Regulação da Expressão Gênica , Glicólise , Lipogênese , Células Madin Darby de Rim Canino/metabolismo , Via de Pentose Fosfato
11.
Cell Microbiol ; 18(6): 784-91, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26687707

RESUMO

Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Interferon beta/metabolismo , Treonina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58/metabolismo , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Interferon beta/genética , Células Madin Darby de Rim Canino/virologia , Mutação , Fosforilação , Regiões Promotoras Genéticas , Receptores Imunológicos , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/genética , Replicação Viral
12.
Arch Virol ; 160(6): 1397-405, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25797195

RESUMO

The Madin-Darby canine kidney (MDCK) cell line is typically used to analyze pathological features after canine influenza virus (CIV) infection. However, MDCK cells are not the ideal cell type, because they are kidney epithelial cells. Therefore, we generated an immortalized canine tracheal epithelial cell line, KU-CBE, to more reliably study immune responses to CIV infection in the respiratory tract. KU-CBE cells expressed the influenza virus receptor, α-2,3-sialic acid (SA), but not α-2,6-SA. KU-CBE and MDCK cells infected with H3N2 CIV demonstrated comparable virus growth kinetics. Gene expression levels of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-ß were estimated in both KU-CBE and MDCK cells infected with CIV by real-time reverse transcription polymerase chain reaction (qRT-PCR). Of these cytokines, IL-4, IL-10, TNF-α, and IFN-ß mRNAs were detected in both cell lines. Gene expression of IL-4, IL-10, and TNF-α was not significantly different in the two cell lines. However, MDCK cells exhibited a significantly higher level of IFN-ß mRNA than KU-CBE cells at 18 h post infection. Additionally, the protein concentrations of these four cytokines were determined by enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants obtained from the two CIV-infected cell lines. MDCK cells produced significantly higher amounts of IL-4 and IFN-ß than KU-CBE cells. However, KU-CBE cells produced a significantly higher amount of TNF-α than MDCK cells. These data indicated that the newly developed canine tracheal epithelial cells exhibited different cytokine production patterns compared to MDCK cells when infected with CIV. Inflammation of the respiratory tract of dogs induced by CIV infection may be attributed to the elevated expression level of TNF-α in canine tracheal epithelial cells.


Assuntos
Citocinas/fisiologia , Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N2 , Infecções por Orthomyxoviridae/veterinária , Mucosa Respiratória/citologia , Traqueia/citologia , Animais , Linhagem Celular , Citocinas/biossíntese , Doenças do Cão/imunologia , Cães , Interferon beta/biossíntese , Interferon beta/fisiologia , Interleucina-10/biossíntese , Interleucina-10/fisiologia , Interleucina-1beta/biossíntese , Interleucina-1beta/fisiologia , Interleucina-2/biossíntese , Interleucina-2/fisiologia , Interleucina-4/biossíntese , Interleucina-4/fisiologia , Interleucina-6/biossíntese , Interleucina-6/fisiologia , Interleucina-8/biossíntese , Interleucina-8/fisiologia , Células Madin Darby de Rim Canino/imunologia , Células Madin Darby de Rim Canino/virologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Mucosa Respiratória/fisiopatologia , Mucosa Respiratória/virologia , Traqueia/fisiopatologia , Traqueia/virologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/fisiologia
13.
Vaccine ; 31(48): 5693-9, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24113260

RESUMO

Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10⁻5, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log10 HAU/100 µL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log10 HAU/100 µL and TCID50 of 1 × 109 and 2.37 × 109 infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.


Assuntos
Apoptose , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Células Madin Darby de Rim Canino/fisiologia , Células Madin Darby de Rim Canino/virologia , Carga Viral , Animais , Adesão Celular , Sobrevivência Celular , Células Imobilizadas , Cães , Cultura de Vírus/métodos
14.
PLoS One ; 8(3): e59892, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555825

RESUMO

Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/ß induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.


Assuntos
Fator Regulador 7 de Interferon/genética , Células Madin Darby de Rim Canino/virologia , Orthomyxoviridae/fisiologia , Cultura de Vírus , Replicação Viral , Animais , Linhagem Celular Tumoral , Cães , Biblioteca Gênica , Humanos , Vacinas contra Influenza/biossíntese , Interferon Tipo I/metabolismo , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética
15.
J Sci Food Agric ; 93(9): 2239-41, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23355221

RESUMO

BACKGROUND: Berries are known to have many kinds of biological activities. We focused on their antiviral effect, which has not yet been well evaluated. RESULTS: We compared the anti-influenza viral effects of berries belonging to the genus Vaccinium - 35 species of blueberry (Vaccinium cyanococcus), the Natsuhaze (Vaccinium oldhamii), bilberry (Vaccinium myrtillus) and cranberry (Vaccinium oxycoccos)- with those belonging to the genus Ribes, i.e. blackcurrant (Ribes nigrum). Only Elliott and Legacy among Northern Highbush varieties but many Rabbiteye varieties such as Austin, Baldwin, Brightblue, Festival, T-100 and Tifblue showed anti-influenza viral activity. Natsuhaze, bilberry, cranberry and blackcurrant had high antiviral effects. A relationship was observed between the antiviral effect and total polyphenol content. CONCLUSIONS: Antiviral effects were found to differ markedly between berry species. Rabbiteye varieties tended to have higher antiviral effects than Northern, Southern and Half Highbush blueberry varieties. We also found that Natsuhaze, which has recently been harvested in Japan as a potential functional food, had an antiviral effect comparable to that of bilberry, cranberry and blackcurrant. There was a positive relationship between antiviral activity and polyphenol content, indicating the possibility that polyphenol is one of the key factors in the antiviral effects of berries.


Assuntos
Antivirais/análise , Frutas/química , Alimento Funcional/análise , Vírus da Influenza A/crescimento & desenvolvimento , Polifenóis/análise , Ribes/química , Vaccinium/química , Adsorção/efeitos dos fármacos , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Mirtilos Azuis (Planta)/química , Mirtilos Azuis (Planta)/crescimento & desenvolvimento , Mirtilos Azuis (Planta)/metabolismo , Linhagem Celular , Cães , Europa (Continente) , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Japão , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Viabilidade Microbiana/efeitos dos fármacos , Nova Zelândia , Extratos Vegetais/farmacologia , Polifenóis/biossíntese , Polifenóis/farmacologia , Ribes/crescimento & desenvolvimento , Ribes/metabolismo , Especificidade da Espécie , Estados Unidos , Vaccinium/crescimento & desenvolvimento , Vaccinium/metabolismo , Ligação Viral/efeitos dos fármacos , Meio Selvagem
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