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1.
RNA ; 2021 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-33888553

RESUMEN

Around half of the genome in mammals is composed of transposable elements (TEs) such as DNA transposons and retrotransposons. Several mechanisms have evolved to prevent their activity and the detrimental impact of their insertional mutagenesis. Despite these potentially negative effects, TEs are essential drivers of evolution, and in certain settings, beneficial to their hosts. For instance, TEs have rewired the antiviral gene regulatory network and are required for early embryonic development. However, due to structural similarities between TE-derived and viral nucleic acids, cells can misidentify TEs as invading viruses and trigger the major antiviral innate immune pathway, the type I interferon (IFN) response. This review will focus on the different settings in which the role of TE-mediated IFN activation has been documented, including cancer and senescence. Importantly, TEs may also play a causative role in the development of complex autoimmune diseases characterised by constitutive type I IFN activation. All these observations suggest the presence of strong but opposing forces driving the coevolution of TEs and antiviral defence. A better biological understanding of the TE replicative cycle as well as of the antiviral nucleic acid sensing mechanisms will provide insights into how these two biological processes interact and will help to design better strategies to treat human diseases characterised by aberrant TE expression and/or type I IFN activation.

2.
Antimicrob Agents Chemother ; 60(5): 2981-92, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26953209

RESUMEN

Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs.


Asunto(s)
Fosfatos de Dinucleósidos/genética , Hepacivirus/genética , Antivirales/farmacología , Western Blotting , Línea Celular , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Farmacorresistencia Viral/genética , Genotipo , Humanos , Replicón/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sofosbuvir/farmacología , Replicación Viral/efectos de los fármacos , Replicación Viral/genética
3.
J Clin Microbiol ; 54(10): 2470-84, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27385709

RESUMEN

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.


Asunto(s)
Genoma Viral , Genotipo , Hepacivirus/genética , Hepatitis C/diagnóstico , Hepatitis C/virología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Farmacorresistencia Viral , Hepacivirus/clasificación , Humanos , Reino Unido
4.
Nucleic Acids Res ; 42(7): 4527-45, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24470146

RESUMEN

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-ß, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.


Asunto(s)
Fosfatos de Dinucleósidos/fisiología , Enterovirus Humano B/fisiología , Secuencia Rica en GC/fisiología , ARN Viral/química , Replicación Viral , Composición de Base , Línea Celular , Enterovirus Humano B/genética , Mutación
5.
Nucleic Acids Res ; 42(5): 3314-29, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24335283

RESUMEN

RNA viruses infecting vertebrates differ fundamentally in their ability to establish persistent infections with markedly different patterns of transmission, disease mechanisms and evolutionary relationships with their hosts. Although interactions with host innate and adaptive responses are complex and persistence mechanisms likely multi-factorial, we previously observed associations between bioinformatically predicted RNA secondary formation in genomes of positive-stranded RNA viruses with their in vivo fitness and persistence. To analyse this interactions functionally, we transfected fibroblasts with non-replicating, non-translated RNA transcripts from RNA viral genomes with differing degrees of genome-scale ordered RNA structure (GORS). Single-stranded RNA transcripts induced interferon-ß mediated though RIG-I and PKR activation, the latter associated with rapid induction of antiviral stress granules. A striking inverse correlation was observed between induction of both cellular responses with transcript RNA structure formation that was independent of both nucleotide composition and sequence length. The consistent inability of cells to recognize RNA transcripts possessing GORS extended to downstream differences from unstructured transcripts in expression of TNF-α, other interferon-stimulated genes and induction of apoptosis. This functional association provides novel insights into interactions between virus and host early after infection and provides evidence for a novel mechanism for evading intrinsic and innate immune responses.


Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , ARN Viral/química , Animales , Línea Celular , Humanos , Inmunidad Innata/genética , Interferón beta/biosíntesis , Conformación de Ácido Nucleico , Transducción de Señal , Transfección
6.
Nucleic Acids Res ; 41(12): 6316-31, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23630317

RESUMEN

Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1-7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.


Asunto(s)
Aptitud Genética , Norovirus/genética , ARN Viral/química , Replicación Viral/genética , Animales , Células Cultivadas , Cricetinae , Genoma Viral , Cinética , Ratones , Mutación , Norovirus/crecimiento & desarrollo , Norovirus/fisiología , Conformación de Ácido Nucleico , Fenotipo , Cultivo de Virus
7.
Viruses ; 16(2)2024 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-38399949

RESUMEN

The type-I interferon (IFN) response constitutes the major innate immune pathway against viruses in mammals. Despite its critical importance for antiviral defence, this pathway is inactive during early embryonic development. There seems to be an incompatibility between the IFN response and pluripotency, the ability of embryonic cells to develop into any cell type of an adult organism. Instead, pluripotent cells employ alternative ways to defend against viruses that are typically associated with safeguard mechanisms against transposable elements. The absence of an inducible IFN response in pluripotent cells and the constitutive activation of the alternative antiviral pathways have led to the hypothesis that embryonic cells are highly resistant to viruses. However, some findings challenge this interpretation. We have performed a meta-analysis that suggests that the susceptibility of pluripotent cells to viruses is directly correlated with the presence of receptors or co-receptors for viral adhesion and entry. These results challenge the current view of pluripotent cells as intrinsically resistant to infections and raise the fundamental question of why these cells have sacrificed the major antiviral defence pathway if this renders them susceptible to viruses.


Asunto(s)
Interferón Tipo I , Virus , Animales , Mamíferos , Antivirales , Inmunidad Innata
8.
J Clin Microbiol ; 50(9): 2910-7, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22740708

RESUMEN

Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and -20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.


Asunto(s)
Enterovirus/clasificación , Enterovirus/aislamiento & purificación , Parechovirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Rhinovirus/clasificación , Rhinovirus/aislamiento & purificación , Enterovirus/genética , Humanos , Tamizaje Masivo/métodos , Datos de Secuencia Molecular , Parechovirus/genética , ARN Viral/genética , ARN Viral/aislamiento & purificación , Rhinovirus/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Transcripción Genética , Virología/métodos
9.
J Clin Microbiol ; 49(7): 2620-4, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21593263

RESUMEN

Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5'-untranslated regions (5'UTR). Amplification dynamics (threshold cycle [C(T)]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). C(T) values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame.


Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Infecciones por Enterovirus/diagnóstico , Enterovirus/aislamiento & purificación , Parechovirus/aislamiento & purificación , Infecciones por Picornaviridae/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virología/métodos , Niño , Preescolar , Enterovirus/genética , Infecciones por Enterovirus/virología , Humanos , Parechovirus/genética , Infecciones por Picornaviridae/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo , Adulto Joven
10.
J Virol ; 84(11): 5494-507, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20237087

RESUMEN

Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1(WT), the JFH1(N415D) was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1(WT) under MAb AP33 selective pressure. MAb AP33 neutralized JFH1(T416A) and JFH1(I422L) more efficiently than the WT virus, while neutralization of JFH1(N417S) and JFH1(G418D) was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1(N415D), highlighting the importance of the E2 412-423 region in virus entry.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Hepacivirus/inmunología , Mutación , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Secuencia Conservada/genética , Humanos , Internalización del Virus
11.
J Virol ; 83(12): 6149-60, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19321602

RESUMEN

Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.


Asunto(s)
Antígenos CD/metabolismo , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/inmunología , Proteínas del Envoltorio Viral/genética , Sustitución de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Sitios de Unión , Línea Celular , Epítopos/inmunología , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Hepatitis C/inmunología , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Pruebas de Neutralización , Análisis de Secuencia de Proteína , Tetraspanina 28 , Proteínas del Envoltorio Viral/metabolismo
12.
Bio Protoc ; 9(21): e3417, 2019 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-33654916

RESUMEN

Controlled differentiation of embryonic stem cells is an essential tool in stem cell research. In this protocol, we describe a simple differentiation protocol involving the induction of embryoid body formation in mouse embryonic stem cells (mESC) using hanging droplets, followed by differentiation into a neuronal lineage.

13.
Elife ; 82019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31012846

RESUMEN

When mammalian cells detect a viral infection, they initiate a type I interferon (IFNs) response as part of their innate immune system. This antiviral mechanism is conserved in virtually all cell types, except for embryonic stem cells (ESCs) and oocytes which are intrinsically incapable of producing IFNs. Despite the importance of the IFN response to fight viral infections, the mechanisms regulating this pathway during pluripotency are still unknown. Here we show that, in the absence of miRNAs, ESCs acquire an active IFN response. Proteomic analysis identified MAVS, a central component of the IFN pathway, to be actively silenced by miRNAs and responsible for suppressing IFN expression in ESCs. Furthermore, we show that knocking out a single miRNA, miR-673, restores the antiviral response in ESCs through MAVS regulation. Our findings suggest that the interaction between miR-673 and MAVS acts as a switch to suppress the antiviral IFN during pluripotency and present genetic approaches to enhance their antiviral immunity.


Asunto(s)
Inmunidad Innata , Virus de la Influenza A/inmunología , Interferones/metabolismo , Células Madre Embrionarias de Ratones/inmunología , Transducción de Señal , Animales , Ratones , Proteoma/análisis
14.
Noncoding RNA ; 5(1)2019 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-30909383

RESUMEN

As part of their innate immune response against viral infections, mammals activate the expression of type I interferons to prevent viral replication and dissemination. An antiviral RNAi-based response can be also activated in mammals, suggesting that several mechanisms can co-occur in the same cell and that these pathways must interact to enable the best antiviral response. Here, we will review how the classical type I interferon response and the recently described antiviral RNAi pathways interact in mammalian cells. Specifically, we will uncover how the small RNA biogenesis pathway, composed by the nucleases Drosha and Dicer can act as direct antiviral factors, and how the type-I interferon response regulates the function of these. We will also describe how the factors involved in small RNA biogenesis and specific small RNAs impact the activation of the type I interferon response and antiviral activity. With this, we aim to expose the complex and intricate network of interactions between the different antiviral pathways in mammals.

15.
Cell Rep ; 23(11): 3275-3285, 2018 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-29898398

RESUMEN

Type I interferons (IFNs) are central components of the antiviral response. Most cell types respond to viral infections by secreting IFNs, but the mechanisms that regulate correct expression of these cytokines are not completely understood. Here, we show that activation of the type I IFN response regulates the expression of miRNAs in a post-transcriptional manner. Activation of IFN expression alters the binding of the Microprocessor complex to pri-miRNAs, reducing its processing rate and thus leading to decreased levels of a subset of mature miRNAs in an IRF3-dependent manner. The rescue of Microprocessor function during the antiviral response downregulates the levels of IFN-ß and IFN-stimulated genes. All these findings support a model by which the inhibition of Microprocessor activity is an essential step to induce a robust type I IFN response in mammalian cells.


Asunto(s)
Interferón Tipo I/metabolismo , Precursores del ARN/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferón beta/genética , Interferón beta/metabolismo , MicroARNs/metabolismo , Poli I-C/genética , Poli I-C/metabolismo , Procesamiento Postranscripcional del ARN
16.
Virus Res ; 118(1-2): 55-61, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16387382

RESUMEN

White Spot Syndrome Virus (WSSV) is a highly pathogenic and prevalent virus affecting crustacea. A number of WSSV envelope proteins, including vp28, have been proposed to be involved in viral infectivity based on the ability of specific antibodies to attenuate WSSV-induced mortality in vivo. In the present study, a series of monoclonal and polyclonal antibodies targeting vp28 were tested for their ability to neutralize WSSV infectivity, with the purpose of identifying epitopes potentially involved in vp28-mediated infection of shrimp. Surprisingly, when used as protein A-purified immunoglobulin, none of the antibodies tested were capable of inhibiting WSSV infectivity. This included one polyclonal preparation that has been previously shown to inactivate WSSV, when used as whole rabbit serum. Moreover, strong inactivation of WSSV by some rabbit sera was observed, in a manner independent of anti-vp28 antibodies. These results underscore the problems associated with using heterogeneous reagents (e.g. whole rabbit antiserum) in viral neutralization experiments aimed at defining proteins involved in infection by WSSV. In light of this, the potential of anti-vp28 antibodies to specifically neutralize WSSV should be reconsidered.


Asunto(s)
Penaeidae/virología , Suero/fisiología , Proteínas del Envoltorio Viral/fisiología , Inactivación de Virus , Virus del Síndrome de la Mancha Blanca 1 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Pruebas de Neutralización , Conejos
17.
Dis Aquat Organ ; 70(1-2): 167-70, 2006 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-16875404

RESUMEN

It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture.


Asunto(s)
Penaeidae/virología , Proteínas del Envoltorio Viral/farmacología , Virus del Síndrome de la Mancha Blanca 1/patogenicidad , Animales , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Mortalidad , Penaeidae/inmunología , Organismos Libres de Patógenos Específicos , Factores de Tiempo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/administración & dosificación
18.
Antiviral Res ; 83(2): 148-55, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19397930

RESUMEN

The human pathogen hepatitis C virus (HCV) is associated with chronic liver disease. The recent development of the cell culture infectious HCV (HCVcc) system has opened up avenues for detailed studies on the life cycle of the virus and its interaction with the host cell. Current methods to quantitate virus infectivity in cell culture are time-consuming and labor-intensive. This study describes the generation of a cell-based secreted alkaline phosphatase (SEAP) reporter assay to facilitate in vitro studies of HCV infection and replication. This assay is based on a novel reporter cell line stably expressing the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted alkaline phosphatase via a recognition sequence of the viral NS3/4A serine protease. The SEAP reporter from a similar construct has previously been shown to be released from the fusion protein and be secreted into the extracellular culture medium following cleavage by the viral NS3/4A protease. The reporter cell line enabled rapid and sensitive quantification of HCV infection and viral replication in cell culture. The utility of this system for investigating virus entry, and for high throughput screening of entry inhibitors and other antiviral compounds was demonstrated using several inter- and intra-genotypic chimeras of HCV.


Asunto(s)
Hepacivirus/patogenicidad , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hepacivirus/crecimiento & desarrollo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sensibilidad y Especificidad , Factores de Tiempo , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismo
19.
J Gen Virol ; 90(Pt 1): 48-58, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19088272

RESUMEN

Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1(DeltaE1E2)-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1(W529A) was unaffected by the presence of neutralizing antibodies that inhibit E2-CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.


Asunto(s)
Antígenos CD/fisiología , Hepacivirus/fisiología , Hepatocitos/virología , Receptores Virales/fisiología , Sustitución de Aminoácidos/genética , Anticuerpos Antivirales/inmunología , Línea Celular Tumoral , Hepacivirus/genética , Humanos , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Tetraspanina 28 , Proteínas del Envoltorio Viral/genética , Replicación Viral
20.
J Gen Virol ; 89(Pt 3): 653-659, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18272755

RESUMEN

The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1-6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Proteínas del Envoltorio Viral/inmunología , Antígenos CD , Línea Celular , Epítopos/química , Epítopos/inmunología , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Humanos , Pruebas de Neutralización , Retroviridae/genética , Retroviridae/inmunología , Tetraspanina 28 , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Virión/genética , Virión/inmunología , Virión/metabolismo
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