RESUMEN

The occlusion bodies of Autographa californica multiple nucleopolyhedrovirus are proteinaceous formations with significant biotechnological potential owing to their capacity to integrate foreign proteins through fusion with polyhedrin, their primary component. However, the strategy for successful heterologous protein inclusion still requires further refinement. In this study, we conducted a comparative assessment of various conditions to achieve the embedding of recombinant proteins within polyhedra. Two baculoviruses were constructed: AcPHGFP (polh+), with GFP as a fusion to wild type (wt) polyhedrin and AcΔPHGFP (polh+), with GFP fused to a fragment corresponding to amino acids 19 to 110 of polyhedrin. These baculoviruses were evaluated by infecting Sf9 cells and stably transformed Sf9, Sf9POLH, and Sf9POLHE44G cells. The stably transformed cells contributed another copy of wt or a mutant polyhedrin, respectively. Polyhedra of each type were isolated and characterized by classical methods. The fusion PHGFP showed more-efficient incorporation into polyhedra than ΔPHGFP in the three cell lines assayed. However, ΔPHGFP polyhedron yields were higher than those of PHGFP in Sf9 and Sf9POLH cells. Based on an integral analysis of the studied parameters, it can be concluded that, except for the AcΔPHGFP/Sf9POLHE44G combination, deficiencies in one factor can be offset by improved performance by another. The combinations AcPHGFP/Sf9POLHE44G and AcΔPHGFP/Sf9POLH stand out due to their high level of incorporation and the large number of recombinant polyhedra produced, respectively. Consequently, the choice between these approaches becomes dependent on the intended application.

Asunto(s)

Biotecnología , Nucleopoliedrovirus , Spodoptera , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Animales , Células Sf9 , Biotecnología/métodos , Spodoptera/virología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión , Cuerpos de Oclusión Viral/metabolismo , Cuerpos de Oclusión Viral/genética , Línea Celular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Baculoviruses have shown great potential as gene delivery vectors in mammals, although their effectiveness in transferring genes varies across different cell lines. A widely employed strategy to improve transduction efficiency is the pseudotyping of viral vectors. In this study, we aimed to develop a stable Sf9 insect cell line that inducibly expresses the G-protein of the vesicular stomatitis virus to pseudotype budded baculoviruses. It was obtained by inserting the VSV-G gene under the control of the very strong and infection-inducible pXXL promoter and was subsequently diluted to establish oligoclonal lines, which were selected by the fusogenic properties of VSV-G and its expression levels in infected cells and purified budded virions. Next, to enhance the performance of the cell line, the infection conditions under which functional pseudotyped baculoviruses are obtained were optimized. Finally, different baculoviruses were pseudotyped and the expression of the transgene was quantified in mammalian cells of diverse origins using flow cytometry. The transduction efficiency of pseudotyped baculovirus consistently increased across all tested mammalian cell lines compared with control viruses. These findings demonstrate the feasibility and advantages of improving gene delivery performance without the need to insert the pseudotyping gene into the baculoviral genomes.

Asunto(s)

Baculoviridae , Técnicas de Transferencia de Gen , Animales , Baculoviridae/genética , Línea Celular , Terapia Genética , Regiones Promotoras Genéticas , Vectores Genéticos/genética , Transducción Genética , Proteínas del Envoltorio Viral/genética , Mamíferos/genética , Mamíferos/metabolismo

RESUMEN

Poxins are poxviral proteins that act by degrading 2´3´-cGAMP, a key molecule of cGAS-STING axis that drives and amplifies the antiviral response. Previous works have described some poxin homologous among lepidopteran and baculoviral genes. In particular, P26, a poxin homologous from AcMNPV retains the 2´3´-cGAMP degradation activity in vitro. In this work, we demonstrated that the antiviral activity triggered by baculovirus was disrupted by the transient expression of P26 in murine and human cell lines, and the effect of this action is not only on IFN-ß production but also on the induction of IFN-λ. Besides, we proved P26 functionality in a stable-transformed cell line where the protein was constitutively expressed, preventing the production of IFN-ß induced by baculovirus and resulting in an improvement in the transduction efficiency by the attenuation of the antiviral activity. Finally, we incorporated P26 into budded virions by capsid display or passive incorporation, and the results showed that both strategies resulted in an improvement of 3-17 times in the efficiency of transgene expression in murine fibroblasts. Our results suggest that the incorporation of P26 to budded baculoviral vectors is a very promising tool to modulate negatively the innate antiviral cellular response and to improve the efficiency of gene delivery in mammalian cells. KEY POINTS: • P26 affects baculovirus-induced IFN-ß and IFN-λ production in mammalian cells. • Murine fibroblasts expressing P26 are more susceptible to transduction by baculovirus. • Incorporation of P26 into the virion improves gene delivery efficiency of baculovirus.

RESUMEN

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.

Asunto(s)

Reoviridae , Compartimentos de Replicación Viral , Animales , ARN/metabolismo , Reoviridae/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo

RESUMEN

The South American soybean pest, Rachiplusia nu (Guenée), is naturally infected by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Rachiplusia nu nucleopolyhedrovirus (RanuNPV). We compared their pathogenicity to fourth-instar R. nu larvae, by evaluating time to death and virus spread throughout the tissues in single and mixed infections. Bioassays showed that generalist AcMNPV had a faster speed of kill than specific RanuNPV, while the mixed-virus treatment did not statistically differ from AcMNPV alone. Histopathology evidenced similar tissue tropism for both viruses, but co-inoculation resulted in mostly AcMNPV-infected cells. In sequential inoculations, however, the first virus administered predominated over the second one. Implications on baculovirus interactions and biocontrol potential are discussed.

Asunto(s)

Lepidópteros , Mariposas Nocturnas , Nucleopoliedrovirus , Animales , Larva , Spodoptera , Virulencia

RESUMEN

The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-ß) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

Asunto(s)

Baculoviridae/genética , Interferón beta/genética , Interferones/genética , Interleucinas/genética , Proteínas de la Membrana/genética , Nucleotidiltransferasas/genética , Animales , Baculoviridae/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , ADN Viral/genética , ADN Viral/inmunología , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica , Células HEK293 , Especificidad del Huésped , Humanos , Interferón beta/inmunología , Interferones/inmunología , Interleucinas/inmunología , Proteínas de la Membrana/inmunología , Ratones , Células 3T3 NIH , Nucleotidiltransferasas/inmunología , Células Sf9 , Transducción de Señal , Spodoptera , Transducción Genética

RESUMEN

In the present study, we developed a complete process to produce in insect cells a high amount of the ectodomain of rabies virus glycoprotein G (GE) as suitable antigen for detecting anti-rabies antibodies. Using the baculovirus expression vector system in Sf9 insect cells combined with a novel chimeric promoter (polh-pSeL), the expression level reached a yield of 4.1 ± 0.3 mg/L culture, which was significantly higher than that achieved with the standard polh promoter alone. The protein was recovered from the cell lysates and easily purified in only one step by metal ion affinity chromatography, with a yield of 95% and a purity of 87%. Finally, GE was successfully used in an assay to detect specific antibodies in serum samples derived from rabies-vaccinated animals. The efficient strategy developed in this work is an interesting method to produce high amounts of this glycoprotein.

RESUMEN

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.

Asunto(s)

Interacciones Huésped-Patógeno , Nucleopoliedrovirus/fisiología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Técnicas de Inactivación de Genes , Cuerpos de Inclusión Viral/ultraestructura , Larva/virología , Lepidópteros/virología , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Unión Proteica , Proteínas Virales/genética , Replicación Viral

RESUMEN

Here, we developed a diagnostic ELISA for foot-and-mouth disease using recombinant occlusion bodies (rOBs) of baculovirus. We fused Δ3AB1-3, a polypeptide derived from non-structural proteins of foot-and-mouth disease virus, to polyhedrin (POLH), the major constituent of OBs, under polh promoter. To further assess the most convenient strategy to improve yields, we designed two recombinant baculoviruses, vPOLH and vPOLHE44G. These carried the sequence of the fusion protein POLH-Δ3AB1-3 with an additional copy in cis of polh or polh E44G , respectively, under p10 promoter. Our results show that both viruses expressed POLH-Δ3AB1-3, which was detected by western blot in purified rOBs with anti-POLH and anti-3AB1 antibodies. We also found that vPOLHE44G produced larger polyhedra and a significant increase of antigen yield (p < 0.01). Furthermore, the chimeric protein POLH-Δ3AB1-3 was recognized by sera from experimentally infected animals, showing that translational fusion to POLH does not alter the antigenicity of Δ3AB1-3. Finally, the rOBs were successfully used in an ELISA test to differentiate infected from vaccinated animals. Taken together, these results demonstrate the great potential of rOBs to develop diagnostic schemes adaptable to animal infectious diseases.

RESUMEN

Mal de Río Cuarto virus (MRCV) is a member of the Fijivirus genus, within the Reoviridae family, that replicates and assembles in cytoplasmic inclusion bodies called viroplasms. In this study, we investigated interactions between ten MRCV proteins by yeast two-hybrid (Y2H) assays and identified interactions of non-structural proteins P6/P6, P9-2/P9-2 and P6/P9-1. P9-1 and P6 are the major and minor components of the viroplasms respectively, whereas P9-2 is an N-glycosylated membrane protein of unknown function. Interactions involving P6 and P9-1 were confirmed by bimolecular fluorescence complementation (BiFC) in rice protoplasts. We demonstrated that a region including a predicted coiled-coil domain within the C-terminal moiety of P6 was necessary for P6/P6 and P6/P9-1 interactions. In turn, a short C-terminal arm was necessary for the previously reported P9-1 self-interaction. Transient expression of these proteins by agroinfiltration of Nicotiana benthamiana leaves showed very low accumulation levels and further in silico analyses allowed us to identify conserved PEST degradation sequences [rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] within P6 and P9-1. The removal of these PEST sequences resulted in a significant increase of the accumulation of both proteins.

Asunto(s)

Interacciones Huésped-Patógeno , Cuerpos de Inclusión/virología , Hojas de la Planta/virología , Protoplastos/virología , Reoviridae/genética , Proteínas no Estructurales Virales/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia Conservada , Expresión Génica , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Oryza/virología , Enfermedades de las Plantas/virología , Hojas de la Planta/metabolismo , Hojas de la Planta/ultraestructura , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteolisis , Protoplastos/metabolismo , Protoplastos/ultraestructura , Reoviridae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Nicotiana/virología , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo

RESUMEN

Baculoviruses are able to enter into mammalian cells, where they can express a transgene that is placed under an appropriate promoter, without producing infectious progeny. ORF109 encodes an essential baculovirus protein that participates in the interaction of the baculovirus with mammalian cells. To date, the mechanisms underlying this interaction are not yet known. We demonstrated that although a Ac109 knock out virus maintained its ability to enter into BHK-21 cells, there was a marked reduction in the expression efficiency of the nuclear transgene. Moreover, the amount of free cytoplasmic viral DNA, which was detected by transcription of a reporter gene, was severely diminished. These results suggest Ac109 could be involved in maintaining the integrity of the viral nucleic acid.

Asunto(s)

Eliminación de Gen , Nucleopoliedrovirus/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Animales , Línea Celular , Cricetinae , Técnicas de Inactivación de Genes , Genes Reporteros , Nucleopoliedrovirus/aislamiento & purificación , Nucleopoliedrovirus/fisiología , Cultivo de Virus , Replicación Viral

RESUMEN

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 core gene has been previously characterized as an essential late gene. Our results showed that budded virions could be detected in supernatants of infected Sf-9 cells, even when ac109 knockout viruses displayed a single-cell infection phenotype. Moreover, confocal microscopy analysis revealed that budded virions can enter the cytoplasm but are unable to enter the cell nucleus. This defect could be repaired by complementing ac109 in trans. In addition, polyhedra of normal size could be detected in Sf-9 nuclei infected with ac109 knockout viruses. However, electron microscopy demonstrated that these occlusion bodies were empty. Altogether, these results indicate that ac109 is required for infectivity of both phenotypes of virus.

Asunto(s)

Núcleo Celular/virología , Nucleopoliedrovirus/metabolismo , Proteínas Virales/metabolismo , Virión/metabolismo , Virión/fisiología , Animales , Línea Celular , Nucleopoliedrovirus/genética , Spodoptera , Proteínas Virales/genética

RESUMEN

The in vivo subcellular localization of Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

Asunto(s)

Reoviridae , Spodoptera/virología , Proteínas no Estructurales Virales/análisis , Proteínas no Estructurales Virales/fisiología , Animales , Línea Celular , Membrana Celular/química , Membrana Celular/virología , Núcleo Celular/química , Núcleo Celular/virología , Citoplasma/química , Citoplasma/virología , Citoesqueleto/virología , Genoma Viral , Proteínas Fluorescentes Verdes/genética , Microscopía Confocal , Proteínas Recombinantes de Fusión/análisis , Reoviridae/genética , Reoviridae/fisiología , Spodoptera/ultraestructura , Fracciones Subcelulares/química , Fracciones Subcelulares/virología , Proteínas no Estructurales Virales/genética

RESUMEN

Baculovirus occlusion-derived viruses (ODVs) and budded viruses (BVs) are morphologically and functionally distinct. ODVs are responsible for primary infection in insect hosts because of their high per os infectivity. On the contrary, BVs poorly infect endothelial gut cells, but propagate the infection in the tissues of insects with a high efficiency. P74 is one of the most important proteins from ODVs, and it participates in the attachment of this viral phenotype to endothelial cells in the midgut. We evaluated the possibility of pseudotyping BVs of Autographa californica multiple nucleopolyhedrovirus with two versions of P74 and its effect on their oral infectivity. Both recombinant BVs contained P74 and replicated similarly to wild-type viruses. Nevertheless, the presence of P74 on the BV's surface does not enhance the oral infectivity of this phenotype, suggesting that the presence of P74 in the membrane of budded virions interferes with their mechanism of infecting midgut cells.

Asunto(s)

Baculoviridae/patogenicidad , Lepidópteros/virología , Proteínas del Envoltorio Viral/metabolismo , Factores de Virulencia/metabolismo , Animales , Baculoviridae/genética , Línea Celular , Proteínas del Envoltorio Viral/genética , Factores de Virulencia/genética

RESUMEN

We describe a point mutation in the AcMNPV polyhedrin gene that produces abnormally large cubic polyhedra in packaging cell lines. A polyhedrin mutant baculovirus in which the single change E44G was introduced confirmed that this mutation and no other alterations in the AcMNPV genome was responsible for the abnormal phenotype. Although baculoviral VP39 protein was detected inside mutant polyhedra, electron microscopy demonstrated that only a proportion of the large crystals allow occlusion of virions. When compared with wild-type polyhedra, the mutant inoculum showed reduced oral infectivity for Rachiplusia nu larvae. Hence, the amino acid 44 substitution in the AcMNPV polyhedrin protein alters polyhedrin assembly and affects viral occlusion and infectivity.

Asunto(s)

Baculoviridae/genética , Baculoviridae/ultraestructura , Mutación Missense , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/ultraestructura , Sustitución de Aminoácidos/genética , Animales , Larva/virología , Lepidópteros/virología , Mutagénesis Sitio-Dirigida , Virulencia

RESUMEN

Oral infection of insect larvae with baculovirus is an advantageous methodology for producing high levels of recombinant proteins and for achieving plague control. However, many recombinant baculoviruses express a foreign protein in lieu of the polyhedrin and hence do not form occlusion bodies (occ-), resulting in extremely reduced per os infectivity in larvae. To overcome this limitation, stably transformed insect cell lines expressing polyhedrin capable of occluding occ- recombinant baculovirus by trans-complementation were developed to obtain oral inoculum for insect larvae infection. First, the optimum regulatory region of polyhedrin promoter was determined utilizing chloramphenicol acetyl transferase (CAT) as the reporter gene. After infection with occ- baculovirus, the higher expression levels of CAT were achieved when a region of 2735bp that contained sequences known to have transcriptional enhancer functions were present upstream the polyhedrin coding sequence. This regulatory region was selected to drive polyhedrin expression in insect cell lines. Transfection of Sf9 cells with plasmid carrying polyhedrin gene and stable cell lines established by selection with blasticidin showed polyhedrin expression and, moreover, crystalline polyhedron-like structures were visualized by optic microscopy. Oral infectivity was demonstrated by fluorescence detection in Rachiplusia nu larvae infected with occluded AcGFPpolh- baculovirus obtained using the system presented here.

Asunto(s)

Baculoviridae/fisiología , Ingeniería de Proteínas/métodos , Spodoptera/microbiología , Spodoptera/fisiología , Proteínas Estructurales Virales/metabolismo , Animales , Línea Celular , Larva , Proteínas de la Matriz de Cuerpos de Oclusión , Proteínas Recombinantes/metabolismo , Spodoptera/citología , Transfección , Proteínas Estructurales Virales/genética

RESUMEN

Hepatitis C virus (HCV) displays high genetic diversity. Inter-host sequence variability may mainly reflect a neutral drift evolution. In contrast, intra-host evolution may be driven by an adaptive selection to host responses to infection. Here, HCV E2 intra-host evolution in two patients during the course and follow-up of successive treatments with IFN-alpha and IFN-alpha/ribavirin was investigated. Phylogenetic analyses suggested that adaptive pressures prompt a continuous selection of viral variants derived from the previous ones (intra-lineage evolution) and/or a swapping of viral lineages during the course of the infection (inter-lineage evolution). Selection would act not only on the phenotypic features of hypervariable region 1 (HVR1) but also on those of the flanking regions. The pressures operate mainly at the amino acid level, but they also appeared to act on nucleotide sequences. Moreover, HVR1 heterogeneity seemed to be strongly constrained. This work contributes to the knowledge of HCV intra-host evolution during chronicity.

Asunto(s)

Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/virología , Interferón-alfa/farmacología , Ribavirina/farmacología , Proteínas del Envoltorio Viral/genética , Antivirales/uso terapéutico , ADN Viral/genética , Quimioterapia Combinada , Evolución Molecular , Variación Genética , Hepacivirus/clasificación , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes , Ribavirina/uso terapéutico , Resultado del Tratamiento , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

Hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) genome was directly sequenced from 12 chronically infected patients who had not responded to interferon (IFN) treatment. Due to the quasispecies nature of HCV circulating genomes, serum samples from four patients showing different evolutionary characteristics were further analysed. Serial samples from each patient were taken before, soon after and 14-23 months after a 6 month IFN treatment. HVR1 from each sample was amplified, cloned and the clones sequenced. For each patient, a phylogenetic analysis of the clones was performed and quasispecies complexity and genetic distances were calculated. The amino acid sequences and predicted antigenic profiles were analysed. The pre-treatment samples of the different patients presented dissimilar genetic quasispecies composition. For three of the patients, we showed that, regardless of the complexity or diversity of the viral populations before treatment, they evolved towards genetic diversification following selective pressure. Once the environment became stable, the entire population tended towards homogeneity. The fourth patient represented a case where different components of the quasispecies coexisted for long periods without replacement. We propose herein that the evolution of HVR1 of E2 is more likely to be directed by selection of clearly different subpopulations (modification of quasispecies equilibrium) than by a continuous mechanism related to the successive accumulation of point mutations. The prevalence of a quasispecies shift mechanism was revealed by the cloning analysis during the follow-up period of the evolutionary process.

Asunto(s)

Evolución Molecular , Hepacivirus/genética , Hepatitis C Crónica/virología , Proteínas Virales/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antivirales/uso terapéutico , Hepacivirus/clasificación , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Datos de Secuencia Molecular , Proteínas Recombinantes , Análisis de Secuencia de ADN , Resultado del Tratamiento , Proteínas Virales/química

RESUMEN

The Hepatitis A virus (HAV) has been classified in seven different genotypes, which include human (I, II, III, and VII) and simian (IV, V, and VI) groups. The sequence analysis of HAV strains contributes to the molecular epidemiology of the virus. Although the infection with HAV is endemic in Argentina and vaccination is being implemented in this country, using both IA and IB strains, there are very few data on the genotypes of the circulating viruses. On the basis of the sequences of 20 isolates collected in Buenos Aires during a 2-year period (extended to 3 years by two additional specimens), we observed the presence of a single sub-genotype, IA, but with a high genetic diversity. We analyzed the VP1-2A junction and also the VP3-VP1 region. Most of the Argentine isolates grouped in at least two clusters. One of these was related to South American strains, thus suggesting a co-circulation of related isolates in neighbor countries. The other cluster was composed only of Argentine specimens. Other sequences were more scattered along the phylogenetic tree. However, we demonstrated that a consistent genetic relatedness of sequences could only be inferred on the basis of a more extensive sequencing of each isolate.

Asunto(s)

Virus de la Hepatitis A/genética , Hepatitis A/virología , Adulto , Secuencia de Aminoácidos , Argentina/epidemiología , Cápside/genética , Variación Genética , Genotipo , Hepatitis A/epidemiología , Virus de la Hepatitis A/clasificación , Virus de la Hepatitis A/aislamiento & purificación , Humanos , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Proteínas Estructurales Virales/genética