RESUMO
Abstract INTRODUCTION: Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS: HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS: The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS: HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.
Assuntos
Humanos , Replicação Viral/imunologia , Vírus da Febre Amarela/imunologia , Montagem de Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Replicação Viral/genética , Vírus da Febre Amarela/genética , Montagem de Vírus/genética , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde , Células HEK293 , Vacinas de Partículas Semelhantes a Vírus/genética , Citometria de FluxoRESUMO
BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Viral , Genoma Viral , Análise de Sequência de RNA/métodos , Montagem de Vírus , Técnicas de Amplificação de Ácido Nucleico/métodos , Valores de Referência , Software , República Centro-Africana , Reprodutibilidade dos Testes , Alphavirus/genética , Mengovirus/genética , Biologia Computacional , Mapeamento de Sequências ContíguasRESUMO
Primary cultures were made from adult mouse spinal ganglia for depicting an ultrastructural description of rabies virus (RABV) infection in adult mouse sensory neuron cultures; they were infected with rabies virus for 24, 36, and 48 h. The monolayers were processed for transmission electron microscopy and immunochemistry studies at the end of each period. As previously reported, sensory neurons showed great susceptibility to infection by RABV; however, in none of the periods evaluated were assembled virions observed in the cytoplasm or seen to be associated with the cytoplasmic membrane. Instead, fibril matrices of aggregated ribonucleoprotein were detected in the cytoplasm. When infected culture lysate were inoculated into normal animals via intra-cerebral route it was observed that these animals developed clinical symptoms characteristic of infection and transmission electron microscopy revealed assembled virions in the cerebral cortex and other areas of the brain. Sensory neurons infected in vitro by RABV produced a large amount of unassembled viral ribonucleoprotein. However, this intracellular material was able to produce infection and virions on being intra-cerebrally inoculated. It can thus be suggested that the lack of intracellular assembly in sensory neurons forms part of an efficient dissemination strategy.
Assuntos
Animais , Camundongos , Gânglios Espinais/virologia , Neurônios Aferentes/virologia , Vírus da Raiva/ultraestrutura , Raiva/virologia , Células Cultivadas , Modelos Animais de Doenças , Imunofluorescência , Gânglios Espinais/ultraestrutura , Microscopia Eletrônica de Transmissão , Neurônios Aferentes , Vírus da Raiva/fisiologia , Fatores de Tempo , Montagem de VírusRESUMO
Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374