RESUMO
We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependant RNA polymerase.
Assuntos
Vírus da Bronquite Infecciosa/genética , Plasmídeos/genética , Genética Reversa , Animais , Técnicas de Cultura de Células , Chlorocebus aethiops , Infecções por Coronavirus/veterinária , DNA Complementar , Regulação Viral da Expressão Gênica , Genoma Viral , Recombinação Homóloga , RNA Viral , Genética Reversa/métodos , Transfecção , Vaccinia virus/genética , Células Vero , Ensaio de Placa Viral , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/isolamento & purificação , Replicação ViralRESUMO
Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts.
Assuntos
Membrana Celular/virologia , Galinhas/virologia , Retículo Endoplasmático/virologia , Gammacoronavirus/patogenicidade , Animais , Células Cultivadas , Retículo Endoplasmático/química , Gammacoronavirus/classificação , Gammacoronavirus/fisiologia , Técnicas de Cultura de Órgãos , Fenótipo , Traqueia/virologia , Virulência , Replicação ViralRESUMO
RNA viruses are known for a high mutation rate and rapid genomic evolution. As such an RNA virus population does not consist of a single genotype but is rather a collection of individual viruses with closely related genotypes-a quasispecies, which can be analyzed by next-generation sequencing (NGS). This diversity of genotypes provides a mechanism in which a virus population can evolve and adapt to a changing environment. Sample preparation is vital for successful sequencing. The following protocol describes the process of generating a high-quality RNA preparation from IBV grown in embryonated eggs and then partially purified and concentrated through a 30% sucrose cushion for NGS.
Assuntos
Vírus da Bronquite Infecciosa/isolamento & purificação , RNA Viral/isolamento & purificação , Animais , Embrião de Galinha , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Bronquite Infecciosa/genética , RNA Viral/genética , Análise de Sequência de RNARESUMO
We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA; this has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.
Assuntos
Vírus da Bronquite Infecciosa/genética , Animais , Técnicas de Cultura de Células , Galinhas , Chlorocebus aethiops , DNA Viral/genética , DNA Viral/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Genes Dominantes , Genes Virais , Engenharia Genética , Recombinação Homóloga , Vírus da Bronquite Infecciosa/isolamento & purificação , Genética Reversa , Seleção Genética , Transfecção , Vaccinia virus/isolamento & purificação , Vaccinia virus/fisiologia , Células Vero , Cultura de VírusRESUMO
Infectious bronchitis virus (IBV) is an economically important virus infecting chickens, causing large losses to the poultry industry globally. While vaccines are available, there is a requirement for novel vaccine strategies due to high strain variation and poor cross-protection. This requires a more detailed understanding of virus-host cell interactions to identify candidates for targeted virus attenuation. One key area of research in the positive sense RNA virus field, due to its central role in virus replication, is the induction of cellular membrane rearrangements by this class of viruses for the assembly of virus replication complexes. In our recent work, we identified the structures induced by IBV during infection of cultured cells, as well as primary cells and ex vivo organ culture. We identified structures novel to the coronavirus family, which strongly resemble replication sites of other positive sense RNA viruses. We have begun to extend this work using recombinant IBVs, which are chimera of different virus strains to study the role of viral proteins in the induction of membrane rearrangements.