RESUMO
Fowl adenovirus 9 (FAdV-9) has one of the largest genomes (45 kb) so far sequenced from all adenoviruses studied. Genus-specific genes located within the early (E) regions at the right and left ends of the viral genome have unknown functions except for ORF8 (Gam-1 gene), ORF22 and ORF1 (dUTPase gene). ORF19, located at the right end of the genome (nts 34,220-36,443), is predicted to encode a lipase protein and its homologs are also found in all FAdV genomes so far sequenced. The role of ORF19 in virus replication and virulence is unknown. To study ORF19 and explore its potential as a locus for foreign gene insertion, we generated one ORF19-deleted mutant virus (rFAdV-9Δ19-SwaI) and three FAdV-9Δ19-based recombinant viruses replacing ORF19 as follows: rFAdV-9Δ19-CAT and enhanced-green fluorescent protein (EGFP) cassette (CMV promoter-EGFP-poly A) in a rightward (rFAdV-9Δ19-EGFP-R) and leftward orientation (rFAdV-9Δ19-EGFP-L). All recombinant viruses were stable after three passages. In chicken hepatoma cells, rFAdV-9Δ19-SwaI, rFAdV-9Δ19-CAT and rFAdV-9Δ19-EGFP-R replicated at titers similar to that of the wild-type virus, whilst rFAdV-9Δ19-EGFP-L replicated at a much lower titer. Interestingly, FAdV-9Δ19-SwaI replicated at higher titers in cells and in embryonated eggs, respectively than those of wild-type and recombinant viruses. These observations suggest ORF19 is nonessential for replication and can be used as a novel cloning site for engineering FAdV-9-based recombinant viruses and rFAdV-9Δ19-SwaI could be used to determine its role for virus replication in vivo.
Assuntos
Aviadenovirus/fisiologia , Expressão Gênica , Proteínas Recombinantes/biossíntese , Proteínas Virais/metabolismo , Replicação Viral , Animais , Aviadenovirus/genética , Linhagem Celular , Galinhas , Deleção de Genes , Vetores Genéticos , Instabilidade Genômica , Proteínas Recombinantes/genética , Carga Viral , Proteínas Virais/genéticaRESUMO
Fowl adenovirus 4 (FAdV-4) is associated with economically important poultry diseases. Recent studies of fully sequenced genomes of FAdV-4 isolates suggest potential genomic regions associated with virulence and amenable for manipulation and vector development. Direct manipulation of viral genomes is cumbersome, as opposed to that of infectious clones-viral genomes cloned into plasmid or cosmid vectors. In this work, we generated an infectious clone, pFAdV-4 ON1, containing the entire viral genome of a nonpathogenic FAdV-4 (ON1 isolate). pFAdV-4 ON1 was used for targeted deletion of open reading frames (ORFs) 16 and 17 and replacement with the enhanced green fluorescence protein (EGFP) expression cassette to generate recombinant viruses. These viruses were viable, and EGFP was expressed in infected cells. Their replication, however, was significantly reduced with respect to that of the wild-type virus. These observations suggest the potential utility of FAdV-4 as a vaccine vector and the importance of ORFs 16 and 17 for virus replication at wild-type levels. To our knowledge, this is the first report of an infectious clone based on the FAdV-4 genome, and our results demonstrate its utility for studies of virulence determinants and as a platform for either vaccine or gene delivery vectors.
Assuntos
Adenoviridae/genética , Galinhas/virologia , Vetores Genéticos , Vacinas Virais , Adenoviridae/patogenicidade , Adenoviridae/fisiologia , Animais , Linhagem Celular Tumoral , Genoma Viral , Proteínas de Fluorescência Verde/genética , Fases de Leitura Aberta/genética , Recombinação Genética , Transgenes/genética , Replicação ViralRESUMO
Nonpathogenic fowl adenoviruses (FAdVs) are amenable for engineering multivalent vaccine platforms due to large stretches of nonessential DNA sequences in their genomes. We describe the generation of FAdV-9-based vaccine platforms by targeted homologous recombination in an infectious clone (pPacFAdV-9 or wild type FAdmid) containing the entire viral genome in a cosmid vector. The viral DNA is subsequently released from the cosmid by restriction enzyme digestion followed by transfection in a chicken hepatoma cell line (CH-SAH). Virus is harvested, propagated, and verified for foreign gene expression.
Assuntos
Aviadenovirus/genética , Cosmídeos/genética , Vacinas Virais/genética , Animais , Linhagem Celular Tumoral , Galinhas , Vetores Genéticos/genética , Genoma Viral , TransfecçãoRESUMO
Nanomaterial-based artificial enzymes or nanozymes exhibit superior properties such as stability, cost effectiveness and ease of preparation in comparison to conventional enzymes. However, the lower catalytic activity of nanozymes limits their sensitivity and thereby practical applications in the bioanalytical field. To overcome this drawback, herein we propose a very simple but highly sensitive, specific and low-cost dual enhanced colorimetric immunoassay for avian influenza A (H5N1) virus. 3,3´,5,5´- Tetramethylbenzidine (TMBZ) was used as a reducing agent to produce gold nanoparticles (Au NPs) with blue colored solution from a viral target-specific antibody-gold ion mixture at first step. The developed blue color from the sensing design was further amplified through catalytic activity of Au NPs in presence of TMBZ-hydrogen peroxide (H2O2) solution in second step. Hence, the developed dual enhanced colorimetric immunosensor enables the detection of avian influenza virus A (H5N1) with a limit of detection (LOD) of 1.11 pg/mL. Our results confirmed that the developed assay has superior sensitivity than the conventional ELISA method, plasmonic-based bioassay and commercial flu diagnostic kits. Proposed sensing method further showed its capability to detect viruses, avian influenza A (H4N6) and A (H9N2) virus, in blood samples with limit of detection of 0.0269 HAU and 0.0331 HAU respectively.
RESUMO
Fowl adenoviruses (FAdVs) are promising vectors for poultry vaccines and gene therapy. The commonly used human cytomegalovirus (CMV) promoter in recombinant FAdV-9 viruses (recFAdV-9s) leads to foreign gene expression that elicits an antibody response. Despite its strength, studies have shown that the CMV promoter is prone to silencing by methylation hampering the in vivo application of vectors containing this promoter. Therefore, to improve our virus vector system and circumvent potential limitations of silencing, we engineered recFAdV-9s with foreign gene expression cassettes carrying the CMV enhancer/chicken ß-actin (CAG) or the human elongation factor 1 alpha (EF1α) promoters with or without the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE). Chicken hepatoma cells (CH-SAH) infected with recFAdV-9s carrying either CAG or EF1α promoters expressed higher levels of foreign protein than those infected with recFAdV-9 carrying the CMV promoter. Incorporation of the WPRE element rendered lower gene expression regardless of promoter type. Surprisingly, most chickens inoculated with recFAdV-9 containing the CMV promoter had the highest antibody response to foreign protein compared to other promoters. Our findings suggest the importance of promoter selection for candidate virus vector vaccines based on humoral immune response rather than foreign protein expression levels in vitro.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Regiões Promotoras Genéticas/genética , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/fisiologia , GalinhasRESUMO
Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.
Assuntos
Hepatócitos/virologia , Vírus Oncolíticos/fisiologia , Orthoreovirus Aviário/fisiologia , Replicação Viral , Morte Celular , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Humanos , Modelos Moleculares , Proteínas Estruturais Virais/químicaRESUMO
N-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (NB). Since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if N-myc expression status would determine virotherapy efficacy to high-risk NB. We showed that induction of exogenous N-myc in a non-N-myc-amplified cell line background (TET-21N) increased susceptibility to oncolytic vesicular stomatitis virus (mutant VSVΔM51) and alleviated the type I IFN-induced antiviral state. Cells with basal N-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust IFN-mediated antiviral state. The same effects were also observed in NB cell lines with and without N-myc amplification. Microarray analysis showed that N-myc overexpression in TET-21N cells downregulated IFN-stimulated genes (ISGs) with known antiviral functions. Furthermore, virus infection caused significant changes in global gene expression in TET-21N cells overexpressing N-myc. Such changes involved ISGs with various functions. Therefore, the present study showed that augmented susceptibility to VSVΔM51 by N-myc at least involves downregulation of ISGs with antiviral functions and alleviation of the IFN-stimulated antiviral state. Our studies suggest the potential utility of N-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk NB using IFN-sensitive oncolytic viruses.